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Host Range, Phylogenetic, and Pathogenic Diversity of Corynespora cassiicola (Berk. and Curt.) Wei

Permanent Link: http://ufdc.ufl.edu/UFE0022669/00001

Material Information

Title: Host Range, Phylogenetic, and Pathogenic Diversity of Corynespora cassiicola (Berk. and Curt.) Wei
Physical Description: 1 online resource (102 p.)
Language: english
Creator: Dixon, Linley
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: disease, fungi, papaya, phylogenetics, population, spot, target, tomato
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The fungus Corynespora cassiicola (Berk. & Curt.) Wei is a pathogen, endophyte, and saprophyte. It can be found growing on at least 530 plant species from 380 genera, primarily in the tropics. Isolates from diverse hosts were collected or solicited from locations in American Samoa, Brazil, Malaysia, Micronesia, and Florida, Mississippi, and Tennessee within the United States. Outgroup taxa including C. citricola, C. melongenea, C. olivaceae, C. proliferata, C. sesamum, and C. smithii were solicited from culture collections. A multilocus phylogenetic analysis using 143 isolates was performed to investigate how genetic diversity correlates with host-specificity, growth rate, and geographic distribution. Phylogenetic trees were congruent from the rDNA ITS region, two random hypervariable loci (Cs caa5 and Cs ga4), and the actin encoding locus CC act1, indicating asexual propagation. Fifty isolates had different pathogenicity profiles when tested against eight known C. cassiicola hosts: basil, bean, cowpea, cucumber, papaya, soybean, sweet potato, and tomato. Phylogenetic lineage correlated with pathogenicity profiles, host originality, and growth rate, but not with geographic location. Common fungal genotypes were widely distributed geographically indicating long distance and global dispersal of clonal lineages. This research reveals an abundance of previously unrecognized diversity within the species and provides evidence for redefining species distinctions within Corynespora, which will aid in future disease control strategies.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Linley Dixon.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Datnoff, Lawrence E.
Local: Co-adviser: Pernezny, Kenneth L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022669:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022669/00001

Material Information

Title: Host Range, Phylogenetic, and Pathogenic Diversity of Corynespora cassiicola (Berk. and Curt.) Wei
Physical Description: 1 online resource (102 p.)
Language: english
Creator: Dixon, Linley
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: disease, fungi, papaya, phylogenetics, population, spot, target, tomato
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The fungus Corynespora cassiicola (Berk. & Curt.) Wei is a pathogen, endophyte, and saprophyte. It can be found growing on at least 530 plant species from 380 genera, primarily in the tropics. Isolates from diverse hosts were collected or solicited from locations in American Samoa, Brazil, Malaysia, Micronesia, and Florida, Mississippi, and Tennessee within the United States. Outgroup taxa including C. citricola, C. melongenea, C. olivaceae, C. proliferata, C. sesamum, and C. smithii were solicited from culture collections. A multilocus phylogenetic analysis using 143 isolates was performed to investigate how genetic diversity correlates with host-specificity, growth rate, and geographic distribution. Phylogenetic trees were congruent from the rDNA ITS region, two random hypervariable loci (Cs caa5 and Cs ga4), and the actin encoding locus CC act1, indicating asexual propagation. Fifty isolates had different pathogenicity profiles when tested against eight known C. cassiicola hosts: basil, bean, cowpea, cucumber, papaya, soybean, sweet potato, and tomato. Phylogenetic lineage correlated with pathogenicity profiles, host originality, and growth rate, but not with geographic location. Common fungal genotypes were widely distributed geographically indicating long distance and global dispersal of clonal lineages. This research reveals an abundance of previously unrecognized diversity within the species and provides evidence for redefining species distinctions within Corynespora, which will aid in future disease control strategies.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Linley Dixon.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Datnoff, Lawrence E.
Local: Co-adviser: Pernezny, Kenneth L.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022669:00001


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1 HOST RANGE, PHYLOGENETIC, AND PATHOGENIC DIVERSITY OF Corynespora cassiicola (Berk. & Curt.) Wei By LINLEY JOY SMITH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Linley Joy Smith

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3 To Peter, for making me laugh.

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4 ACKNOWLEDGMENTS Funding and support was m ade possible by the USDA Special Grant Program for Tropical and Subtropical Agriculture Research, the Univ ersity of Florida, IFAS, EREC, the Florida Tomato Committee, the University of Guam, Guam Cooperative Extens ion, and the USDA IPM 3-D and Hatch funds. I would like to thank Drs. Ken Pernezny, Pam Roberts, Jeffrey Rollins, and Jay Scott for their support while serving on my supervisory committee. I would also like to express appreciation to my major a dvisor, Dr. Lawrence Datnoff, for his commitment and help throughout the course of my Ph.D. I would especially like to thank Dr. Robert Schlub for his willingness to help in every step of the process and for his unwavering support, encouragement, and friendship. Special thanks to my helpful coworkers in Guam, especially Roger Brown and Lauren Gutierrez. Most importantly, my heartfelt appreciation goe s to my parents for their unconditional love and support. Finally, I thank my husband for en couraging me to pursue this opportunity, an ocean and continent away, for coming to Gainesville for me, and for keeping me smiling throughout.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........6 LIST OF FIGURES.........................................................................................................................7 ABSTRACT.....................................................................................................................................8 CHAP TER 1 INDEX OF PLANT HOSTS OF Corynespora cassiicola .....................................................10 Introduction................................................................................................................... ..........10 Methods..................................................................................................................................12 Literature Survey and Host Index.................................................................................... 12 Guam and Florida Surveys..............................................................................................13 Results.....................................................................................................................................14 Discussion...............................................................................................................................16 2 GENETIC AND PATHOGENIC DIVERSITY OF CORYNESPORA CASSIICOLA ...........48 Introduction................................................................................................................... ..........48 Methods..................................................................................................................................52 Collection and Solicitation of Fungal Isolates................................................................. 52 Primer Development for Ra ndom Hypervariable Loci................................................... 54 Fungal Cultures and Extraction of Genomic DNA......................................................... 55 Phylogenetic Analyses.....................................................................................................57 Pathogenicity Analyses...................................................................................................59 Growth Rate Analyses..................................................................................................... 60 Results.....................................................................................................................................61 Phylogenetic Analyses.....................................................................................................61 Pathogenicity Analyses...................................................................................................65 Growth Rate Analyses..................................................................................................... 66 Discussion...............................................................................................................................67 LIST OF REFERENCES...............................................................................................................90 BIOGRAPHICAL SKETCH.......................................................................................................102

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6 LIST OF TABLES Table page 1-1 Taxonomic grouping of Corynespora cassiicola host species. ......................................... 20 1-2 Occurrence and fungalhost interaction of Corynespora cassiicola identified during 2004-2005 Gua m and Florida surveys............................................................................... 21 2-1 Isolate designations, geog raphic location of isolation, hos t of isolation, phylogenetic lineage (PL), type of growth on a sso ciated host, and species of Corynespora used in the phylogenetic analyses.................................................................................................. 72 2-2 Summary of sequence data from four lo ci used to confirm the phylogenetic lineage of Corynespora cassiicola isolates....................................................................................76 2-3 Pathogenicity profiles for 50 Corynespora cassiicola isolates. .........................................77 2-4 Growth rate of Corynespora cassiicola isolates at 23C. .................................................. 79 2-5 Growth rate of Corynespora cassiicola isolates at 33C. .................................................. 81

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7 LIST OF FIGURES Figure page 1-1 Corynespora cassiicola isolate from Cucumis sativus ......................................................45 1-2 Various symptoms caused by Corynespora cassiicola on naturally infected leaves......... 46 2-1 Fifty percent majority rule consensu s tree-phylogram from Bayesian inference analysis of combined data from rDNA ITS, Cc-ga4, Cc-caa5, and Cc-act1 sequences...................................................................................................................... .....83 2-2 Fifty percent majority rule consensu s tree-phylogram from Bayesian inference analysis of rDNA ITS locus. ............................................................................................84 2-3 Fifty percent majority rule consensu s tree-phylogram from Bayesian inference analysis of the Cc-caa5 locus............................................................................................85 2-4 Fifty percent majority rule consensu s tree-phylogram from Bayesian inference analysis of the Cc-ga4 locus..............................................................................................86 2-5 Fifty percent majority rule consensu s tree-phylogram from Bayesian inference analysis of the Cc-act1 locus. .......................................................................................... 87 2-6 UPGMA dendrogram of 50 Corynespora cassiicola isolates based on pathogenicity profiles on eight crop plants:..............................................................................................88 2-7 Demonstration of the C. cassiicola disease rating system ................................................. 89

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8 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy HOST RANGE, PHYLOGENETIC, AND PATHOGENIC DIVERSITY OF Corynespora cassiicola (Berk. & Curt). Wei By Linley Joy Smith August 2008 Chair: Lawrence E. Datnoff Major: Plant Pathology The fungus Corynespora cassiicola (Berk. & Curt.) Wei is a pathogen, endophyte, and saprophyte. It can be found growing on at least 5 30 plant species from 380 genera, primarily in the tropics. Isolates from dive rse hosts were collected or solicited from locations in American Samoa, Brazil, Malaysia, Micronesia, and Florid a, Mississippi, and Tennessee within the United States. Outgroup taxa including C. citricola C melongenea C. olivaceae, C. proliferata C. sesamum and C. smithii were solicited from culture co llections. A multilocus phylogenetic analysis using 143 isolates was performed to investigate how genetic dive rsity correlates with host-specificity, growth rate, a nd geographic distribution. Phyl ogenetic trees were congruent from the rDNA ITS region, two random hypervariable loci ( Cs caa5 and Cs ga4), and the actin encoding locus CC act1, indicating asexual propagation. Fifty isolates had different pathogenicity profiles when tested against eight known C. cassiicola hosts: basil, bean, cowpea, cucumber, papaya, soybean, sweet potato, and toma to. Phylogenetic lineage correlated with pathogenicity profiles, host originality, and growth rate, but not with geographic location. Common fungal genotypes were widely distributed geographically indicating long distance and global dispersal of clonal lineages. This re search reveals an abundance of previously

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9 unrecognized diversity within the species and provides evid ence for redefining species distinctions within Corynespora, which will aid in future disease control strategies.

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10 CHAPTER 1 INDEX OF PLANT HOSTS OF CORYNESP ORA CASSIICOLA Introduction Corynespora cassiicola (Berk. & Curt.) W ei has been commonly reported as a plant pathogenic foliar fungus with a wide host range w ithin tropical and subtropical areas (Holliday 1980; Farr et al 1980; Romruensukharom et al 2005). In addition to being a pathogen, on some hosts C. cassiicola is also reported to grow as an endophyte or saprophyte (Collado 1999; Gond et al 2007; Promputtha et al. 2007; Suryanarayanan et al. 2002; Kingsland 1985; Hyde et al. 2001; Lee et al. 2004; Lumyong et al. 2003). Though the diseases attributed to C. cassiicola are mainly foliar, it may also cause fruit, stem and root diseases (J ones et al. 1991). The generalization that individual C. cassiicola isolates have a wide host range is not supported by the literature because hos t specific isolates, isolates pathogenic to select hosts, and weak pathogens or secondary invaders of senescent ti ssue are known to exist (Onesirosan et al. 1974; Cutrim and Silva et al. 2003; Kingsland 1985; Pere ira et al. 2003). Rarely reported outside the tropics and subtropics, there ar e occasional reports of the f ungus from temperate regions, particularly on soybean (Boosalis and Hamilton 1957; Malvick 2004; Raffel et al. 1999; Seaman et al. 1965). Disease symptoms attributed to C. cassiicola include necrosis, of ten with a surrounding yellow halo (Pernezny and Simone 1993) due to the production of a host specific protein toxin, cassiicolin (Barthe et al. 2007; Kurt 2004). With respect to foliage, young and mature leaves can be affected, although the pathogen is more commonly associated w ith older leaves (Pernezny et al. 2008). Substantial crop losses have been observed in many countries on numerous hosts: southern United States on ornamentals (Alfieri et al. 1984, 1994; Chase 1981,1982, 1984, 1986, 1987, 1993; El-Gholl and Schubert 1990; El-Gho ll et al. 1997; Miller and Alfieri 1973;

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11 McRitchie and Miller 1973; Simone 2000, 2000), cucumber (Abul-Hayja et al. 1978; Blazquez 1967; Strandburg 1971), and tomato (Bliss et al. 1973; Blazquez 1972; Jones and Jones 1984; Pernezny et al. 1996, 2002; Smith et al. 2006, Smith et al. 2008b); Midwestern United States on soybean (Boosalis and Hamilton 1957), cowpea (Olive and Bain 1945) and sesame (Stone and Jones 1960); India on ornamentals (Cheeran 1968; Mallaiah et al. 1981 ; Mehrotra 1987, 1997; Silva et al. 2000; Singh et al. 1982), Hevea rubber trees (Atan and Hamid 2003; Silva et al. 1998), cotton (Lakshmanan et al. 1990), and weed s (Philip et al. 1972); Brazil on ornamentals (Da Silva et al. 2005; Leite and Barreto 2000; Pohltronieri 2003) and weeds (Pereira et al. 2003); Philippines, Nigeria, and U.S. Virgin Is lands on papaya (Quimio and Abilay 1979; Oluma and Amuta 1999; Bird et al. 1966); and Microne sia and Asia on ornamentals (Florence and Sharma 1987; Hasama et al. 1991), cucurbit s (Yudin and Schlub 1998; Tsay and Kuo 1991), tomato (Schlub and Yudin 2002), and pepper (Kwon et al. 2001). Most regions report C. cassiicola diseases on only a few host species, despite the broad host range of the fungus, prompting questions pertaining to isolate host specificity and distribution. Addressing such questions will have implications for disease control and quarantine. The host -specificity and severity of the fungus on Lantana camara in Brazil led to the discovery of a new forma specialis, C. cassiicola f. sp. lantanae, and the use of the isolate as a bioherbicide (Pereira et al. 2003 ). Based on the vast number of weeds that serve as hosts, and past demonstration of host-specificity in some is olates, there is great pot ential for the discovery of additional isolates useful for biological control. Further information on the fungal-host interaction and host range of individual isolates will be useful in the st udy of disease epidemics. The objective of this study was to compile a list of C. cassiicola hosts into a single document, thereby aiding further research on the hos t range of individual isol ates. Prior to this

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12 study, the most complete host list ing is in the fungal database of the ARS/USDA Systematic Mycology and Microbiology Labor atory, which included 257 plan t host species (Farr et al. 2008). This study will provide a more complete index for use by those engaged in phylogenetic analysis of Corynespora spp. and in disease management. Awareness of the potential host range of the fungal species is vital to the determination of the host-speci ficity of individual isolates. The host range of individual isolates has direct implications for disease management, including the identification of potential inoculum sources, recomme ndations for intercropping and crop rotation, weed management, biological control candidacy, and isolate choice for resistance breeding. In order to obtain an estimate of the comp leteness of the list of hosts known to harbor C. cassiicola surveys were conducted to identify hosts in Guam and Florida. Guam is an ideal location to discover new hosts due to its tropi cal climate, wet and dry seasons, and lack heretofore of a Corynespora host survey (Schlub and Yudin 2002). Florida was included because outbreaks of target spot on tomato caused by C. cassiicola are common and it represents a subtropical environment located an ocean and a continent away from Guam. Methods Literature Survey and Host Index An index of plant hosts of C. cassiico la was compiled from a search of world literature for any reference regarding its presen ce on plant tissue. All plant-f ungus associations were included such as pathogenic, endophytic, and saprophytic. Resources included ar ticles in refereed journals, graduate student theses, books, and web-based resources such as annual reports, production guides, and plant clinic lists. The final list of susceptible hosts of C. cassiicola was compiled from the literature and personal observation from surveys in Florida and Guam.

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13 All plant species, genera, and families were named and classified according to the USDA Germplasm Resources Information Network (GRIN) taxonomy, which follows the APGII system. In some cases, the host name given in th e original citation was ch anged to be consistent with GRIN taxonomy. In a few cases, neither the species cited nor a proper synonym was identified using GRIN taxonomy and the species na me was kept as originally cited. Only one reference was provided per host, with emphasis on citing the first known report of that host. For some hosts, the only reference that could be found was a website, and in those cases the website is listed. The number of plan t host species was conservative ly determined by counting only unique species within each genus. Ge nera with unidentified species (e.g. Crossandra spp.) were counted only once when no other named speci es were present within that genus. Guam and Florida Surveys Surveys for the pres ence of C. cassiicola were conducted throughout Guam and Florida. The Guam survey was conducted for one year beginning in January of 2004 and the Florida survey was conducted for one year beginning in January of 2005. Survey areas focused on roadsides, nurseries, and farms. During the c ourse of the survey, leaves from plants with characteristic C. cassiicola foliage disease symptoms were co llected and placed in individual plastic bags. Known hosts of C. cassiicola were sampled more intensely through the additional collection of old and young asymptomatic leaves. An effort was made to sample from an equal number of individual plants and unique plant species in Florida and Guam. To induce sporulation, leaf tissu e was placed abaxial side up in the moisture chamber for 10 days. Moisture chambers were created on the lab bench by placing 10 ml of sterilized distilled water on a paper towel in a 150 mm petri plate. A plant species was identified as a host of C. cassiicola if characteristic structures of the fungus developed within 10 days. An isolate was labeled a pathogen if coni diophores arose from a necrot ic spot and an endophyte if

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14 conidiophores arose from healthy, green tissue. Petri plates were inspected under a dissecting microscope daily for spores and conidiophores of C. cassiicola. Structures were confirmed based on microscopic morphological features su ch as percurrent proliferation of the conidiophores and pseudoseptation. Single spore isolates were obt ained for long-term storage by needle transfer of spores to antibiotic V8 agar agar slants (340 ml V8 juice, 660 ml water, 3 g CaCO3, 17 g agar, 100 g/ml ampicillin or kanamycin). Slants were left at room temperature until colonies reached at least 5 cm in diameter, covered with autoclaved mineral oil, and stored at 5o C until further study. Results Over 900 individual plants were surveyed in both Gua m and Florida from 320 unique plant species in Guam and 289 unique plant species in Florida. Compilation of Corynespora cassiicola hosts from the literature and surveys conducted in Guam and Florida resulted in an index of 530 plant species from 380 genera. The majority of index host species for C cassiicola are herbaceous Eudicotyledonae, but 52 Monocot yledonae, eight Magnoliids, five Filicopsida (ferns), and one cycad are also represented. No hosts were found within the Anthocerotophyta (hornworts), Bryophyta (mosses), Equisetops ida (horsetails, s phenophytes), Lycopsida (lycophytes), or Marchantiomorpha (liverworts) (Table 1-1). Hosts were found in two plant divisions: Filicopsida and Spermatopsida. The five hosts in the Filicopsida include Arachniodes aristata ( Davalliaceae ), Athyrium niponicum ( Dryopteridaceae ), Adiantum cuneatum ( Pteridaceae ), Davallia repens ( Davalliaceae ), and Platycerium spp. ( Pteridaceae ). The plant division Spermatophyta ( Cycadales, Magnoliidae, Monocotolydonae, and Tricolpates) contains 99% of the host spec ies (Table 1-1). There are eight species from the Magnoliidae. Three species are from the Piperaceae ( Piper betle P.

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15 hispidinervum and Perperomia obtusifolia). Three species are from the Magnoliales in the family Annonaceae ( Annona reticulata A. squamosa and Asimina triloba ). Two species are from the Laurales in the Hernandiaceae ( Hernandia ovigera) and the Lauraceae ( Ocotea leucoxylon ) (Table 1-2). The 52 host species from the Monocotolydonae are from 16 families: Araceae (13 species), Poaceae (9 species), Arecaceae (7 species), Dioscoreaceae (5 species, all from the genus Dioscorea), Orchidaceae (4 species), Agavaceae (3 species), Musaceae (2 species), Alismataceae (1 species), Asparagaceae (1 species), Bromeliaceae (1 species), Commelinaceae (1 species), Heliconiaceae (1 species), Hemerocallidaceae (1 species), Marantaceae (1 species), Restonaceae (1 species), and Strelitziaceae (1 species), in decreasing order of host species numbers. The remaining 464 host species are Eudicots. Families that contain the largest number of hosts include Fabaceae (70 species), Lamiaceae (33 species), Malvaceae (32 species), Asteraceae (26 species), Apocynaceae (21 species), Acanthaceae (20 species), Euphorbiaceae (20 species), Verbenaceae (17 species), Convolvulaceae (14 species), Cucurbitaceae (13 species), and Solanaceae (13 species), in decreasing order of host species numbers. Between the two surveys, 91 new hosts specie s were identified, 87 of which were found in the survey conducted on Guam. New hosts were found in 32 families, of which three families had never been reported to harbor the fungus: Hernandiaceae, Moringaceae and Mutingiaceae. Ten new host species were found to harbor the fungus in the survey conducted in F lorida ( Cerinthe major Corchorus aestuans Fatshedera lizei Hibiscus rosa-sinensis Jatropha spp., Salvia farinacea Salvia microphylla Salcia officinalis Sida spinosa, and Stachytarpheta

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16 jamaicensis). Six new hosts were found in both Guam and Florida ( Corchorus aestuans, Salvia farinacea S. microphylla S. officinalis Sida spinosa, and Stachytarpheta jamaicensis ). From the Guam and Florida surveys, C. cassiicola was more often identified as a pathogen than as an endophyte on 191 and 121 plant species, respectively. On 48 hosts, the fungus was identified as both a pathogen and an endophyte. Endophytic isolates of C. cassiicola were most likely recovered from young leaves and pa thogenic isolates from older leaves. Discussion The index produced here contains 530 C. cassiico la host plant species. Four hundred thirty nine species were identified from the literature and 91 new species were identified from the field surveys conducted in Guam and Florida. The number of new hosts found to harbor the fungus in Guam was 87 and in Florida was 10, with six new hosts found in both Guam and Florida. This suggests that there are many add itional host species remaining to be discovered. Although most of the literature on C. cassiicola relates to the diseases it causes, in this study the fungus was often isolated from asymptom atic tissue, indicative of endophytic growth. There are likely many additional endophytic hosts that remain to be discovered considering only healthy leaves from previous ly reported hosts were sampled. The extent to which C. cassiicola was occurring as an endophyte was not appreciated prior to this su rvey. During the course of the Guam survey, C. cassiicola often sporulated from healthy tissue when placed in a moisture chamber instead of necrotic tissue. In these cases, C. cassiicola was likely not the cause of the necrosis because other fungi were ofte n found to sporulate in those areas. There seems to be no clear demarcation as to the presence of C. cassiicola on a particular host and its ability to grow endophytically or pathogenically. Publications on C. cassiicola are usually restricted to a description of symptoms on a particular host or as part of a list of fungi from an endophyte study. Kochs postulates are ra rely completed, and when they are, often the

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17 fungus is not pathogenic on the host it was is olated from without wounding (Kingsland 1985; Pernezny et al. 1996). This study recorded 48 cas es from the Guam and Florida surveys where plants were found to harbor pathogenic isolates of C. cassiicola and in other locations harbor endophytic isolates. It may be that the fungus has the ability to delay symptoms by growing initially as an endophyte. Pathogenic isolates were often found on older leaves indicating that endophytic isolates may become pathogens as the hos t tissue ages or begins senescence. Despite the symptomless nature of an e ndophytic relationship with the host, it is likely that the potential exists for the fungus to switch to an opportunist ic pathogen and/or a saprophyte on the same host because individual hosts were found to har bor both pathogenic and endophytic isolates. The likelihood of finding the fungus as an e ndophyte or as a pathogen may depend on the plant family. In this study, plant families more likely found harboring the fungus growing as an endophyte were Araceae Bignoniaceae, Convolvulaceae Crassulaceae Elaeocarpaceae Hernandaceae Magnoliaceae, Meliaceae, and Moraceae. Magnolia liliifera ( Magnoliaceae ) was recently reported as hosting a Corynespora spp. endophyte with ribosomal DNA (ITS15.8S-ITS2) sequence homology to C. cassiicola (Promputtha et al. 2007) and was therefore included in our list. In the Guam survey, Hernandia sp. ( Magnoliaceae ) was also found to support endophytic growth of C. cassiicola. Families that were likely to support pathogenic growth of the fungus in these surveys were Acanthaceae, Amaranthaceae Apocynaceae, Asteraceae Begoniaceae, Boragniaceae, Gesnariaceae, Lamiaceae and Verbenaceae. Throughout the survey, it was diffi cult to determine whether the Corynespora species observed were in fact C. cassiicola At least one hundred an d thirteen species of Corynespora are currently described, but a m onograph is needed, including mol ecular analyses, in order to assess the validity of these species (Sivanesan 1996). Most species have been named according

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18 to host identity, and only a few species have been described in culture. In addition, single isolates exhibit considerable morphological plasticity that depends on humidity, light, temperature, and substrate; therefore, morphol ogical differences need to be compared with molecular differences. Although the hosts included in this index are restri cted to those reported for C. cassiicola some may actually be hosts of other Corynespora species due to misidentification. Likewise, there may be hosts reported to harbor other species of Corynespora that may, in fact, be harboring C. cassiicola because the morphological distinctions between species are based on overlapping, variable, morphol ogical characters. Phylogenetic analyses of the isolates should help to clarify these issues. Despite these complications, this is the first step taken to consolid ate our knowledge of the potential host range of C. cassiicola, which is vital for further stud ies of the biology of individual isolates and ultimately in future studies of Corynespora species evolution. Although there is no teleomorphic stage currently known for C. cassiicola the Ascomycete species Corynesporasca caryote and Pleomassaria swidae have unknown Corynespora species anamorphs (Sivanesan 1996; Tanaka et al. 2008). There is no evidence to suggest that C. cassiicola is reproducing other than by asexual spores. However, eviden ce for sexual recombination needs to be tested between isolates within and among host species. Insight into the evolut ionary potential of the fungus will lead to a better understanding of how to control its diseases (McDonald 2004). The literature search and surveys elucidated several characteristics of C. cassiicola that warrant further investigation: (1) the inability of some isolates recovered from symptomatic tissue to re-infect the original hosts; (2) the ability to be endophytic, path ogenic, and saprophytic on individual hosts; (3) the wide host range of th e fungal species, yet restricted host ranges of individual isolates; (4) the ability to grow on some members of a plant taxonomic group and not

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19 others; (5) a lack of unde rstanding of the diversity within th e fungal species and how it relates to host range; (6) the taxonomic va lidity of the 113 species of Corynespora considering the high morphological plasticity of indivi dual isolates. Future research should attempt to address these issues and the organization of the plant hosts in a single publication will facilitate this.

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20 Table 1-1. Taxonomic grouping of Corynespora cassiicola host species. Plant Group Number of Host Species in the Index Number of Host Species Sampled in Guam Number of Host Species Sampled in Florida Anthocerotphyta (hornworts) 0 2 3 Bryophyta (mosses) 0 5 2 Filicopsida (ferns) 5 14 21 Spermatopsida (seed plants) 525 299 263 Conifers 0 3 6 Cycads 1 4 5 Gnetales 0 2 1 Angiosperms 524 290 251 Magnoliids 8 6 4 Monocotyledons 52 61 38 Eudicots 464 223 209

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21Table 1-2. Occurrence and f ungal-host interaction of Corynespora cassiicola identified during 2004-2005 Guam and Florida surveys. Host Fungal-Host Interaction LocationReference Acanthaceae Juss. (dicot) Acanthus ilicifolius L. endophytic GU Sadaba et al. 1995 Aphelandra squarrosa Nees pathogenic FL, GU Chase 1982 Asystasia spp. Blume Alfieri et al. 1984 Asystasia gangetica (L.) T. Anders. pathogenic GU Alfieri et al. 1984 Crossandra spp. Salisb. pathogenic FL Alfieri et al. 1994 Eranthemum pulchellum Andrews pathogenic FL Alfieri et al. 1994 Fittonia spp. Coem. pathogenic FL Chase 1982 Fittonia albivenis (Lindl. ex hort. Veitch) Brummitt endophytic, pathogenic FL, GU Chase 1982 Hygrophila spp. R. Br. FL Alfieri et al. 1994 Justicia spp. L. Ellis 1957 Justicia brandegeeana Wasshausen & L.B. Sm. pathogenic FL, GU Alfieri et al. 1994 Justicia carnea Lindl. pathogenic GU Ellis 1957 Justicia ventricosa Wall. ex Hook. Zhuang 2001 Meisosperma oppositifolium endophytic GU Smith et al. 2007 Pachystachys coccinea (Aubl.) Nees Urtiaga 1986 Pachystachys lutea Nees pathogenic FL, GU Alfieri et al. 1994 Peristrophe spp. Nees Alfieri et al. 1994 Pseuderanthemum spp. Radlk. El-Gholl et al. 1997 Pseuderanthemum carruthersii (Seem.) Guillaumin pathogenic GU El-Gholl et al. 1997 Ruellia humboldtiana (Nees) Lindau endophytic, pathogenic FL Urtiaga 2004 Strobilanthes dyerianus M.T. Mast. pathogenic GU Coile and Dixon 1994 Thunbergia fragrans Roxb. Zhuang 2001 Warpuria clandestina Stapf. pathogenic GU Ellis 1957 Actinidiaceae Gilg & Werderm. (dicot) Actinidia chinensis Planch. Peregrine and Ahmad 1982 Adoxaceae E. Mey. (dicot) Viburnum spp. L. Alfieri et al. 1994 Viburnum odoratissimum Ker Gawl. endophytic FL, GU Alfieri et al. 1994

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22Table 1-2. Continued Host Fungal-Host Interaction LocationReference Agavaceae Dumort. (monocot) Agave sisalana Perrine Ellis 1957 Cordyline fruticosa (L.) Chev. endophytic GU Situmorang and Budimen 1984 Dracaena spp. Vand. ex L. Alfieri et al. 1984 Dracaena reflexa Lam. endophytic, pathogenic FL Alfieri et al. 1994 Alismataceae Vent. (monocot) Echinodorus spp. Rich. ex Engelm. Alfieri et al. 1994 Amaranthaceae Juss. (dicot) Achyranthes aspera L. CABI, Herb. IMI 191361 Alternanthera ficoidea (L.) P. Beauv. pathogenic GU first report Amaranthus spp. L. Alfieri et al. 1994 Amaranthus spinosus L. pathogenic FL, GU Alfieri et al. 1994 Amaranthus tricolor L. Peregrine and Ahmad 1982 Celosia argentea L. var. cristata (L.) Kuntze pathogenic GU first report Digera muricata (L.) Mart. Sarma and Nayudu 1970 Anacardiaceae R. Br. (dicot) Lannea coromandelica (Houtt.) Merr. CABI, Herb. IMI 266196 Mangifera indica L. Rajak and Pandey 1985 Schinus spp. L. endophytic, pathogenic FL Alfieri et al. 1984 Spondias purpurea L. pathogenic FL Freire 2005 Vernicia montana Lour. endophytic FL Ellis 1957 Annonaceae Juss. (dicot) Annona reticulata L. Peregrine and Ahmad 1982 Annona squamosa L. endophytic GU first report Asimina triloba (L.) Dunal CABI, Herb. IMI 364250 Apiaceae Lindl. (dicot) Foeniculum vulgare Mill. Peregrine and Ahmad 1982

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23Table 1-2. Continued Host Fungal-Host Interaction LocationReference Apocynaceae Juss. (dicot) Adenium obesum (Forssk.) Roem. & Schult. El-Gholl 1997 Allamanda spp. L. endophytic FL Alfieri et al. 1984 Allamanda cathartica L. pathogenic GU Alfieri et al. 1994 Alstonia scholaris (L.) R. Br. endophytic, pathogenic FL Suryanarayanan et al. 2002 Calotropis procera (Aiton) W. T. Aiton CABI, Herb. IMI 173980 Carissa spp. L. pathogenic FL Alfieri et al. 1994 Catharanthus roseus (L.) G. Don pathogenic FL, GU McGovern 1994 Conopharyngia longiflora (Benth.) Stapf Kranz 1963 Cryptolepis buchananii Schult. CABI, Herb. IMI 221003 Funastrum clausum (Jacq.) Schltr. Urtiaga 2004 Hoya spp. R. Br. pathogenic FL Alfieri et al. 1994 Mandevilla spp. Lindl. Alfieri et al. 1984 Mandevilla splendens (Hook. f.) Woodson pathogenic FL, GU Alfieri et al. 1994 Nerium oleander L. pathogenic FL Alfieri et al. 1994 Plumeria rubra L. forma acutifolia (Poir.) Woodson endophytic, pathogenic GU Ellis 1957 Rauvolfia serpentina (L.) Benth. ex Kurz CABI, Herb. IMI 122395 Tabernaemontana divaricata (L.) R. Br. ex Roem. & Schult. CABI, Herb. IMI 209321 Tabernaemontana sananho Ruiz & Pav. Urtiaga 2004 Tacazzea spp. Decne. Ellis 1957 Telosma cordata (Burm. f.) Merr. endophytic, pathogenic GU first report Thevetia peruviana (Pers.) K. Schum. CABI, Herb. IMI 231448 Trachelospermum jasminoides (Lindl.) Lem. pathogenic FL Alfieri et al. 1984 Vinca spp. L. Alfieri et al. 1994 Aquifoliaceae Bercht. & J. Presl (dicot) Ilex vomitoria Sol. ex Aiton endophytic FL Alfieri et al. 1994 Araceae Juss. (monocot) Aglaonema spp. Schott pathogenic FL Alfieri et al. 1994 Alocasia macrorrhizos (L.) G. Don endophytic, pathogenic GU Mercado et al. 1997 Amorphophallus paeoniifolius (Dennst.) Nicolson Puzari and Saikia 1981

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24Table 1-2. Continued Host Fungal-Host Interaction LocationReference Anthurium spp. Schott pathogenic Alfieri et al. 1994 Anthurium andraeanum Linden ex Andr pathogenic GU Alfieri et al. 1994 Anubias afzelii Schott El-Gholl 1997 Caladium bicolor (Aiton) Vent. endophytic, pathogenic GU first report Colocasia esculenta (L.) Schott endophytic GU Onesirosan et al. 1974 Dieffenbachia spp. Schott endophytic FL Alfieri et al. 1994 Epipremnum pinnatum (L.) Engl. pathogenic FL Alfieri et al. 1984 Philodendron bipinnatifidum Schott ex Endl. endophytic GU first report Syngonium podophyllum Schott pathogenic GU Coile and Dixon 1994 Xanthosoma sagittifolium (L.) Schott endophytic GU Ellis 1957 Zantedeschia spp. Spreng. Raabe et al. 1981 Zantedeschia aethiopica (L.) Spreng. Raabe et al. 1981 Araliaceae Juss. (dicot) Fatshedera spp. Guillaumin Alfieri et al. 1984 Fatshedera lizei (hort. ex Cochet) Guillaumin endophytic FL first report Polyscias balfouriana L.H.Bailey Alfieri et al. 1984 Polyscias fruticosa (L.) Harms pathogenic FL Alfieri et al. 1994 Polyscias scutellaria (Burm. f.) Fosberg pathogenic GU first report Arecaceae Bercht. & J. Presl (monocot) Attalea butyracea (Mutis ex L. f.) Wess. Boer Urtiaga 2004 Calyptronoma plumeriana (Mart.) Lourteig Delgado-Rodriguez and Mena-Portales 2004 Cocos nucifera L. CABI, Herb. IMI 317357 Dypsis lutescens (H. Wendl.) Beentje & J. Dransf. endophytic, pathogenic FL Alfieri et al. 1994 Elaeis guineensis Jacq. Ellis 1957 Licuala ramsayi (Mueler) Domin. Shivas and Alcorn 1996 Rhopalostylis sapida H. Wendl and Drude McKenzie et al. 2004 Asparagaceae Juss. (monocot) Asparagus officinalis L. Urtiaga 2004 Asteraceae Bercht. & J. Presl (dicot) Ageratum conyzoides L. pathogenic GU Smith and Schlub 2004

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25Table 1-2. Continued Host Fungal-Host Interaction LocationReference Aspilia africana (Pers.) C. D. Adams pathogenic Onesirosan et al. 1974 Bidens spp. L. Alfieri et al. 1984 Bidens alba (L.) DC. pathogenic FL, GU Alfieri et al. 1984 Calyptocarpus vialis Less. pathogenic GU Smith and Schlub 2004 Chromolaena odorata (L.) R. M. King & H. Rob. pathogenic GU CABI, Herb. IMI 147913 Chrysanthemum spp. L. endophytic FL Turner 1971 Chrysanthemum indicum L. Peregrine and Ahmad 1982 Elephantopus mollis Kunth endophytic GU first report Elephantopus scaber L. CABI, Herb. IMI 199985 Elephantopus tomentosus L. Zhuang 2001 Emilia sonchifolia (L.) DC pathogenic GU McKenzie 1990 Gaillardia aristata Pursh pathogenic GU Ellis 1957 Lactuca sativa L. pathogenic GU Ellis 1957 Liatris spp. Gaertn. ex Schreb. endophytic, pathogenic FL Alfieri et al. 1994 Melanthera biflora (L.) Wild Ellis 1957 Mikania micrantha Kunth pathogenic GU Smith et al. 2007 Pseudelephantopus spicatus (B. Juss. ex Aubl.) C. F. Baker endophytic GU first report Pseudogynoxys chenopodioides (Kunth) Cabrera endophytic, pathogenic FL Alfieri et al. 1994 Sphagneticola trilobata (L.) Pruski endophytic GU Alfieri et al. 1994 Symphyotrichum novi-belgii (L.) G. L. Nesom Dixon 1997 Synedrella nodiflora (L.) Gaertn. pathogenic GU Onesirosan et al. 1974 Tithonia rotundifolia (Mill.) S. F. Blake Wei 1950 Tridax procumbens L. pathogenic GU first report Verbesina turbacensis Kunth Urtiaga 2004 Vernonia cinerea (L.) Less. pathogenic GU Cutrim and Silva 2003 Zinnia violacea Cav. Urtiaga 2004 Balsaminaceae A. Rich. (dicot) Impatiens balsamina L. pathogenic GU Wei, 1950 Impatiens noli-tangere L. pathogenic FL CABI, Herb. IMI 124564 Impatiens sultanii Hook. f. Urtiaga 2004

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26Table 1-2. Continued Host Fungal-Host Interaction LocationReference Impatiens walleriana Hook. f. Alfieri et al. 1994 Begoniaceae C. Agardh (dicot) Begonia spp. L. Chase 1982 Begonia coccinea Hook. pathogenic GU Chase 1982 Begonia cucullata Willd. pathogenic GU first report Bignoniaceae Juss. (dicot) Bignonia spp. L. Orieux and Felix 1968 Crescentia cujete L. pathogenic FL Alfieri et al. 1994 Handroanthus serratifolius (Vahl) S. Grose Mendes et al. 1998 Newbouldia laevis (P. Beauv.) Seem. ex Bureau endophytic, pathogenic GU Ellis 1957 Radermachera sinica (Hance) Hemsl. endophytic FL Alfieri et al. 1994 Radermachera xylocarpa (Roxb.) K. Schum. endophytic FL Suryanarayanan et al. 2002 Stereospermum colais (Buch.-Ham. ex Dillwyn) Mabb. endophytic FL Murali et al. 2007 Tabebuia spp. Gomes ex DC. Mendes et al. 1998 Tabebuia aurea (Silva Manso) Benth. & Hook. f. ex S. Moore pathogenic FL Alfieri et al. 1984 Tabebuia heterophylla (DC.) Britton endophytic GU Alfieri et al. 1994 Tabebuia pallida (Lindl.) Miers pathogenic FL Alfieri et al. 1994 Tabebuia odontodiscus (Bureau & K. Schum.) Toledo Mendes et al. 1998 Tecoma capensis (Thunb.) Lindl. Urtiaga 2004 Boraginaceae Juss. (dicot) Cerinthe major L. pathogenic FL first report Cordia collococca L. Urtiaga 2004 Cordia curassavica (Jacq.) Roem. & Schult. Urtiaga 2004 Cordia obliqua Willd. Murali et al. 2007 Cordia wallichii G. Don. Murali et al. 2007 Cordia subcordata Lam. pathogenic GU first report Tournefortia argentea L. f. pathogenic GU first report Brassicaceae Burnett (dicot) Brassica rapa L. Peregrine and Ahmad 1982

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27Table 1-2. Continued Host Fungal-Host Interaction LocationReference Bromeliaceae Juss. (monocot) Ananas comosus (L.) Merr. Blazquez 1968 Burseraceae Kunth (dicot) Bursera simaruba (L.) Sarg. endophytic, pathogenic FL Alfieri et al. 1994 Canarium album (Lour.) Raeusch. Zhang and Ji 2005 Cannabaceae Martinov (dicot) Trema micrantha (L.) Blume Arnold 1986 Trema orientalis (L.) Blume CABI, Herb. IMI 256125 Capparaceae Juss. (dicot) Capparis spp. L. CABI, Herb. IMI 259297 Caprifoliaceae Juss. (dicot) Lonicera japonica Thunb. endophytic FL Alfieri et al. 1984 Lonicera sempervirens L. Alfieri et al. 1994 Caricaceae Dumort. (dicot) Carica papaya L. pathogenic FL, GU Beaver 1981 Vasconcellea cauliflora (Jacq.) A. DC. Urtiaga 2004 Vasconcellea pubescens A. DC. Johnston 1960 Celastraceae R. (dicot) Celastrus paniculatus Willd. CABI, Herb. IMI 302698 Elaeodendron glaucum (Rottb.) Pers. Murali et al. 2007 Euonymus spp. L. Alfieri et al. 1994 Salacia senegalensis (Lam.) DC. Ellis 1957 Combretaceae R. Br. (dicot) Anogeissus latifolia (Roxb. ex DC.) Wall. ex Guill. & Perr. Suryanarayanan et al. 2002 Terminalia arjuna (Roxb. ex DC.) Wight & Arn. CABI, Herb. IMI 302839 Terminalia catappa L. endophytic GU first report Terminalia crenulata Roth. Murali et al. 2007 Terminalia elliptica Willd. Suryanarayanan et al. 2002

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28Table 1-2. Continued Host Fungal-Host Interaction LocationReference Commelinaceae Mirb. (monocot) Commelina benghalensis L. pathogenic GU Cutrim and Silva 2003 Convolvulaceae Juss. (dicot) Evolvulus glomeratus Nees & Mart. endophytic GU Alfieri et al.1994 Ipomoea alba L. endophytic, pathogenic GU McKenzie 1990 Ipomoea aquatica Forssk. endophytic GU McKenzie 1990 Ipomoea batatas (L.) Lam. endophytic, pathogenic FL, GU Silva et al. 2003 Ipomoea indica (Burm.) Merr. endophytic, pathogenic GU first report Ipomoea littoralis (L.) Blume endophytic, pathogenic GU first report Ipomoea obscura (L.) Ker Gawl. endophytic, pathogenic GU Smith and Schlub 2004 Ipomoea pes-caprae (L.) R. Br. endophytic GU Hawaiian Ecosys tems at Risk (HEAR) 2008 Ipomoea triloba L. endophytic, pathogenic GU Smith and Schlub 2004 Lepistemon spp. Blume Onesirosan et al. 1974 Merremia aegyptia (L.) Urb. endophytic, pathogenic GU first report Merremia peltata (L.) Merr. endophytic, pathogenic GU first report Operculina turpethum (L.) Silva Manso GU first report Stictocardia tiliifolia (Desr.) Hallier f. endophytic GU first report Cornaceae Bercht. & J. Presl (dicot) Alangium chinense (Lour.) Harms Guo 1992 Cornus florida L. Alfieri et al. 1994 Crassulaceae J. St.-Hil. (dicot) Crassula ovata (Mill.) Druce Alfieri et al. 1994 Kalanchoe spp. Adans. endophytic FL Alfieri et al. 1994 Kalanchoe pinnata (Lam.) Pers. endophytic GU first report Kalanchoe thyrsiflora Harv. endophytic, pathogenic GU first report Sedum spp. L. Chase 1982 Cucurbitaceae Juss. (dicot) Citrullus lanatus (Thunb.) Matsum. & Nakai pathogenic GU Sobers 1966 Coccinia grandis (L.) Voigt endophytic, pathogenic GU Philip et al. 1972 Cucumis anguria L. endophytic GU Cutrim and Silva 2003

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29Table 1-2. Continued Host Fungal-Host Interaction LocationReference Cucumis melo L. endophytic, pathogenic GU Ellis and Holliday 1971 Cucumis sativus L. pathogenic FL, GU Wei 1950 Cucurbita spp. L. Grand 1985 Cucurbita maxima Duchesne Williams and Liu 1976 Cucurbita moschata Duchesne Minter et al. 2001 Cucurbita pepo L. pathogenic GU Cutrim and Silva 2003 Lagenaria siceraria (Molina) Standl. endophytic, pathogenic GU Ellis 1957 Luffa acutangula (L.) Roxb. endophytic, pathogenic GU Onesirosan et al. 1974 Luffa aegyptiaca Mill. Onesirosan et al. 1974 Momordica charantia L. pathogenic GU Alfieri et al. 1994 Sechium edule (Jacq.) Sw. endophytic, pathogenic FL, GU Alfieri et al. 1984 Davalliaceae M. R. Schomb. (dicot) Arachniodes aristata (G. Forst.) Tindale endophytic, pathogenic GU Anderson and Dixon 2004 Davallia spp. Sm. Alfieri et al. 1994 Davallia repens (L. f.) Kuhn pathogenic GU Alfieri et al. 1994 Dioscoreaceae R. Br. (monocot) Dioscorea alata L. CABI, IMI 229871 Dioscorea bulbifera L. endophytic, pathogenic GU Onesirosan et al. 1974 Dioscorea cayenensis Lam. CABI IMI 83832 Dioscorea esculenta (Lour.) Burkill endophytic, pathogenic GU Onesirosan et al. 1974 Dioscorea pentaphylla L. Peregrine and Ahmad 1982 Dryopteridaceae Herter (fern) Athyrium niponicum (Mett.) Hance endophytic GU El-Gholl 1997 Ebenaceae Grke (dicot) Diospyros montana Roxb. Murali et al. 2007 Elaeocarpaceae Juss. ex DC. (dicot) Elaeocarpus joga Merr. endophytic GU first report Elaeocarpus tuberculatus Roxb. Suryanarayanan et al. 2002 Muntingia calabura L. endophytic GU first report

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30Table 1-2. Continued Host Fungal-Host Interaction LocationReference Ericaceae Juss. (dicot) Oxydendrum arboreum (L.) DC. Alfieri et al. 1994 Rhododendron spp. L. Alfieri et al. 1984 Rhododendron canescens (Michx.) Sweet Rhododendron obtusum (Lindl.) Planch. endophytic, pathogenic FL E llis and Holliday 1971 Vaccinium corymbosum L. pathogenic FL Hongn et al. 2007 Erythroxylaceae Kunth (dicot) Erythroxylum monogynum Roxb. Murali et al. 2007 Euphorbiaceae Juss. (dicot) Acalypha macrostachya Jacq. Urtiaga 2004 Bridelia ferruginea Benth. Ellis 1957 Chamaesyce hirta (L.) Millsp. pathogenic GU first report Codiaeum variegatum (L.) A. Juss. endophytic, pathogenic GU CABI, IMI 179212 Cnidoscolus aconitifolius (Mill.) I. M. Johnst. Peregrine and Ahmad 1982 Croton bonplandianus Baill. endophytic, pathogenic FL, GU Sarma and Nayudu 1970 Croton fragrans Kunth. Urtiaga 2004 Drypetes alba Poit. Mercado 1984 Euphorbia spp. L. Ellis 1957 Euphorbia cyathophora Murray endophytic, pathogenic GU Barreto and Evans 1998 Euphorbia pulcherrima Willd. ex Klotzsch pathogenic FL Chase 1986 Euphorbia milii Des Moulins pathogenic GU Smith et al. 2007 Givotia rottleriformis Griff. Murali et al. 2007 Hevea brasiliensis (Willd. ex A. Juss.) Mll. Arg. Silva et al. 1995 Hura crepitans L. Urtiaga 1986 Jatropha spp. L. pathogenic FL first report Jatropha gossypiifolia L. pathogenic GU Smith et al. 2007 Manihot spp. Mill. Malvick 2004 Manihot carthagenensis (Jacq.) Mll. Arg. Onesirosan et al. 1974 Manihot esculenta Crantz endophytic, pathogenic GU Ellis 1957

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31Table 1-2. Continued Host Fungal-Host Interaction LocationReference Phyllanthus amarus Schumach. & Thonn. endophytic, pathogenic GU Mathiyazhagan et al. 2004 Phyllanthus emblica L. Prakash and Garg 2007 Tragia spp. L. Ellis 1957 Fabaceae Lindl. (dicot) Acacia spp. Mill. Situmorang and Budimen 1984 Acacia auriculiformis A. Cunn. ex Benth. endophytic, pathogenic GU first report Afzelia africana Sm. ex Pers. Dade 1940 Albizia lebbeck (L.) Benth. endophytic GU first report Albizia zygia (DC.) J. F. Macbr. Ellis 1957 Alysicarpus vaginalis (L.) DC. endophytic GU first report Arachis hypogaea L. Vyas et al. 1985 Bauhinia spp. L. Alfieri et al. 1994 Bauhinia galpinii N. E. Br. pathogenic GU Smith and Schlub 2004 Bauhinia purpurea L. pathogenic FL, GU Ellis 1957 Bauhinia racemosa Lam. Suryanarayanan et al. 2002 Butea monosperma (Lam.) Taub. Murali et al. 2007 Caesalpinia granadillo Pittier Urtiaga 2004 Cajanus cajan (L.) Millsp. Lenn 1990 Calopogonium mucunoides Desv. pathogenic GU Onesirosan et al. 1974 Cassia fistula L. endophytic GU Suryanarayanan et al. 2002 Clitoria ternatea L. pathogenic GU first report Crotalaria goreensis Guill. & Perr. Hyde and Alcorn 1993 Crotalaria juncea L. GU Wei 1950 Crotalaria micans Link Shaw 1984 Crotalaria pallida Aiton Turner 1971 Crotalaria retusa L. endophytic, pathogenic GU first report Crotalaria spectabilis Roth Malvick 2004 Cyamopsis tetragonoloba (L.) Taub. Spencer 1962 Dalbergia spp. L. f. Ellis 1957

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32Table 1-2. Continued Host Fungal-Host Interaction LocationReference Dalbergia latifolia Roxb. endophytic Suryanarayanan et al. 2002 Dalbergia lanceolaria L. f. Murali et al. 2007 Delonix regia (Bojer ex Hook.) Raf. CABI, Herb. IMI 314022 Desmodium spp. Desv. Lenn, 1990 Desmodium incanum DC. pathogenic GU Smith and Schlub 2004 Desmodium tortuosum (Sw.) DC. pathogenic GU Smith and Schlub 2004 Desmodium triflorum (L.) DC. pathogenic GU Smith and Schlub 2004 Erythrina spp. L. Delgado-Rodriguez et al. 2002 Gliricidia sepium (Jacq.) Kunth ex Walp. Boa and Lenn 1994 Glycine max (L.) Merr. pathogenic FL, GU Olive et al. 1945 Glycine soja Siebold & Zucc. Lenn 1990 Hymenaea courbaril L. Urtiaga 2004 Lens culinaris Medik. Khare 1991 Lupinus albus L. Sobers 1966 Lupinus angustifolius L. Sobers 1966 Lupinus luteus L. Sobers 1966 Lupinus pilosus L. Malvick 2004 Macrolobium spp. Schreb. Kranz 1963 Macroptilium atropurpureum (Moc. & Sess ex DC.) Urban pathogenic GU first report Macroptilium lathyroides (L.) Urban pathogenic GU Smith and Schlub 2004 Mimosa diplotricha C. Wright Silva 1995 Mimosa pudica L. endophytic, pathogenic GU Smith and Schlub 2004 Mucuna pruriens (L.) DC. Sobers 1966 Phaseolus lunatus L. Malvick 2004 Phaseolus vulgaris L. pathogenic GU Wei 1950 Pisum sativum L. pathogenic GU first report Pithecellobium dulce (Roxb.) Benth. pathogenic GU first report Psophocarpus tetragonolobus (L.) DC. Ellis 1957 Pterocarpus indicus Willd. Situmorang and Budimen 1984

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33Table 1-2. Continued Host Fungal-Host Interaction LocationReference Pueraria montana (Lour.) Merr. Peregrine and Ahmad 1982 Ricinus communis L. Spencer and Walters 1968 Saraca indica L. CABI, Herb. IMI 210811 Senna alata (L.) Roxb. Wei 1950 Senna occidentalis (L.) Link pathogenic GU first report Senna surattensis (Burm. f.) H. S. Irwin & Barneby pathogenic GU first report Senna tora (L.) Roxb. Situmorang and Budimen 1984 Sesamum indicum L. endophytic GU Wei 1950 Spathodea campanulata P. Beauv. pathogenic GU Smith et al. 2007 Teramnus labialis (L. f.) Spreng. pathogenic GU Smith et al. 2007 Trifolium repens L. Cho and Shin 2004 Trigonella foenum-graecum L. Komaraiah and Reddy 1986 Tylosema esculentum (Burch.) A. Schreib. Alfieri et al. 1994 Vicia spp. L. Alfieri et al. 1984 Vigna mungo (L.) Hepper Gowda et al. 2001 Vigna radiata (L.) R. Wilczek Malvick 2004 Vigna unguiculata (L.) Walp. subsp. sesquipedalis (L.) Verdc. pathogenic GU Seaman et al. 1965 Vigna umbellata (Thunb.) Ohwi & H. Ohashi Peregrine and Ahmad 1982 Wisteria sinensis (Sims) DC. endophytic FL Alfieri et al. 1984 Fagaceae Dumort. (dicot) Quercus ilex L. Collado et al. 1999 Gesneriaceae Rich. & Juss. (dicot) Aeschynanthus longicaulis Wall. ex R. Br. Chase 1982 Aeschynanthus radicans Jack pathogenic GU Chase 1982 Columnea spp. L. Chase 1982 Episcia cupreata (Hook.) Hanst. pathogenic FL Alfieri et al. 1994 Gloxinia perennis (L.) Fritsch Brooks 2002 Nematanthus spp. Schrad. Chase 1982 Saintpaulia ionantha H. Wendl. pathogenic GU Smith et al. 2007

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34Table 1-2. Continued Host Fungal-Host Interaction LocationReference Sinningia speciosa (Lodd. et al.) Hiern pathogenic FL Alfieri et al. 1994 Streptocarpus spp. Lindl. Alfieri et al. 1994 Streptocarpus rexii (Bowie ex Hook.) Lindl. pathogenic FL, GU Alfieri et al. 1994 Heliconiaceae Nakai (monocot) Heliconia caribaea Lam. Urtiaga 2004 Hemerocallidaceae R. Br. (monocot) Hemerocallis spp. L. Peregrine and Ahmad 1982 Hernandiaceae Blume (dicot) Hernandia spp. L. endophytic GU first report Hernandia ovigera L. endophytic GU first report Hydrangeaceae Dumort. (dicot) Hydrangea spp. L. Alfieri et al. 1984 Hydrangea macrophylla (Thunb.) Ser. pathogenic FL Sobers 1966 Lamiaceae Martinov (dicot) Ajuga spp. L. Alfieri et al. 1984 Ajuga reptans L. pathogenic FL Alfieri et al. 1984 Anisochilus carnosus (L. f.) Wall. ex Benth. CABI, Herb. IMI 151008 Coleus barbatus (Andrews) Benth. pathogenic FL, GU Fernandes and Barreto 2003 Congea tomentosa Roxb. Peregrine and Ahmad 1982 Clerodendrum inerme (L.) Gaertn. Ahmad 1969 Clerodendrum infortunatum L. CABI, Herb. IMI 112265 Clerodendrum speciosissimum Van Geert ex C. Morren Urtiaga 1986 Hyptis suaveolens (L.) Poit. endophytic GU Smith et al. 2007 Leucas aspera (Willd.) Link Sarma and Nayudu 1970 Mentha arvensis L. endophytic, pathogenic GU Cheeran 1968 Mentha piperita L. Williams and Liu 1976 Moluccella spp. L. Alfieri et al. 1984 Moluccella laevis L. Alfieri et al. 1984 Monarda punctata L. Alfieri et al. 1994

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35Table 1-2. Continued Host Fungal-Host Interaction LocationReference Ocimum basilicum L. endophytic, pathogenic GU Taba et al. 2002 Ocimum tenuiflorum L. Sarma and Nayudu 1970 Origanum vulgare L. pathogenic FL, GU Perilla frutescens (L.) Britton pathogenic GU Hasama et al. 1991 Plectranthus amboinicus (Lour.) Spreng. pathogenic GU Miller 1991 Plectranthus barbatus Andrews Smith et al. 2007 Plectranthus parviflorus Willd. pathogenic FL Alfieri et al. 1994 Premna serratifolia L. pathogenic GU first report Premna tomentosa Willd. Murali et al. 2007 Rosmarinus officinalis L. Alfieri et al. 1994 Salvia spp. L. Peregrine and Ahmad 1982 Salvia farinacea Benth. pathogenic FL, GU first report Salvia leucantha Cav. pathogenic FL Riley 1960 Salvia microphylla Kunth pathogenic FL, GU first report Salvia officinalis L. pathogenic FL, GU first report Salvia splendens Sellow ex Schult. pathogenic FL Chase 1982 Solenostemon scutellarioides (L.) Codd pathogenic FL, GU Alfieri et al. 1994 Stachys floridana Shuttlew. ex Benth. Alfieri et al. 1994 Thymus vulgaris L. pathogenic FL Silva 1995 Tectona grandis L. f. Murali et al. 2007 Teucrium canadense L. El-Gholl 1997 Lauraceae Juss. (dicot) Ocotea leucoxylon (Sw.) Laness. Delgado-Rodriguez et al. 2002 Lecythidaceae A. Rich. (dicot) Careya arborea Roxb. Murali et al. 2007 Lecythis ollaria Loefl. Urtiaga 2004 Loganiaceae R. Br. ex Mart. (dicot) Buddleja asiatica Lour. pathogenic GU Smith and Schlub 2004 Strychnos potatorum L. f. Murali et al. 2007

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36Table 1-2. Continued Host Fungal-Host Interaction LocationReference Lythraceae J. St.-Hil. (dicot) Lagerstroemia indica L. pathogenic FL Alfieri et al. 1994 Lagerstroemia microcarpa Wight Murali et al. 2007 Lagerstroemia parviflora Roxb. Murali et al. 2007 Pemphis acidula Forst. & Forst. endophytic GU first report Magnoliaceae Juss. (dicot) Magnolia champaca (L.) Baill. ex Pierre CABI, Herb. IMI 254407 Magnolia liliifera (L.) Baill. endophytic FL Promputtha et al. 2007 Malpighiaceae Juss. (dicot) Malpighia glabra L. Poltronieri et al. 2003 Malvaceae Juss. (dicot) Abelmoschus esculentus (L.) Moench pathogenic GU Wei 1950 Abutilon theophrasti Medik. endophytic, pathogenic GU Spencer and Walters 1969 Ceiba pentandra (L.) Gaertn. endophytic GU Mehrotra 1989 Ceiba speciosa (A. St.-Hil.) Ravenna Ferreira 1989 Corchorus aestuans L. pathogenic FL, GU Smith and Schlub 2004 Corchorus capsularis L. pathogenic GU Wei 1950 Corchorus olitorius L. endophytic, pathogenic GU Ellis 1957 Desplatsia spp. Bocq. Ellis 1957 Durio zibethinus L. Williams and Liu 1976 Gossypium barbadense L. endophytic, pathogenic GU Jones 1961 Gossypium hirsutum L. Jones 1961 Grewia tiliifolia Vahl Suryanarayanan et al. 2002 Helicteres isora L. Murali et al. 2007 Hibiscus spp. L. Urtiaga 2004 Hibiscus cannabinus L. Shaw 1984 Hibiscus mutabilis L Kwon and Park 2003 Hibiscus rosa-sinensis L. endophytic FL first report Hibiscus sabdariffa L. endophytic GU Wei 1950

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37Table 1-2. Continued Host Fungal-Host Interaction LocationReference Kydia calycina Roxb. CABI, Herb. IMI 264454 Pavonia spp. Cav. Urtiaga 2004 Pseudobombax septenatum (Jacq.) Dugand Urtiaga 2004 Sida acuta Burm. f. pathogenic GU Smith and Schlub 2004 Sida glomerata Cav. Urtiaga 2004 Sida rhombifolia L. pathogenic GU CABI, Herb. IMI 180198 Sida spinosa L. pathogenic FL, GU first report Sida urens L. Ellis 1957 Sterculia apetala (Jacq.) H. Karst. Urtiaga 2004 Talipariti tiliaceum (L.) Fryxell endophytic GU first report Theobroma cacao L. Duarte et al. 1978 Thespesia populnea (L.) Soland. ex Correa endophytic pathogenic GU first report Triumfetta rhomboidea Jacq. endophytic GU Onesirosan et al. 1974 Waltheria indica L. endophytic GU CABI, Herb. IMI 123575 Urena lobata L. pathogenic GU first report Marantaceae R. Br. (monocot) Maranta leuconeura E. Morren pathogenic FL Alfieri et al. 1994 Marcgraviaceae Bercht. & J. Presl (dicot) Norantea guianensis Aubl. endophytic GU Wei 1950 Meliaceae Juss. (dicot) Chukrasia velutina M. Roem. endophytic GU first report Guarea guidonia (L.) Sleumer Urtiaga 2004 Melia azedarach L. endophytic GU first report Moraceae Gaudich. (dicot) Artocarpus altilis (Parkinson) Fosberg CABI, Herb IMI 351978 Broussonetia spp. L'Hr. ex Vent. Pollack and Stevenson 1973 Broussonetia papyrifera (L.) L'Hr. ex Vent. endophytic GU Alfieri et al. 1994 Ficus spp. L. Ellis 1957 Ficus benjamina L. endophytic GU Chase 1984

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38Table 1-2. Continued Host Fungal-Host Interaction LocationReference Ficus elastica Roxb. ex Hornem. endophytic GU Chase 1987 Ficus exasperata Vahl Onesirosan et al. 1974 Ficus hispida L. f. CABI, Herb IMI 311137 Ficus lyrata Warb. endophytic FL Alfieri et al. 1994 Ficus racemosa L. Gilson 2002 Ficus religiosa L. CABI, Herb. IMI 217075 Moringaceae Martinov (dicot) Moringa oleifera Lam. endophytic GU Smith et al. 2007 Muntingiaceae C. Bayer et al. (dicot) Muntingia calabura L. pathogenic GU first report Musaceae Juss. (monocot) Musa sapientum L. Blazquez 1968 Musa acuminata Colla Lumyong et al. 2003 Myrsinaceae R. Br. (dicot) Ardisia foetida Willd. Urtiaga 2004 Myrtaceae Juss. (dicot) Eucalyptus spp. L'Hr. Eucalyptus grandis W. Hill ex Maiden C.M.I. No. 303 Eucalyptus tereticornis Sm. Vittal and Dorai 1994 Eugenia uniflora L. pathogenic GU CABI, Herb IMI 99533 Psidium guajava L. Alfieri et al. 1984 Syzygium aromaticum (L.) Merr. & L. M. Perry Saikia and Sarbhoy 1981 Syzygium cumini (L.) Skeels pathogenic GU Sarbhoy et al. 1971 Syzygium jambos (L.) Alston pathogenic GU Smith et al. 2007 Nyctaginaceae Juss. (dicot) Bougainvillea spectabilis Willd. endophytic GU first report Mirabilis jalapa L. CABI, Herb IMI 259283 Nymphaeaceae Salisb. (dicot) Nymphaea ampla (Salisb.) DC. Urtiaga 2004

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39Table 1-2. Continued Host Fungal-Host Interaction LocationReference Nyssaceae Juss. ex Dumort. (dicot) Nyssa spp. L. Alfieri et al. 1994 Oleaceae Hoffmanns. & Link (dicot) Chionanthus retusus Lindl. & Paxton Alfieri et al. 1994 Jasminum spp. L. Alfieri et al. 1984 Jasminum laurifolium Roxb. forma nitidum (Skan) P. S. Green Alfieri et al. 1994 Jasminum multiflorum (Burm. f.) Andrews Alfieri et al. 1994 Jasminum sambac (L.) Aiton CABI Herb. IMI 111858 Jasminum simplicifolium G. Forst. pathogenic FL Alfieri et al. 1994 Ligustrum lucidum W. T. Aiton Alfieri et al. 1994 Ligustrum japonicum Thunb. Alfieri et al. 1994 Ligustrum sinense Lour. endophytic GU Alfieri et al. 1994 Orchidaceae Juss. (monocot) Cattleya spp. Lindl. Simone 2000 Dendrobium spp. Sw. Alfieri et al. 1994 Phalaenopsis spp. Blume Alfieri et al. 1994 Vanilla planifolia Andrews Urtiaga 2004 Passifloraceae Juss. ex Roussel (dicot) Passiflora spp. L. Pernezny and Simone 1993 Passiflora edulis Sims endophytic FL Alfieri et al. 1994 Passiflora foetida L. pathogenic GU Smith et al. 2007 Passiflora suberosa L. endophytic GU first report Pedaliaceae R. Br. (dicot) Josephinia imperatricis Vent. Hyde and Alcorn 1993 Martynia annua L. CABI, Herb IMI 264260 Sesamum indicum L. Riley 1960 Piperaceae Giseke (dicot) Piper betle L. endophytic, pathogenic GU Acharya et al. 2003

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40Table 1-2. Continued Host Fungal-Host Interaction LocationReference Piper hispidinervum C. DC. Poltronieri et al. 2003 Peperomia obtusifolia (L.) A. Dietr. pathogenic FL Chase 1982 Poaceae Barnhart (monocot) Arundinaria pygmaea (Miq.) Asch. & Graebn. LSU Ag Center 2008 Bambusa vulgaris Schrad. ex J. C. Wendl. endophytic GU first report Dendrocalamus spp. Nees Lu et al. 2000 Oryza sativa L. CABI, Herb IMI 280017 Ottochloa nodosa (Kunth) Dandy Situmorang and Budimen 1984 Panicum repens L. Situmorang and Budimen 1984 Pennisetum glaucum (L.) R. Br. Lenn 1990 Megathyrsus maximus (Jacq.) B. K. Simon & S. W. L. Jacobs endophytic GU Smith and Schlub 2004 Sorghum bicolor (L.) Moench Mendes et al. 1998 Polypodiaceae Bercht. & J. Presl (fern) Platycerium spp. Desv. pathogenic FL Alfieri et al. 1994 Polygonaceae Juss. (dicot) Coccoloba fallax Lindau Urtiaga 2004 Pteridaceae E. D. M. Kirchn. (fern) Adiantum spp. L. Situmorang and Budimen 1984 Adiantum tenerum Sw. pathogenic FL Alfieri et al. 1984 Restionaceae R. Br. (monocot) Ischyrolepis subverticillata Steud. Lee et al. 2004 Rhamnaceae Juss. (dicot) Colubrina retusa (Pittier) Cowan Urtiaga 2004 Ziziphus cyclocardia S.F. Blake pathogenic FL Urtiaga 2004 Ziziphus mauritiana Lam. pathogenic GU first report Ziziphus xylopyrus (Retz.) Willd. Murali et al. 2007 Rosaceae Juss. (dicot) Malus pumila Mill. CABI, Herb IMI 284207 Pyrus communis L. Alfieri et al. 1984

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41Table 1-2. Continued Host Fungal-Host Interaction LocationReference Rubiaceae Juss. (dicot) Guettarda speciosa L. endophytic GU first report Ixora coccinea L. CABI, Herb. IMI 129296 Ixora nigricans R. Br. ex Wt. & Am. Murali et al. 2007 Morinda citrifolia L. endophytic GU first report Nauclea diderrichii (De Wild.) Merr. CABI, Herb. IMI 126192 Pentas lanceolata (Forssk.) Deflers pathogenic GU first report Spermacoce spp. L. Situmorang and Budimen 1984 Rutaceae Juss. (dicot) Aegle marmelos Gond et al. 2007 Naringi crenulata (Roxb.) Nicolson Murali et al. 2007 Salicaceae Mirb. (dicot) Casearia decandra Jacq. Urtiaga 2004 Sapindaceae Juss. (dicot) Acer negundo L. El-Gholl 1997 Acer rubrum L. Alfieri et al. 1994 Cupaniopsis anacardioides (A. Rich.) Radlk. Alfieri et al. 1994 Dodonaea viscosa Jacq. Singh et al. 1982 Litchi chinensis Sonn. Matayba scrobiculata (Kunth) Radlk. Urtiaga 2004 Saxifragaceae Juss. (dicot) Saxifraga stolonifera Curtis El-Gholl 1997 Tolmiea spp. Torr. & A. Gray Alfieri et al. 1984 Tolmiea menziesii (Pursh) Torr. & Gray pathogenic FL Alfieri et al. 1984 Scrophulariaceae Juss. (dicot) Alectra sessiliflora (Vahl) Kuntze Urtiaga 2004 Antirrhinum majus L. Alfieri et al. 1994 Buchnera americana L. pathogenic GU Smith and Schlub 2004 Digitalis spp. L. Alfieri et al. 1994

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42Table 1-2. Continued Host Fungal-Host Interaction LocationReference Paulownia spp. Siebold & Zucc. Mehrotra 1997 Paulownia tomentosa (Thunb.) Steud. endophytic GU Mehrotra 1997 Russelia equisetiformis Schltdl. & Cham. endophytic, pathogenic FL, GU Alfieri et al. 1984 Simaroubaceae DC. (dicot) Ailanthus excelsa Roxb. CABI, Herb IMI 337615 Solanaceae Juss. (dicot) Capsicum annuum L. endophytic GU Kwon et al. 2001 Capsicum frutescens L. Pernezny and Simone 1993 Nicotiana glutinosa L. Tsay and Kuo 1991 Nicotiana tabacum L. pathogenic GU Fajola and Alasoadura 1973 Petunia hybrida hort. ex E. Vilm. pathogenic GU Alfieri et al. 1994 Petunia integrifolia (Hook.) Schinz & Thell. Peregrine and Ahmad 1982 Solanum erianthum D. Don Shaw 1984 Solanum lycopersicum L. pathogenic FL, GU Wei 1950 Solanum melongena L. endophytic GU Onesirosan et al. 1974 Solanum nigrum L. endophytic FL, GU Sarma and Nayudu 1971 Solanum torvum Sw. endophytic GU Onesirosan et al. 1974 Solanum tuberosum L. Peregrine and Ahmad 1982 Solanum viarum Dunal Casady 1994 Strelitziaceae Hutch. (monocot) Strelitzia spp. Aiton Alfieri et al. 1994 Strelitzia reginae Aiton pathogenic FL, GU Alfieri et al. 1994 Theaceae Mirb. (dicot) Camellia sinensis (L.) Kuntze endophytic GU El-Gholl et al. 1997 Turneraceae Kunth ex DC. (dicot) Turnera ulmifolia L. Urtiaga 2004 Urticaceae Juss. (dicot) Boehmeria nivea (L.) Gaudich. Cecropia peltata L. Minter et al. 2001

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43Table 1-2. Continued Host Fungal-Host Interaction LocationReference Cecropia schreberiana Miq. Minter et al. 2001 Laportea aestuans (L.) Chew Alfieri et al. 1994 Pilea spp. Lindl. Chase 1982 Pilea cadierei Gagnep. & Guillaumin pathogenic GU Alfieri et al. 1994 Pilea microphylla (L.) Liebm. pathogenic GU Smith and Schlub 2004 Pilea nummulariifolia (Sw.) Weddell pathogenic FL, GU Alfieri et al. 1994 Verbenaceae J. St.-Hil. (dicot) Callicarpa americana L. Alfieri et al. 1994 Citharexylum spinosum L. El-Gholl 1997 Clerodendrum buchananii (Roxb.) Walp. pathogenic GU first report Clerodendrum paniculatum L. pathogenic FL Ellis 1957 Clerodendrum quadriloculare (Blanco) Merr. pathogenic GU first report Clerodendrum thomsoniae Balf. pathogenic FL Daughtrey 2000 Gmelina arborea Roxb. endophytic GU Florence and Sharma 1987 Lantana camara L. pathogenic FL, GU Pereira et al. 2003 Petrea spp. L. Ellis 1957 Stachytarpheta angustifolia (Mill.) Vahl. pathogenic GU Ellis 1957 Stachytarpheta cayennensis (Rich.) Vahl pathogenic GU McKenzi 1990 Stachytarpheta jamaicensis (L.) Vahl pathogenic FL, GU Smith and Schlub 2004 Vitex agnus-castus L. Alfieri et al. 1994 Vitex negundo L. CABI, Herb. IMI 244917 Vitex parviflora Juss. pathogenic GU Smith and Schlub 2004 Vitex pinnata L. Ellis 1957 Vitex trifolia L. pathogenic GU McKenzie 1996 Vitaceae Juss. (dicot) Cissus spp L. Alfieri et al. 1994 Cissus alata Jacq. Alfieri et al. 1994 Tetrastigma voinierianum (Baltet) Pierre ex Gagnep. Alfieri et al. 1994 Vitis spp. L. Alfieri et al. 1994

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44 Table 1-2. Continued Host Fungal-Host Interaction LocationReference Zamiaceae Horan. (gymnosperm) Encephalartos spp. Lehm. Alfieri et al. 1994 Host plants are listed alphabetically by family (in bold). Each species is followe d by the first known reported reference. Fu ngal-host interaction refers to the endophytic or pa thogenic nature of the fungus and was only reported for hosts that were collected dur ing the Guam (GU) and Florida (FL) surveys. Location refers to whether the plant species was found as a host of C. cassiicola in FL or GU. Forty of the hosts were found on the CABI online database website (http://194.203.77.76/herbIMI/Disp layResults.asp?strName=Corynespora+cassiicola CABI Databases: Herb. IMI records for Fungus: Corynespora cassiicola).

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45 Figure 1-1. Corynespora cassiicola isolate from Cucumis sativus A) sporulating on naturally infected leaf tissue after 24 hours in the moisture chamber, B) germinating spore on water agar, and C) growing on V8 agar afte r single spore isolation (images are not shown to scale).

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46 Figure 1-2. Various symptoms caused by Corynespora cassiicola on naturally infected leaves of A) Vaccinium corymbosum B) Carica papaya C) Ageratum conyzoides D) Allamanda spp., E) Macroptilium lathyroides, F) Abutilon theophrasti G) Bidens alba, H) Euphorbia cyathophora I) Chromolaena odorata Continued. J) Corchorus aestuans K) Passiflora foetida L) Ipomoea pes-caprae M) Ipomoea obscura N) Lantana camara, O) Merremia peltata P) Bauhinia galpinii Q) Catharanthus roseus R) Phyllanthus amarus S) Hydrangea macrophylla and T) Salvia farinacea Images are not to scale.

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47 Figure 1-2. Continued.

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48 CHAPTER 2 GENETIC AND PATHOGENIC DIVERSITY OF CORYNESPORA CASSIICOLA Introduction Target spot, caused by the fungal pathogen Corynespora cassiicola (Berk. & Curt.) Wei, is common in the tropics, subtropics and greenhouses (Chase 1987). C. cassiico la is reported to infect 530 plant species from 380 genera, includi ng monocots, dicots, ferns, and one cycad (Chapter 1, this dissertation). Isolate characterization is needed to determine which hosts might serve as sources of inoculum for target spot of tomato species and other hosts since there is much variability concerning the host range of i ndividual isolates. Some isol ates show pathogenicity to a wide range of hosts, whereas others exhibit host specificity, and some are only pathogenic when associated with wounding (Chase 1982; Cutrim and Silva 2003; Kingsland 1985; Onesirosan et al. 1973, 1974; Pereira et al. 2003; Poltronieri et al. 2003; Seaman et al. 1965; Smith and Schlub 2004; Smith and Schlub 2005; Spencer and Walters 1969; Volin and Pohronezny 1989). At least two ra ces of the fungus have been distinguished based on their differential pathogenicity response on soybean and cowpea (Olive and Bain 1945; Spencer and Walters 1969). However, isolates from soybea n, sesame, cowpea and cotton in Mississippi were alike in pathogenicity (Jones 1961) A more extensive study found eight different pathogenicity profiles among 28 isolates from soybean in Mexic o, cucumber in Florida, and diverse hosts in Nigeria (Onesirosan et al 1974). Furukawa et al. (2008) found that an isolate from Salvia splendens was not pathogenic to cucumber, green pe pper or hydrangea; howev er isolates from these hosts were pathogenic to Salvia splendens Furukawa et al (2008), therefore, demonstrated that isolates with different pathogenicity profiles can be found on the same host. Since the 1960s, a leaf and fruit spot disease of tomato caused by C. cassiicola has become increasingly serious in tropical countries worldwide (Jones and Jones 1984). It was first

PAGE 49

49 reported in Florida in 1972 and ha s since become one of states most damaging foliage and fruit diseases (Blazquez 1972; Pernezny et al. 1993, 1996, 2000, 2002). Under warm, humid, conditions the disease leads to heavy defoliation and significant losses in yield (Volin and Pohronezny 1989). Currently, there are no resi stant tomato cultivars available, although resistance found in PI 120265 ( Lycopersicon esculentum ) and PI 11215 ( L. pimpinellifolium ) and was controlled by a single recessive gene (Bli ss et al. 1973). Understanding the genetic and pathogenic diversity of the pathogen and its distribution is vital to isolate selection for resistance screening. Kingsland (1985) compared three isolates from tomato, cucumber and papaya debris and found that tomato and cucumber were susceptible to all isolates, but the isolate from papaya debris was not pathogenic on papaya indicating that it was possibl y growing as a saprophyte. In many studies, isolates were found to be non-pa thogenic on the hosts from which they were isolated, further indicating that C. cassiicola can grow as a saprophyte (Chase 1982; Kingsland 1985; Onesirosan et al. 1974; Hyde et al. 2001; Lee et al. 2004). Other studies show that isolates are only secondary invaders, or inva ders of senescent tissue. Isol ates from the ornamental hosts Aeschynanthus pulcher (lipstick vine), Aphelandra squarrosa (zebra plant), azalea and hydrangea were pathogenic on all hosts in cros s-pathogenicity trials when wounded; however, only A. pulcher was susceptible without wounding (Chase 1982). Silva et al. (1998) compared pathogenicity of 16 isolates from rubber trees in Sri Lanka and five isolates from diverse hosts in Australia. Papaya isolates from Australia were pathogenic to tomato and rubber, but not cowpea and eggplan t. Mimosa and thyme isolates from Australia were pathogenic to eggplant, rubber, and tomato, but not cowpea. Isolates from Sri Lanka

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50 collected from different rubber clones were eith er pathogenic to all hosts (cowpea, eggplant, rubber, and tomato), or pathogeni c to all hosts but eggplant. The host specificity and severity of the fungus on Lantana camara in Brazil has led to the discovery that C. cassiicola may be useful as a bioherbicide (Pereira et al. 2003). Based on the vast number of weeds that serve as hosts of th e fungus, there is great potential for the discovery of several more isolates useful for biological cont rol of weeds. Consideri ng the wide variation in isolate pathogenicity that has been previously reported, additional studies are needed to further understand the host range of individual isolates from different hosts and locations. Prior research on the genetic characterization of C. cassiicola is limited to restriction fragment length polymorphism (RFLP) of ITS rDNA and random amplified polymorphic DNA (RAPD) studies. No variati on between five isolates of C. cassiicola collected from mimosa, papaya, and thyme in Australia was found based on RFLP of ITS (S ilva et al. 1995). Silva et al. (1995) concluded that RFLP of the ITS regions of rDNA can be used to distinguish between Corynespora and the morphologically similar genus Helminthosporium but not different isolates of C. cassiicola However, the three isolates from papa ya had identical RAPD patterns, growth rate, isolate color, and pathoge nicity profiles, which were di fferent from the isolates from mimosa and thyme, indicating an ongoing process of host specialization on papaya (Silva et al. 1995). RAPD analyses from 27 isolates collected from Hevea brasiliensis in Sri Lanka revealed correlations between host locati on, host genotype, isolate mor phology, and isolate pathogenicity (Silva et al. 1998). Silva et al (1998) concluded that a progenitor strain may have been spread in India by distribution of live plant material. Prior outbreaks of the disease on the susceptible

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51 rubber clone RRIC 103 in other countries, and the sudden appearance and severity of target spot on the same clone in Sri Lanka in 1985, is evidence for such dissemination. Silva et al. (2003) characterized 42 is olates from bitter gourd, cocoa, manihot, papaya, rubber, sweet potato, tomato, and wing-bean from various regions in India based on RAPD analyses. RAPD groups did not correlate with geographic origin, but isolates obtained from rubber clone RRIC 103 grouped together. This stra in might be responsible for several recent outbreaks on this clone. In addition, all but one of the isolates from rubber clone RRIC 110 clustered in 2 RAPD groups, which may identify th e strain that caused the outbreak on this clone in 1995. Silva et al (2003) concluded that correlation of RAPD groups with pathogenicity was needed to help develop resistant cl ones against all pathogenic isolates. Atan and Hamid (2003) characterized nine C. cassiicola isolates from Hevea brasiliensis in Malaysia using RAPD of genomic DNA and RFLP of amplified ITS regions. RFLP analyses with three restriction enzymes yielded monomorphic patterns. However, isolate OPEN 1 from clone RRIM 2020 had a distinct RFLP pattern from the other eight isolates after digestion with Hae III. RAPD results indicated the presence of at least two genetical ly distinct races that infect rubber. Seven isolates pathogenic to clones RRIM 600, RRIM 2009, and two unidentified rubber clones were molecularly similar and identi fied as Race 1. The remaining two isolates, both pathogenic on clone RRIM 2020, had identical banding patterns and we re considered Race 2. Unfortunately, the majority of the diversity assessments are limited to rubber isolates from Malaysia and Sri Lanka and are ba sed on RAPD techniques, which is problematic with respect to repeatability and homology assessm ent (Isabel et al. 1999). In addition, all the RFLP studies used the ITS rDNA region which has minimal vari ation among isolates (Silva et al. 1995, 1998).

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52 Investigations into th e genetic variation among C. cassiicola isolates using more reliable molecular methods and more dive rse isolates are needed. In this study, we collected and solicited 143 is olates from diverse hosts and locations. To test whether C. cassiicola is panmictic throughout its range, a llelic genealogies were constructed from four loci including the rDNA IT S region, two random hypervariable loci, Cc caa5 and Cc ga4, and the single copy actin -encoding nuclear gene, Cc act1 Fifty of these isolates were spray inoculated on seedlings of eight crop plants to te st pathogenicity profiles. Correlations among an isolates pathogenicity profile, it s host of origin, and genotype we re investigated. The purpose of this research is to gain knowledge of the diversity within the species C. cassiicola because of its implications for resistance breeding and disease management of target spot of basil, bean, cowpea, cucumber, papaya, soybean, sweet potat o, tomato, and potentially other crops. Methods Collection and Solicitation of Fungal Isolates C. cassiico la isolates were collected from diverse plant hosts during 5-day collecting trips to locations in the Pacific: American Samo a (AS), Hawaii (HI), Palau (PW), Pohnepei (PH), Saipan (SN), and Yap (YP) in the summer of 2005. More extensive surveys were conducted to collect the fungus in Florid a (FL) and Guam (GU) betw een 2004-2006 (see Chapter 1). Farms, nurseries, and roadsides were surveyed fo r plants with target spot symptoms. First, second, and third priority was given to crops, weeds, and naturalized or indigenous hosts of C. cassiicola respectively. Symptomatic le aves were put into individual plastic bags in the field and later placed abaxial side up in petri dishes with moistened paper towels in a laboratory. After 24 hours in the moisture chamber, petri plat es were placed under th e dissecting microscope and suspected spores and conidiophores of C. cassiicola were confirmed microscopically.

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53 Single spores were captured at the end of a teasing needle an d transferred to antibiotic V8 agar (340 ml V8 juice, 660 ml water, 3g CaCO3, 17g agar, 100 g/ml Ampicillin or Kanamycin) slants, left at room temperature until the colony re ached at least 5 cm in diameter, whereby it was covered with autoclaved mine ral oil, and stored at 5o C until further study. Sporulation from non-symptomatic leaf material was noted, po ssibly indicating non-pathogenic growth. To obtain globally diverse isolates, individual researchers in Brazil (BZ), Malaysia (MY), Mississippi (MS), and Tennessee (TN) were solicited for additional C. cassiicola cultures. Isolates from BZ on lantana (JMP216), papa ya (DOA16b), soybean (RWB321) and tomato (JMP217) came from Alvaro Almeida, EMBRAPA Isolates CBPP, CLN 16 and CSB1 2 were received from MY off of rubber from Dr. Safiah Atan, Malaysian Rubber Board. Isolate TN13-3 was received from Nashville, TN on greenhouse Afri can violet from Justin S. Clark, University of Tennessee. Isolate MS01 was received from MS on greenhouse tomato leaves from David Ingram, Central MS Research and Extension Center. Isolates of different specie s were also solicited from culture collections to serve as outgroups. Cultures from Commonwealth Agricu ltural Bureaux Interna tional (CABI) in the United Kingdom included C smithii IMI 5649b and C citricola IMI 211585. Cultures from National Institute of Agrobiological Sciences (NIAS) in Japan included C citricola MAFF No. 425231, C melongenea MAFF No. 712045, and C sesamum MAFF No. 305095. Cultures from Centraalbureau voor Schimmelcultures (CBS) in the Netherlands included C proliferata CBS 112393, C citricola CBS 169.77, and C olivaceae CBS 291.74. Cultures were single-spored after they were received. A complete list of isolates used in these st udies, along with the plant host, geographic location and the type of a ssociation with the hos t plant (endophytic or pathogenic growth) can be found in Table 2-1.

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54 Primer Development for Random Hypervariable Loci Three C. cas siicola isolates from long-term stor age (FL31, GU112, and PW56) were chosen based on unique host and location. A sm all piece of mycelium from the monosporic cultures was extracted with tweezers and placed onto a V8 agar plate. The isolates were grown under constant fluorescent light for 7 days. Aerial mycelium was scraped from the agar surface, placed in 1.5 ml microcentrifuge tubes, lyophilized overnight, and then frozen in liquid nitrogen. Genomic DNA was purified using the DNeasy plant Mini Kit (Quiagen, Inc.) according to the manufacturers specifications. Genomic DNA combined from all three isolates was digested with the Sau 3AI restriction enzyme (7.2 l of DNA from each of the three isolates; 2.5 l 10X buffer; 1.0 l 10 U/ l Sau 3A I enzyme; incubated at 37oC for 2 hours). The digested genomic DNA was fractionated to remove fragments less than 400 bp using a Chroma Spin column (Chroma Spin + TE 400, Clonetech Laboratories, Inc.) a ccording to the manufacturers specifications. The digested fractionated DNA was quantified and ligated to Sau 3AI linkers and incubated at 16oC overnight. Excess linkers were removed using the same Chroma Spin column as above. The linkerligated fragments were PCR amplified using SauL-A primers and a program consisting of initial denaturation for 3 min at 94oC, followed by 25 cycles of 94oC for 1 min, 68oC for 1 min, and 72oC for 2 min, and a final amplification at 72oC for 10 min. The amplified genomic PCR library (compos ed of 400-1500 bp fragments) was enriched for fragments containing two differe nt microsatellite repeats, (CAA)n and (GA)n. The denatured genomic PCR library was hybridized to the following biotinylated oligoprobes: [5(CAA)15TATAAGATA-Biotin] and [5(GA)15TATAAGATA-Biotin] (Tepnel Lifecodes Corporation) and incubated at 48oC overnight. The PCR fragments that hybridized to the repeat

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55 probes were captured and eluted using two VECT REX Avidin D matrix columns (cat. No. A2020, Vector Laboratories, Burlingame, CA) accordi ng to the manufacturers specifications. The two mixtures containing genomic fragments enriched for the (CAA)n tri-repeat and the (GA)n direpeat were PCR amplified using SauL-A primer s following the same PCR conditions as above. PCR products from the amplification of the en riched microsatellite library were ligated into a plasmid vector (pCR 2.1-TOPO vector; Invitrogen, Inc.) a nd transformed into E. coli (One ShotTM TOP 10 Cells, Invitrogen, In c.) using the TOPO TA Cl oning Kit (Invitrogen, Inc.) according to the manufacturers instructions. Transformed colonies were lifted and crosslinked onto nylon membranes in an UV chamber (GS Gene LinkerTM, Bio-Rad Laboratories, Inc., Hercules, CA) using the optimal crosslink program. Nylon membranes were hybridized with al kaline phosphatase-label ed repeat probes ((CAA)n and (GA)n) and the Quick-LightTM hybridization Kit (Te pnel Lifecodes Corp.) according to the manufacturers recommendation. Colonies containing plasmids that tested positive for inserts with repeats were sequenced in one direction. Primers were designed to amplify 300-500 base pair fragments flanking lo w repeat number (<10) sequences using Primer3 (v. 0.4.0). Sequences with repeats of less than 10 were likely to be non-va riable microsatellite loci, but may contain polymorphic flanking sequences. Sequences were screened for polymorphisms using five isolates fr om different hosts and locations. Primers that amplified the Cc ga4 and Cc caa5 loci were chosen for further study because they amplified sequences with relatively high levels of polymorphism (>5%). Fungal Cultures and Extraction of Genomic DNA Genom ic DNA from 143 isolates (Table 2-2) in long-term storage was purified and amplified using Extract-N-Amp (Sigma-Aldrich) according to the manufacturers specifications.

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56 The following primers were used for PCR amplification: ITS1 and ITS4 (White et al. 1990) for the internal transc ribed spacer region, including the 5.8 rRNA coding region; ACT512F and ACT-783R (Carbone and Kohn 1999) fo r the single copy nuc lear actin locus Cc act1 ; GA4-F (5-CCT GCT CCG ACT TTG TTG AG-3 ) and GA4-R (5-GTC TGG GAG CAG CAA AGA CT-3) for the random hypervariable Cc ga4 locus; CAA5-F (5-GTC CAC AAG TGG AAC CTC GT-3) and CAA5-R (5-CCT CGT CTG CCA GTT CTT CT-3) for the random hypervariable Cc caa5 locus. Hot-start PCR was performed with a MyCyclerTM thermocycler (BioRad) with a program consisting of initial denaturation for 3 min at 94oC, followed by 30 cycles of 30 sec at 94oC, 30 sec at 58oC, and 30 sec at 72oC, and a final cycle of 5 min at 72oC for the ITS, Cc ga4 and Cc caa5 loci. For the Cc act1 locus, the program was identical except for an annealing temperature of 61oC. PCR products were puri fied using the QIA quick PCR purification Kit (QIAGEN Inc.) according to the manufacturers instructions. The purified products were then quantified on 1% ethidium bromide-stained agarose gels. Sequencing of the DNA samples was done at the University of Florida DNA Sequencing Core Laboratory using ABI Prism BigDye Terminator cycle sequenc ing protocols (part number 4303153) developed by Applied Biosystems (Perkin-Elmer Corp., Foster City, CA). The excess dye-labeled terminators were removed using MultiScreen 96-well filtration system (Millipore, Bedford, MA, USA). The purified extension products were dried in SpeedVac (ThermoSavant, Holbrook, NY, USA) and then suspended in Hi-di formamide. Se quencing reactions were performed using POP-7 sieving matrix on 50-cm capillaries in an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, US A) and were analyzed by ABI Sequencing Analysis software v. 5.2 and KB Basecaller.

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57 Phylogenetic Analyses Four loci (rDNA ITS, Cc caa5, Cc ga4, and Cc act1 ) from 143 isolates were sequenced. Forward and reverse sequences from each P CR product were concatenated in SequencherTM 4.8 and trimmed to include only bases sequenced in bo th directions. Samples with ambiguities were sent for re-sequencing. Multiple alignments fr om each locus were executed separately with Clustal X (1.83.1) and the alignments were insp ected and adjusted manually using MacClade 4.08 OS X (Maddison and Maddison 2005). Data from ITS rDNA, Cc ga4, Cc caa5 and Cc act1 loci were partitioned to facilitate different permutations of combined analysis. A partitionhomogeneity test (incongruence le ngth-difference test or ILD) was implemented to evaluate the homogeneity of different data partition subs ets using PAUP* v4.0b10 (Swafford 2002). The test implemented 1,000 replicates (heuristic search ; random simple sequence additions; TBR; maxtrees = 1,000). Comparisons were evaluated using a threshold of p < 0.001 and were made between all data partitions. With the ILD test indicating the combinability of all molecular data, neighbor joining (NJ) and maximum parsimony (MP) analyses were conducted for each data partition and the combined data set using PAUP* (Swafford 2002). C smithii IMI 5649b, C citricola IMI 211585, C proliferata CBS 112393, C citricola CBS 169.77, and C olivaceae CBS 291.74 were defined as outgroups. Cultures from Nationa l Institute of Agrobiol ogical Sciences (NIAS) in Japan ( C citricola MAFF No. 425231, C melongenea MAFF No. 712045, and C sesamum MAFF No. 305095) were not included as outgroups because they grouped with C. cassiicola isolates in phylogenetic analyses (see Results below). For the NJ analyses, default settings were used except ties were broken randomly by initial seed. Due to long computational time, MP analyses were conducted in th e following manner. An initial heuristic search was conducted w ith one random addition replicate, TBR (tree-

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58 bisection-reconnection) branch swapping, and the MulTre es option (saving a ll optimal trees) in effect. A second heuristic search was conducte d using 1000 random addition replicates with the above settings and saving no more than 10 trees with a sc ore greater than or e qual to the best tree score from the first replicate in the previous an alysis. In all analyses, gaps were treated as missing data. Strict consensus trees were generated from analyses with multiple equally parsimonious trees. For all MP analyses, st atistical support for node s was estimated using maximum parsimony bootstrap (BS) replicates (Felse nstein 1985). For the combined data set, BS estimates were obtained using 1,000 replicates, each with 100 random taxon addition replicates and saving no more than 1,500 trees pe r bootstrap replicate, TBR branch swapping and the MulTrees option in effect. All data were also analy zed by Bayesian inference (BI) methods with MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and Hu elsenbeck 2003). An a ppropriate model of evolution (under the AIC criterion) was selected for each data partition using the program Modeltest v3.4 (Posada and Crandall 1998). All Bayesian analyses (individual loci and combined data) were conducted while retaining the appropriate model for each data partition. Markov Chain Monte Carlo was implemented with four heated chains and trees were sampled every 1,000th generation for one million generations. The first 25 percent of the total number of generations was discarded as burn-in. A 50 percent majority rule consensus tree was generated from the remaining trees, in which the percenta ge of nodes recovered represented their posterior probability (PP). Congruent nodes resultin g from the NJ, MP, and BI analyses of the combined molecular data was used to assign isolates to a phylogenetic lineage (PL). On ly isolates that fell within clades of high support (BS value >70 a nd PP value > 95) were assigned to a PL.

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59 Pathogenicity Analyses Fifty out of the 143 Corynespora isolates were used for pathogenicity profiling (Table 2-3) on eight crop plants. Isolates originally isolated from crop pl ants and from all phylogenetic lineages were chosen. Each isolate was spray-in oculated onto four replicate plants of eightweek-old: basil Italian La rge Leaf (Ba), bean Bush Kentucky Wonder (Be), cowpea California black-eye (Co), cucumber Straigh t 8 (Cu), soybean AG00901 (So), and tomato Rutgers (To) seedlings; 8-week-old sweet potat o Beauregard (Sw) cuttings; and 12-week-old papaya HI Sunrise (Pa) seedlings. Cultivars were chosen based on their known susceptibility in the survey regions. To increase colony sporulation for inoculum preparation, aerial mycelium from 10-day-old V8 agar plates was gently scraped with a gla ss cover slip to flatten mycelium and then placed under constant cool-white fluorescent light (One sirosan et al. 1975). Three days later, the surface of the agar was scraped with a glass cove r slip and the resulting mycelia was blended in 200 ml sterile distilled water fo r two seconds and filtered through three layers of cheesecloth. Spores were counted under a hemacytometer and the concentration was adjusted to 20,000 spores/ml. One drop of Tween 20 per 100 ml was added to the inoculum. Plants were sprayed with the spore suspension until leaf run off (a bout 500 ml), making sure that both leaf surfaces were fully covered. Plants were kept on a mist bench to maintain constant leaf moisture 3 days prior to inoculation and for the remainder of th e experiment. Plants we re rated 7 days after inoculation using the rating system developed by Onesirosan et al. (1973): (0) symptomless, no lesions on leaves or stems; (1) non pathogeni c hypersensitive response, a few to many nonexpanding pinpoint lesions; (2) moderately vi rulent, many expanding lesions, some coalescing, but not resulting in blight; (3) hi ghly virulent, lesions spreading to form large areas of dead tissue resulting in a blighting e ffect. Incidence (I), defined as the number of plants showing symptoms

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60 (with ratings of 1, 2, or 3), and severity (S), defined as the av erage rating for all symptomatic plants, were recorded. The experiment was repeated. Each isolate was assigned a pathogenicity profile (PP), which is a list of susceptible hosts. Hosts were considered susceptible if at least one of th e replicates from the two experiments (total of eight plants) received a rating of 2 or 3. PPs were converted to a binary character matrix so that each isolate received a zer o (non-pathogenic, all reps with ratings of 0 or 1) or a one (pathogenic, at least one rep with a rating of 2 or 3) for each host. Unweighted pair group method with arithmetic mean (UPGMA) trees we re constructed from the binary matrix and internal support for nodes was estimated using bootstrap analyses with 1,000 reps and a UPGMA algorithm. The tree topology was visually compared to the PL designation of each isolate tested (Figure 2-6). PPs were also visually mapped on the four-locus combined BI phylogenetic tree (Figure 2-1). Growth Rate Analyses Seventy-seven isolates were test ed for growth rate at two te mperatures (23 C and 33 C). A sm all piece of aerial mycelium was extracted from the monosporic cultures in long-term storage with tweezers and placed onto a V8 agar plate. After 5 days, the 77 colonies had grown beyond the mineral oil and six 4 mm agar plugs were cut from actively growing mycelium at the colony edge. A single plug was placed in the center of six V8 agar plates. Three replicate plates of each isolate were immediately placed in growth chambers at 23 C and 33 C under 12 hours of alternating fluorescent light (ca. 25 lux) and dark. The average of two colony diameters at 90 degrees from each other was recorded at 48, 72, 96, 120, 144, and 166 hours. Average colony diameter was plotted against time and a line of best fit was generated for each replicate. The slope of the line of best fit (R2>0.98) was used to compare variation within reps to variation between isolates in SAS Statistical Software

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61 (Version 8, 1999). The experiment was repeated with five isolates with no statistically significant variation (data not shown). A co rrelation between isolate growth rate and phylogenetic lineage was tested us ing SAS statistical software. Results Phylogenetic Analyses The General Tim e Reversible model (GTR + I + ) was selected by Modeltest for each of the four gene partitions. The corresponding model for each locus wa s applied to all BI analyses and the combined dataset was partitioned. The final combined dataset contains 2,136 aligned characters used for analyses. Tree topologies re sulting from NJ, MP, and BI analyses recovered essentially the same well-supported nodes. The analyses reveal four ma jor phylogenetic lineages (PL) with high statistical support (BS value >70 and PP value > 95) (Figure 2-1). All major PLs contain isolates from diverse lo cations, indicating their global dispersal. PL1 contains a distinct clade with high stat istical support (designate d PL1.1) containing only isolates collected from papaya from around the world indicating specialization on this host. PL1.2 contains two isolates from Stachytarpheta jamaicensis collected from Guam and Palau, indicating potential specializati on on this host. This supports pathogenicity studies showing isolate specificity to this host (Smith and Schl ub 2005). Isolates from diverse hosts are present in PL1 including crops (basil, bitter melon, eggp lant, cowpea, cucumber, oregano, pumpkin, rubber, soybean, sweet potato, watermelon), ornamentals ( Buddleja Catharanthus Codiaeum Coleus Episcia and Tabebouia ), and weeds ( Bidens, Buchnera Clerodendrum Commelina Lantana, Macroptilium Meisosperma, Vitex ). Tomato isolates are missing from PL1, indicating that isolates in this lineage may not be pathogenic to tomato. Isolates in PL2 are also globa lly distributed and include crops (cucumber, rubber, sweet potato), ornamentals (African violet, Allamanda Catharanthus Pilea ), and weeds ( Piper Pilea ).

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62 There is also a lack of tomato isolates in PL2 indicating that isolates from this lineage may be nonpathogenic on this host. Though PL2 was highl y supported (BS and PP values of 100), its sister relationship to PL1, PL 3, and PL4 remains unresolved. Globally distributed isolates from PL3 incl ude crops (basil, bitter melon, cucumber, pumpkin, soybean, tomato), ornamentals ( Bauhinia, Moringa, Pachystachys Plectranthus, Saintpaulia ), and weeds ( Acanthus Asystasia Calopogonium Coccinia Euphorbia Luffa Passiflora Teramnus ). These are hosts that may harbor is olates pathogenic to tomato. PL5 and PL6 group with PL3 with low support (MPBS value of 60). PL5 contain C. cassiicola isolates from African violet in Guam and Tennessee that ar e very similar in sequence, especially at the Cc-caa5 locus, indicating specialization on this host. African violet isolates from Saipan and Yap are found in PL3. PL6 is highly supporte d and contains isol ates from Brazil on Coleus Palau on cowpea, and Saipan on Asystasia The majority of tomato isolates group in PL 4 from diverse locations including American Samoa, Brazil, Florida, Guam, Mississippi, Palau, a nd Saipan. These twelve tomato isolates also group with isolates from crops (bean, cassa va, cucumber, sweet potato), ornamentals ( Bauhinia Cassia, Coleus Eugenia Ficus Jatropha, Salvia Syzygium ), and common weeds ( Calopogonium Calyptocarpus Chromolaena, Euphorbia Hyptus Lantana Mikania Spathodea ), which are likely inoculum sources for the initiation of disease on tomato. The rDNA ITS region (Figure 2-2) is composed of 1,013 characters, 400 of which are an insertion in the outgroup taxa C. smithii Of the 612 remaining characters, 141 are variable and 107 are informative. The rDNA ITS sequences re veal the m isidentification of three outgroup taxa from the NIAS culture collection. C sesamum 305095, C citricola 425231, and C melongenea 712045 should be reclassified as C cassiicola based on rDNA ITS sequences.

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63 When the outgroup taxa C. citricola C. olivaceae, C. proliferata and C. smithii are removed from the analyses, the rDNA ITS sequences of C. cassiicola contain only three informative characters out of 584 bases. Two of these characters separate the isolat es into three distinct phylogenetic lineages that correlate with the PLs in the combined analysis. These two characters are base pair 158 (C or T), and base pair 497 (A or G) of the C. cassiicola rDNA ITS alignment. Three haplotypes are represented by these two ch aracters: CA, CG, and TG (no haplotype TA); all isolates with haplotype CA group in PL4, isolates with hapl otype TG group in PL1, isolates with haplotype CG group in PL2, PL3, PL5 and PL6. CG is also the ance stral haplotype, present in all outgroups except for C. proliferata (haplotype CA). The third informative character in the rDNA ITS sequences of C. cassiicola is base pair 123, which is a T in the majority of isolates, but a C in isolates PW101 (PL5), RWB321 (PL5), SN64 (PL5.1), and TN13-3 (PL6). It is this character (bp 123) that caused the polymorphic band pattern obs erved by Atan and Hamid (2003) in their RFLP analysis of the rDNA ITS region of rubber isolates using HaeIII (recognition sequence GGCC). The sister relationships between the phyl ogenetic lineages remain unresolved in the analyses of the individual loci and in the combined analyses. In addition, the ITS rDNA region was the only locus that showed good support for C citricola C olivaceae C proliferata and C smithii as sister taxa to the ingroup of C. cassiicola isolates. The phylogenetic placement of the outgroup taxa was not well supported in the combined analyses, or the GA4 locus. The CAA5 locus showed support for C olivaceae C proliferata and C smithii as basal to PL1, PL2, PL3, PL5, and PL6, but PL4 and C. citricola fell basal to that group. The apparent paraphyly of C. cassiicola at the Cc-caa5 locus may be a result of character variation that occurred at this locus

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64 before the species evolved. Additional loci c ontaining characters that reveal the sister relationships of the different PLs ar e needed in future analyses. The Cc-caa5 locus (Figure 2-3) reveals similar tree topologies to the combined analyses with high support for the four major PLs. Differen ces at this locus in PL1 include the lack of PL1.1 that distinguishes papaya isol ates from other isolates in PL1 in the combined analyses. In addition, PL1.2, which includes two rubber isolates from Malaysia, has low support. GU70 and SN59, isolates basal to PL1 in th e combined analyses, groups with other isolates in PL1 at this locus. The Cc-caa5 locus shows strong support for PL2 with the same nine isolates as in the combined analysis. The Cc-caa5 locus does not resolve PL3.1 or PL3.3 as distinct from PL3, although the five isolates in PL 3.2 group together with strong support. This locus does not distinguish isolates FL2920, GU120 GU136 as distinct from other isolates in PL4. PL5 and PL5.1 isolates are group basal to PL3, but with low support. PL6, which includes African violet isolates from Guam and Tennesse e, group with isolate NIAS 712045 w ith high support. Isolates FL50 ( Hydrangea macrophylla ) and FL51 ( Vaccinium corymbosum ) are unresolved at this locus as well as in combined analyses. The Cc-ga4 locus (Figure 2-4) highly suppor ts PL1, PL2, PL4, and PL6, although the sister relationships between th e PLs are unresolved. Isolates in PL3 form a clade with low support. The Cc-ga4 locus did reveal a shared haplotype between papaya isolates with a point mutation from an A to a G at base 74. C cassiicola isolates are not monophyletic at this locus because the outgroups C. proliferata C. olivaceae, and C. smithii fall basal to PL1, PL2, and PL6 with low support. This may be a result of character variation before speciation, a high incidence of homoplasious characters, or the convergent evolution of specific adaptations.

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65 The Cc-act1 locus could not be amplified in the outgroup taxa C citricola C. proliferata and C. smithii perhaps due to mutations in the primer annealing site. The locus did amplify in C olivaceae and shows high variation from C cassiicola isolates (Figure 2-5). There is good support for PL3, PL2, and PL4 at this locus, al though only marginal support for PL1 (BS value of 68). Again, the sister relationships between th e PLs are unresolved. The Cc-act1 locus also reveals a shared haplotype between papaya isolat es with a point mutation from an A to a G at base 229. Pathogenicity Analyses As a result of screening f ifty isolates fo r pathogenicity on eight index hosts, 16 unique pathogenicity profiles (PP) were developed (Table 2-3). The most common PP was CuTo, followed by Pa and CuSwTo. Cucumber was the most susceptible host, with all isolates producing symptoms and an average severity rati ng of 2.3. Tomato was also highly susceptible with 49 out of 50 isolates show ing symptoms with an average severity rating of 1.8. Even though only eight isolates were pathogenic on papaya, the average severity rating was 2.1 indicating that pathogenic isolates were highly virulent. Isolates pathogenic to basil, bean, cowpea, soybean and sweet potato were less virulent on these hosts with average severity ratings less than 1.5. There was a strong correlation be tween PP and PL (Figure 2-6). Seven out of ten isolates with PP CuTo were from PL4 and all isolates with PP CuSwTo and BeCuSwTo were from PL4. In PL4, all isolates but SN37 were highly virule nt on tomato (average severity ratings ranging from 2.5 to 3) and all isolates but GU28 were pa thogenic to cucumber (average severity ratings ranging from 1.3 to 3). In addition, the only is olates pathogenic to bean were from PL4, although these five isolates were weakly virulent (average severity ra tings ranging from 1.3 to 1.9).

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66 In PL3, all isolates were strongly pathogenic to cucumber (average se verity ratings ranging from 2.3 to 2.8) and six out of seven isolates were strongly pathogenic to tomato (average severity ratings ranging from 2.5 to 3). Four out of the six isolates also were pathogenic to basil and all isolates with PP BaCuTo were from PL3. Pathogenicity profile CuSw was unique to isolat es from PL2 and all isolates tested from PL2 had this profile. In additi on, isolates collected in the field from papaya in PL1.1 were specific to papaya in pathogenicity studies, although all isolat es were weakly virulent on cucumber with average severity ratings of 1.3 or less. All isolates from PL1 were pathogenic to cucumber with average severity ratings ranging from 1.3 to 3. Nine out of the 13 isolates from PL1 were pathogenic to cowpea and seven were pathogenic to basil. The only other host susceptible to isolates from PL1 was soybea n, which was only weakly susceptible when inoculated with isolate PW87. Growth Rate Analyses The null hypotheses of no growth rate diffe rences am ong isolates, phylogenetic lineage, and temperatures were rejected (P <0.0001), while the null hypothe ses of no growth rate differences among repetitions was accepted with a probability of 0.7546. The 77 isolates tested all grew faster at 23 C than 33 C. At 23 C, average isolate growth rate (average of three repetitions) was between 0.1479 and 0.474 with an ove rall mean of 0.3855 (Table 2-4). At 33 C, average isolate growth rate was between 0.1382 and 0.4153, with an overall mean of 0.2958 (Table 2-5). At 23 C, there we re 29 significantly different growth rates and at 33 C there were 39 significantly different growth rates. Among the fastest growing isolates at both temperatures were FL37, GU90, GU99, AS67, and HI01. The slowest growing isolates were very different at the two temperatures. Slow growing isolat es at 23 C were PH01, JMP216a, GU120, FL15, and

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67 DOA16b. Slow growing isolates at 33 C were AS119, AS117, JMP217, AS49, GU120, and GU112. Though there were not enough replicates to test for interactions among effects, growth rate alone correlated with lo cation, phylogenetic lineage, and with host. Isolates from Oahu and Palau grew the fastest at both te mperatures. Isolates from Braz il, Florida and Malaysa tended to have slower growth rates at bot h temperatures. Surprisingly, American Samoan isolates grew proportionately much faster at 23 C than 33 C, despite its tropical climate. Isolates from PL6 and PL1 grew the fastest at both temperatures. Isolates from PL 2 and PL4 grew the slowest at 23 C and isolates from PL5 and PL3 grew the slowest at 33 C. All isolates from Clerodendrum Commelina, Ficus Macroptilium pumpkin, and Stachytarpheta were fast growing at both temperatures. In addition, isolates from Allamanda Coleus eggplant, Lantana, and tomato isolates had slower growth ra tes at both temperatures. Discussion The current study presents the first robust, global phylogeny of the species Corynespora cassiicola Based on sequence data from four unique loci, there is eviden ce for high genetic diversity within the species. The highly clonal nature of C. cassiicola is demonstrated in the congruence of the phylogenetic trees from distinct loci. All loci disti nguish four major clonal lineages within C. cassiicola The low level of sequence va riation at the rDNA ITS region within the species relative to other Corynespora species suggests that these lineages are in fact clonal populations, rather than taxon omically distinct species. As reported previously, the pa ttern of distribution of the diversity within the species correlates with the host (Smith et al. 2008a). Identical hapl otypes are widely distributed geographically. The lack of correlation be tween phylogenetic data and location provides evidence for the recent global dispersal of isolat es from all four phylogenetic lineages. In

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68 addition, geographically diverse is olates from the same host plan t shared identical haplotypes, potentially indicating host specialization. For example, isolates co llected from tomato in Brazil, Florida, Guam, Mississippi, Palau, and Saipan had identical haplotype s at all four loci. Isolates collected from Lantana in Florida and Brazil were also iden tical at all four loci. Isolates collected from African violet in Guam and Tenne ssee were unique from all other isolates and nearly identical to each other. Perhaps the mo st compelling evidence for host specialization is the shared identical sequences of all isolates collected from papaya from very diverse locations. Tomato isolates from diverse locations incl uding North and South America and the Pacific Islands are found in only two of the five major phylogenetic lineages (PL3 and PL4). Isolates from other hosts that fall into these same PLs are likely pathogenic to tomato and may serve as source hosts or altern ative hosts for target spot of tomato. Tomato isolates are genetically similar to isolates from common crops (basil, bean, bitter melon, cassava, cu cumber, papaya, pumpkin, soybean, sweet potato), weeds (Acanthus, Calopogonium Calyptocarpus Chromolaena, Coccinia Euphorbia, Lantana, Macroptilium Mikania Momordica, Passiflora, and Teramnus ), and ornamentals ( Asystasia Bauhinia, Cassia, Coleus Eugenia, Euphorbia Ficus Hyptus Jatropha, Luffa Moringa Pachystachys Plectranthus, Saintpaulia Salvia Spathodea, and Syzygium ). Based strictly on these data, control of ta rget spot should involve isolation of tomato fields from these plant species, when possible. Pathogenicity testing, in addi tion to phylogenetics, should be used to determine which hosts might serve as sources of inoculum for the in itiation of target spot of tomato. There are at least sixteen unique pathoge nicity profiles within C. cassiicola on the eight crop plants that were tested. Isolates from the same lineages show similar but not identical prof iles (Figure 2-1 and Figure 2-6). For example, all but two isolates in PL3 and PL4 are pat hogenic to tomato, and

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69 isolates from all other lineages are nonpathogenic to tomato. All is olates pathogenic to basil are from PL1 and PL3, but not all isolates in these cl ades are pathogenic to ba sil. Interestingly, the majority of isolates, excluding the isolates collected from papaya, were pathogenic on cucumber. Though there were no isolates collected from toma to that grouped in PL1 and PL2, isolates from these lineages produced a hypersen sitive response on tomato, showi ng pinpoint lesions that were given a disease rating of one. These data are similar to pathogenicity tests using 18 C. cassiicola isolates from Nigeria, the Southern U.S., and Mexico (Onesirosan et al. 1973) in that both studies found isolates specific to papaya and cucumber. Likewise, bo th studies found that isolates pathogenic to tomato also were likely to be pathogenic on several other hosts. The number of isolates screened compared to the number of unique pathogenicity pr ofiles in both studies i ndicates that gains and losses of pathogenicity are common. Growth rate at different temper atures has provided evidence for isolates adapted to tropical and temperate environments. Using an isolate collected from toma to in Florida, Pernezny et al. (2000) found the best colony growth occurred at 32C, whereas Sobers (1966) reported an optimum growth rate at 24C for Florida isolates collected from hydrangea and azalea. Jones and Jones (1984) report higher disease severity on tomato inoculated a nd maintained at temperatures between 20-23 C. In this study, two temperatur e extremes (23 C and 33 C) were chosen in attempt to discern between isolates adapted to temperate and tropical climates. Though the majority of isolates were collected from tropical c limates, all isolates grew faster at 23 C than 33 C. Growth rate also strongly correlated with phylogenetic lineage. Isol ates from PL2 and PL4 may be more adapted to warmer temperatures, and isolates from PL5 and PL3 might be more adapted to cooler temperatures. Such phys iological traits, incl uding growth rate and

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70 pathogenicity profile, correlate with phylogene tic data and may be useful for isolate classification. These studies have shown that the rDNA ITS sequence will be useful for the initial screening of isolates and for isolate selection for resistance breeding. The rDNA ITS region was useful for the grouping of isolates into three grou ps (PL1 (haplotype TG), PL4 (haplotype CA), and PLs 2, 3, 5, and 6 (haplotype CG)) that correlate with phylogenetic data from the combined four locus data set. For example, isolates fr om PL1 (rDNA ITS haplotype TG) should be used to screen for resistance to target spot in papaya. In contrast, isolates from PL2 and PL4 (rDNA ITS haplotypes CA and CG) should be used to screen for resistance to target spot in tomato. In addition, genotyping by restriction digest of the amplified ITS region is possible now that specific polymorphisms have been identified an d mapped. For example, use of the enzyme HpyCH4V (recognition sequence TGCA) will cut in two positions in haplotypes CA and CG, but only one position in haplotype TG. Additionally, this research found isolates with the same unique genotype found in Atan and Hamids (2003) RFLP analysis of the rDNA ITS region using HaeIII. However, only four of the 143 isolates we sequenced shared this polymorphism at base pair 123, rendering RFLP analysis of the rDNA ITS region using HaeIII ineffective for distinguishing among the majority of isolates. Despite evidence for host specificity (on African violet, Lantana papaya, and Stachytarpheta for example), the combined pathogenici ty and phylogenetic data indicate that there are many hosts with the potential to harbor C. cassiicola isolates pathogenic to susceptible crops such as basil, cucumber, and tomato. Studi es that incorporate many isolates from the same host across diverse locations, the sequencing of additional loci, and subsequent pathogenicity screening, will no doubt reveal additional ge netic diversity and host specificities.

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71 It is hoped that this research will aid ot hers in unraveling the many complexities that remain to be discovered with respect to C. cassiicola and its disease development in the field. For example, more studies are needed to explain why C. cassiicola is rare in Hawaii on all cultivated crops except basil, if there are isolates adapted to tr opical and temperate climates, and how isolate genotype and pathogeni city profiles are corre lated using more diverse isolates and hosts.

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72 Table 2-1. Isolate designations geographic location of isolati on, host of isolation, phylogenetic lineage (PL), type of growth on a ssociated host, and species of Corynespora used in the phylogenetic analyses. Isolate ID Location Host PL Growth Species CABI 211585 New Zealand Poncirus trifoliatus O endophytic C. citricola CBS 169.77 New Zealand Poncirus trifoliatus O endophytic C. citricola NIAS 425231 Japan Ocimum basilicum 1 pathogen C. citricola NIAS 712045 Japan Solanum melongenea ? pathogenic C. melongenae CBS 291.74 Netherlands Tilia spp. O saprophyte C. olivacea CBS 112393 Italy Fagus sylvatica O endophytic C. proliferata NIAS 305095 Japan Sesamum indicum 1 pathogenic C. sesamum CABI 5649b England Fagus sylvatica O saprophyte C. smithii AS49 Amer. Samoa Solanum lycopersicum 3 pathogenic C. cassiicola AS50 Amer. Samoa Solanum lycopersicum 3 pathogenic C. cassiicola AS54 Amer. Samoa Vigna unguiculata 1 saprophyte C. cassiicola AS58 Amer. Samoa Vigna unguiculata 1 saprophyte C. cassiicola AS65 Amer. Samoa Solanum melongenea 6 saprophyte C. cassiicola AS67 Amer. Samoa Commelina benghalensis 1 pathogenic C. cassiicola AS71 Amer. Samoa Cucurbita pepo 1 saprophyte C. cassiicola AS78 Amer. Samoa Ocimum basilicum 1 pathogenic C. cassiicola AS80 Amer. Samoa Ocimum basilicum 3.1 pathogenic C. cassiicola AS81 Amer. Samoa Clerodendrum quadriloculare 1 pathogenic C. cassiicola AS92 Amer. Samoa Cucumis sativus 6 pathogenic C. cassiicola AS98 Amer. Samoa Cucumis sativus 1 pathogenic C. cassiicola AS117 Amer. Samoa Carica papaya fruit 3.1 saprophyte C. cassiicola AS119 Amer. Samoa Cucurbita pepo 3.1 saprophyte C. cassiicola DOA16b Brazil Carica papaya 1.1 pathogenic C. cassiicola JMP216a Brazil Lantana camara 6 pathogenic C. cassiicola JMP217 Brazil Solanum lycopersicum 6 pathogenic C. cassiicola JMP218 Brazil Glycine max 1 pathogenic C. cassiicola RWB321 Brazil Coleus barbatus 4 pathogenic C. cassiicola FL09 FL, USA Lantana camara 6 pathogenic C. cassiicola FL11 FL, USA Carica papaya 1.1 pathogenic C. cassiicola FL12 FL, USA Solanum lycopersicum 6 pathogenic C. cassiicola FL15 FL, USA Salvia farinacea 6 pathogenic C. cassiicola FL21 FL, USA Bauhinia galpinii 6 pathogenic C. cassiicola FL34 FL, USA Tabebouia pallida 1 pathogenic C. cassiicola FL36 FL, USA Catharanthus roseus 2 pathogenic C. cassiicola FL37 FL, USA Clerodendrum paniculatum 1 pathogenic C. cassiicola FL50 FL, USA Hydrangea macrophylla ? pathogenic C. cassiicola FL51 FL, USA Vaccinium corymbosum ? pathogenic C. cassiicola FL62 FL, USA Coleus barbatus 1 pathogenic C. cassiicola FL757 FL, USA Origanum vulgare 1 pathogenic C. cassiicola FL2920 FL, USA Solanum lycopersicum 6 pathogenic C. cassiicola MS31 MS, USA Solanum lycopersicum 6 pathogenic C. cassiicola TN3-3 TN, USA Saintpaulia ionantha 5 pathogenic C. cassiicola GU01 Guam Cassia fistula 6 saprophyte C. cassiicola

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73 Table 2-1. Continued. Isolate ID Location Host PL Growth Species GU06 Guam Hyptus suarelens 6 endophytic C. cassiicola GU08 Guam Lantana camara 1 pathogenic C. cassiicola GU10 Guam Codiaeum variegatum 1 endophytic C. cassiicola GU11 Guam Citrullus vulgaris 1 saprophyte C. cassiicola GU12 Guam Calopogonium mucunoides 6 pathogenic C. cassiicola GU14 Guam Calyptocarpus vialis 6 pathogenic C. cassiicola GU16 Guam Asystasia gangetica 3 pathogenic C. cassiicola GU21 Guam Buddleja asiatica 1 pathogenic C. cassiicola GU23 Guam Ipomoea batatas 6 endophytic C. cassiicola GU25 Guam Buchnera floridana 1 pathogenic C. cassiicola GU28 Guam Solanum lycopersicum 6 pathogenic C. cassiicola GU32 Guam Euphorbia heterophylla 3 endophytic C. cassiicola GU38 Guam Allamanda cathartica 2 pathogenic C. cassiicola GU41 Guam Eugenia uniflora 6 endophytic C. cassiicola GU42 Guam Bidens alba 1 pathogenic C. cassiicola GU44 Guam Jatropha curcas 6 endophytic C. cassiicola GU49 Guam Syzygium jambos 6 endophytic C. cassiicola GU51 Guam Meisosperma oppositifolium 1 endophytic C. cassiicola GU55 Guam Calopogonium mucunoides 3.1 pathogenic C. cassiicola GU65 Guam Passiflora foetida 3 endophytic C. cassiicola GU68 Guam Moringa oleifera 3 endophytic C. cassiicola GU70 Guam Solanum melongenea 1.3 endophytic C. cassiicola GU79 Guam Acanthus ilicifolius 3 endophytic C. cassiicola GU83 Guam Euphorbia heterophylla 6 endophytic C. cassiicola GU90 Guam Stachytarpheta jamaicensis 1 pathogenic C. cassiicola GU92 Guam Carica papaya 1.1 pathogenic C. cassiicola GU93 Guam Capsicum annum 1 endoph ytic C. cassiicola GU98 Guam Spathodea campanulata 6 pathogenic C. cassiicola GU99 Guam Saintpaulia ionantha 5 pathogenic C. cassiicola GU101 Guam Euphorbia milii 6 saprophyte C. cassiicola GU102 Guam Phaseolus vulgaris 6 saprophyte C. cassiicola GU103 Guam Pilea nummulariifolia 2 endophytic C. cassiicola GU104 Guam Macroptilium atropurpureum 1 pathogenic C. cassiicola GU107 Guam Mikania micrantha 6 pathogenic C. cassiicola GU109 Guam Bauhinia galpinii 3 pathogenic C. cassiicola GU110 Guam Plectranthus ambionicus 3 pathogenic C. cassiicola GU111 Guam Manihot esculenta 6 endophytic C. cassiicola GU112 Guam Glycine max 3 endophytic C. cassiicola GU114 Guam Teramnus labialis 3 endophytic C. cassiicola GU115 Guam Vitex parviflora 1 pathogenic C. cassiicola GU120 Guam Coleus barbatus 6 pathogenic C. cassiicola GU128 Guam Solanum lycopersicum 6 pathogenic C. cassiicola GU136 Guam Ficus benjamani 6.1 endophytic C. cassiicola HI01 Oahu, Hawaii Ocimum basilicum 1 pathogenic C. cassiicola CBPP Malaysia Hevea brasiliensis clone unk. 1.2 pathogenic C. cassiicola CLN16 Malaysia Hevea brasiliensis RRIM 2020 1.2 pathogenic C. cassiicola

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74 Table 2-1. Continued. Isolate ID Location Host PL Growth Species CSB12 Malaysia Hevea brasiliensis RRIM 725 2 pathogenic C. cassiicola GU136 Guam Ficus benjamani 6.1 endophytic C. cassiicola HI01 Oahu, Hawaii Ocimum basilicum 1 pathogenic C. cassiicola CBPP Malaysia Hevea brasiliensis clone unk. 1.2 pathogenic C. cassiicola CLN16 Malaysia Hevea brasiliensis RRIM 2020 1.2 pathogenic C. cassiicola CSB12 Malaysia Hevea brasiliensis RRIM 725 2 pathogenic C. cassiicola PH01 Pohnpei Carica papaya 1.1 endophytic C. cassiicola PW01 Palau Carica papaya 1.1 pathogenic C. cassiicola PW12 Palau Carica papaya 1.1 pathogenic C. cassiicola PW17 Palau Carica papaya 1.1 pathogenic C. cassiicola PW20 Palau Carica papaya 1.1 pathogenic C. cassiicola PW25 Palau Carica papaya 1.1 pathogenic C. cassiicola PW27 Palau Carica papaya 1.1 pathogenic C. cassiicola PW34 Palau Carica papaya 1.1 pathogenic C. cassiicola PW37 Palau Carica papaya 1.1 pathogenic C. cassiicola PW38 Palau Carica papaya 1.1 pathogenic C. cassiicola PW43 Palau Carica papaya 1.1 pathogenic C. cassiicola PW48 Palau Carica papaya 1.1 pathogenic C. cassiicola PW53 Palau Carica papaya 1.1 pathogenic C. cassiicola PW56 Palau Carica papaya 1.1 pathogenic C. cassiicola PW57 Palau Solanum lycopersicum 6 pathogenic C. cassiicola PW63 Palau Solanum lycopersicum 6 pathogenic C. cassiicola PW69 Palau Piper betle 2 endophytic C. cassiicola PW79 Palau Pilea microphylla 2 pathogenic C. cassiicola PW80 Palau Saintpaulia ionantha 1 pathogenic C. cassiicola PW83 Palau Saintpaulia ionantha 1 pathogenic C. cassiicola PW87 Palau Cucumis sativus 1 pathogenic C. cassiicola PW89 Palau Chromolaena odorata 6 endophytic C. cassiicola PW91 Palau Luffa acutangula 1 endophytic C. cassiicola PW92 Palau Catharanthus roseus 1 pathogenic C. cassiicola PW94 Palau Stachytarpheta jamaicensis 1 pathogenic C. cassiicola PW99 Palau Momordica charantia 3 pathogenic C. cassiicola PW101 Palau Vigna unguiculata 4 saprophyte C. cassiicola SN03 Saipan Momordica charantia 1 pathogenic C. cassiicola SN05 Saipan Ipomoea batatas 1 pathogenic C. cassiicola SN06 Saipan Luffa acutangula 3.1 endophytic C. cassiicola SN07 Saipan Carica papaya 1.1 endophytic C. cassiicola SN18 Saipan Carica papaya 1.1 pathogenic C. cassiicola SN24 Saipan Solanum lycopersicum 6 pathogenic C. cassiicola SN27 Saipan Solanum lycopersicum 6 pathogenic C. cassiicola SN30 Saipan Solanum lycopersicum 6 pathogenic C. cassiicola SN37 Saipan Vigna unguiculata 6 saprophyte C. cassiicola SN40 Saipan Cucumis sativus 6 pathogenic C. cassiicola SN43 Saipan Saintpaulia ionantha 3 pathogenic C. cassiicola SN48 Saipan Coccinia grandis 3 endophytic C. cassiicola SN53 Saipan Carica papaya 1.1 pathogenic C. cassiicola

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75 Table 2-1. Continued. Isolate ID Location Host PL Growth Species SN59 Saipan Lantana camara 1.4 pathogenic C. cassiicola SN64 Saipan Asystasia gangetica 4.1 pathogenic C. cassiicola SN69 Saipan Pachystachys lutea 3 pathogenic C. cassiicola YP01 Yap Carica papaya 1.1 pathogenic C. cassiicola YP08 Yap Carica papaya 1.1 pathogenic C. cassiicola YP17 Yap Carica papaya 1.1 pathogenic C. cassiicola YP26 Yap Cucumis sativus 1 pathogenic C. cassiicola YP27 Yap Cucumis sativus 2 pathogenic C. cassiicola YP29 Yap Cucumis sativus 1 pathogenic C. cassiicola YP41 Yap Saintpaulia ionantha 2 pathogenic C. cassiicola YP42 Yap Solanum lycopersicum 3 pathogenic C. cassiicola YP51 Yap Vigna unguiculata 1 saprophyte C. cassiicola YP59 Yap Ipomoea batatas 2 endophytic C. cassiicola Information on Corynespora cassiicola isolates used in this study including location, original host, phylogenetic lineage (PL), type of growth in associa tion with the host (endophytic or pathogenic), and the Corynespora species. The first eight isolates were solic ited from culture collections as outgroups (O). Three isolates from the NIAS culture collection (305095, 425231, and 712045) are likely misidentified because they grouped with C. cassiicola isolates according to sequence data. They are labeled here according to the original culture collection desi gnations, though they shoul d be re-classified as C. cassiicola The remaining isolates were collected as part of this study or solicited from other researchers and are listed according to geographic location.

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76 Table 2-2. Summary of sequence da ta from four loci used to c onfirm the phylogenetic lineage of Corynespora cassiicola isolates. Locus Total Variable Informative Tree ScoreNo. MP Trees Combineda 2136 248 1743307430 rDNA ITS 1013 135 1001589990 rDNA ITSb 584 4 34 1 Cc-ga4 414 31 25409530 Cc-ga4b 414 28 25369560 Cc-caa5 366 38 3452 40 Cc-caa5b 366 37 3246 12 Cc-act1 343 44 1549 11 Cc-act1b 343 16 1517 4a Combined loci: rDNA ITS, Cc-ga4, Cc-caa5, and Cc-act1 b Locus analyzed with only C. cassiicola taxa represented (no outgroups).

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77Table 2-3. Pathogenicity profiles for 50 Corynespora cassiicola isolates. Path Pro PL Isolate Host Ba Be Co Cu Pa So Sw To I S I S I S I S I S I S I S I S BaCoCu 1 AS78 Ba 52.6 041.8 62.3 0011 21 BaCoCu 1 AS58 Co 62.3 032.3 63 00071 BaCoCu 1 YP29 Cu 72.1 031.7 83 0000BaCoCu 1 AS71 Pu 82.1 022 71.3 011 021 BaCoCuSo 1 PW87 Cu 52.2 051.6 82.5 021.5 071 BaCu 1 HI01 Ba 42.5 000 73 0011 81 BaCu 1 SN05 Sw 71.9 11 00 62.7 00021 BaCuTo 3 AS50 To 52.2 021 62.7 011 21 83 BaCuTo 3 YP42 To 62.3 0072.6 00082.6 BaCuTo 3.1 AS80 Ba 51.8 0082.5 011 082.8 BaCuTo 3.1 AS117 Sap 71.9 021 82.8 021 81 72.9 BeCoCuSw 4 SN37 Co 71 51.6 51.4 82.8 021 31.3 81 BeCuSwTo 4 JMP217 To 51 71.9 082.9 0012 82.5 BeCuSwTo 4 GU102 Be 071.3 082.6 0021.5 82.5 BeCuSwTo 4 SN40 Cu 061.3 082.5 0012 82.6 BeCuTo 4 PW57 To 061.7 082.1 021 083 CoCu 1 AS98 Cu 051 61.8 82.6 00071 CoCu 1 YP26 Cu 071 41.5 72.4 0081 81 CoCu 1 JMP218 So 0072.1 83 0071 71 CoCu 1 GU08 La 0031.7 73 00081 Cu 1 AS54 Co 011 083 011 81 11 Cu 1 YP51 Co 00083 0071 11 CuPa 1.1 DOA16b Pa 71 0081.1 82.3 081 11 CuPa 1.1 FL11 Pa 051 081.3 72.6 0081 CuPa 1.1 PH01 Pa 11 11 071.1 61.8 0081 CuSo 3 GU112 So 0051 82.8 012 11 21 CuSw 2 YP27 Cu 11 021 62.8 0051.4 21 CuSw 2 YP59 Sw 21 011 72.6 0061.4 71 CuSw 2 SN59 La 11 51 082.8 0071.3 71 CuSwTo 4 PW63 To 41 071 82.6 0012 83 CuSwTo 4 SN24 To 00082.5 0021.5 82.6 CuSwTo 4 SN27 To 51 0083 0051.2 82.9

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78 Path Pro PL Isolate Host Ba Be Co Cu Pa So Sw To I S I S I S I S I S I S I S I S CuSwTo 4 GU23 Sw 0 0 7 1 82.4 00 51.8 82.9 CuSwTo 4 FL09 La 0 1 1 0 81.4 02 1 31.3 82.8 CuTo 3 AS49 To 0 0 0 42.8 00 11 72.6 CuTo 3.1 AS119 Pu 0 0 0 82.3 01 1 71 82.5 CuTo 4 FL12 To 0 5 1 5 1 83 02 1 11 83 CuTo 4 FL2920 To 4 1 0 0 82 00 082.6 CuTo 4 MS31 To 0 7 1 8 1 82.8 00 11 82.3 CuTo 4 GU128 To 0 1 1 0 82.9 02 1 11 82.5 CuTo 4 SN30 To 0 0 0 83 02 1 21 83 CuTo 4 AS92 Cu 2 1 1 1 0 82.8 00 11 82.9 CuTo 4 JMP216a La 7 1 0 0 71.3 00 21 83 CuTo 5 PW101 Co 0 0 4 1 72.9 00 071.3 Pa 1.1 GU92 Pa 2 1 0 0 11 72.1 0 11 71 Pa 1.1 PW01 Pa 0 0 0 81 82.4 0 081 Pa 1.1 PW12 Pa 0 0 1 1 11 41.4 2 1 011 Pa 1.1 SN03 Pa 1 1 0 0 81 62.3 0 11 11 Pa 1.1 YP01 Pa 0 7 1 0 11 81.9 0 081 To 4 GU28 To 0 0 0 81 00 11 83 Path Pro (Pathogenicity Profile) : A list of susceptible hosts, or plants with an average disease rating greater than 1. PL : Phylogenetic lineage designation based on combined se quence analysis of ITS rDNA, CAA5, GA4, and ACT. Isolate : Corynespora cassiicola isolate code. Host : Original host the isolate was collected from. Ba ( Ocimum basilicum ), Be ( Phaseolus vulgarus ), Co ( Vigna unquiculata ), Cu ( Cucumis sativus ), La ( Lantana camara ), Pa ( Carica papaya), Pu ( Cucurbita pepo), Sw ( Ipomoea batatas ), To ( Solanum lycopersicum ). I (Incidence): Number of plants (out of 8 reps) that s howed symptoms seven days after inoculation with 20,000 C. cassiicola spores per ml. S (Severity): Average rating of symptomatic plants (these rated 1, 2, or 3). Plants were rated with the following scale: (0) symptomless; (1 ) non pathogenic hypersensitive response, a few to many non-expanding pinpoint lesions; (2) moderately virulent, many expanding lesio ns, some coalescing, but not resulting in blight; (3 ) highly virulent, lesions spreading to fo rm large areas of dead tissue resulting in a blighting effect.

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79 Table 2-4. Growth rate of Corynespora cassiicola isolates at 23C. Iso. ID PL Location Host Avg GR LSD GU99 6 Guam Saintpaulia 0.4743a AS81 1 Samoa Clerodendron 0.4639ab GU90 1 Guam Stachytarpheta 0.4528abc HI01 1 Oahu Basil 0.4521abcd PW94 1 Palau Stachytarpheta 0.4521abcd FL37 1 Florida Clerodendron 0.4514abcd GU104 1 Guam Macroptilium 0.4507abcde AS67 1 Samoa Commelina 0.4479bcde AS54 1 Samoa Bean 0.4444bcdef GU08 1 Guam Lantana 0.4438bcdef AS71 1 Samoa Pumpkin 0.4410bcdef SN03 1 Saipan Bitter melon 0.4389cdef YP26 1 Yap Cucumber 0.4375cdef GU136 4 Guam Ficus 0.4375cdef PW80 1 Palau Saintpaulia 0.4375cdef SN05 1 Saipan SwPotato 0.4375cdef AS58 1 Samoa Bean 0.4368cdef YP29 1 Yap Cucumber 0.4361cdef YP51 1 Yap Bean 0.4326cdef SN37 4 Saipan Bean 0.4313cdef GU115 1 Guam Vitex 0.4278defg PW92 1 Palau Catharanthus 0.4264efg AS78 1 Samoa Basil 0.4229fgh GU21 1 Guam Buddleja 0.4215fgh AS80 3 Samoa Basil 0.4202fgh PW91 1 Palau Luffa 0.4202fgh AS50 3 Samoa Tomato 0.4063ghi FL34 1 Florida Tabebouia 0.4055ghi YP08 1 Yap Papaya 0.4000hij PW79 2 Palau Pilea 0.3951ijk SN59 1 Saipan Lantana 0.3951ijk RWB321 5 Brazil Coleus 0.3945ijk JMP218 1 Brazil Soybean 0.3924ijkl CSB12 2 Malaysia Rubber 0.3917ijklm SN06 3 Saipan Luffa 0.3903ijklmn PW37 1 Palau Papaya 0.3896ijklmn FL2920 4 Florida Tomato 0.3882ijklmn SN07 1 Saipan Papaya 0.3868ijklmno PW101 5 Palau Bean 0.3833ijklmno PW99 3 Palau Bitter melon 0.3833ijklmno SN64 5 Saipan Asystasia 0.3822ijklmno GU109 3 Guam Bauhinia 0.3820ijklmno AS98 1 Samoa Cucumber 0.3819ijklmno CLN16 1 Malaysia Rubber 0.3778jklmnop PW89 4 Palau Chromolaena 0.3778jklmnop YP59 2 Yap SwPotato 0.3778jklmnop

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80 Table 2-4. Continued. Iso. ID PL Location Host Avg GR LSD AS49 3 Samoa Tomato 0.3771jklmnopq GU107 4 Guam Mikania 0.3729klmnopqr CBPP 1 Malaysia Rubber 0.3695lmnopqrs AS119 3 Samoa Papaya 0.3687lmnopqrst YP42 3 Yap Tomato 0.3674mnopqrstu YP01 1 Yap Papaya 0.3667nopqrstu AS92 4 Samoa Cucumber 0.3632opqrstu GU112 3 Guam Bean 0.3632opqrstu FL12 4 Florida Tomato 0.3556pqrstuv GU98 4 Guam Spathodea 0.3549pqrstuv GU92 1 Guam Papaya 0.3529qrstuvw FL09 4 Florida Lantana 0.3521rstuvw YP17 1 Yap Papaya 0.3521rstuvw GU102 4 Guam Bean 0.3507rstuvwx GU38 2 Guam Allamanda 0.3507rstuvwx PW57 4 Palau Tomato 0.3500rstuvwx YP41 2 Yap Saintpaulia 0.3480stuvwxy AS117 3 Samoa Papaya 0.3458stuvwxy SN40 4 Saipan Cucumber 0.3444tuvwxy AS65 4 Saipan Eggplant 0.3437uvwxy PW01 1 Palau Papaya 0.3368vwxyz GU41 4 Guam Eugenia 0.3361vwxyz YP27 2 Yap Cucumber 0.3340vwxyz FL36 2 Florida Catharanthus 0.3312vwxyz JMP217 4 Brazil Tomato 0.3299wxyz SN24 4 Saipan Tomato 0.3264xyz DOA16b 1 Brazil Papaya 0.3250yz FL15 4 Florida Salvia 0.3146z GU120 4 Guam Coleus 0.2792A JMP216a 4 Brazil Lantana 0.2535B PH01 1 Pohnpei Papaya 0.1479C PL: Phylogenetic Lineage based on sequence data from 4 loci. Avg GR: Average slope (growth ra te) of three replicate plates. LSD: Average slope (growth rate) values followed by different letters ar e significantly different from one another according to l east significant difference test ( P <0.05).

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81 Table 2-5. Growth rate of Corynespora cassiicola isolates at 33C. Iso. ID PL Location Host Avg GR LSD AS71 1 Samoa Pumpkin 0.4153a FL37 1 Florida Clerodendron 0.3972b AS78 1 Samoa Basil 0.3965b SN37 4 Saipan Bean 0.3917bc PW92 1 Palau Catharanthus 0.3813bcd SN03 1 Saipan Bitter melon 0.3799bcd GU90 1 Guam Stachytarpheta 0.3778cd GU99 6 Guam Saintpaulia 0.3771cde AS67 1 Samoa Commelina 0.3764cde PW79 2 Palau Pilea 0.3722def PW80 1 Palau Saintpaulia 0.3715defg HI01 1 Oahu Basil 0.3680defgh GU136 4 Guam Ficus 0.3674defghi YP51 1 Yap Bean 0.3653defghi SN05 1 Saipan SwPotato 0.3597efghij GU115 1 Guam Vitex 0.3577fghij GU21 1 Guam Buddleja 0.3576fghij YP26 1 Yap Cucumber 0.3569fghij YP29 1 Yap Cucumber 0.3548fghij AS54 1 Samoa Bean 0.3542ghijk AS58 1 Samoa Bean 0.3542ghijk GU104 1 Guam Macroptilium 0.3542ghijk PW91 1 Palau Luffa 0.3514hijkl GU08 1 Guam Lantana 0.3500ijklm AS98 1 Samoa Cucumber 0.3451jklmno SN07 1 Saipan Papaya 0.3368klmno YP08 1 Yap Papaya 0.3354lmno AS92 4 Samoa Cucumber 0.3327mnop PW94 1 Palau Stachytarpheta 0.3292nopq JMP218 1 Brazil Soybean 0.3285nopq GU98 4 Guam Spathodea 0.3278nopq PW89 4 Palau Chromolaena 0.3278nopq YP17 1 Yap Papaya 0.3236opqr PW37 1 Palau Papaya 0.3224opqr GU107 4 Guam Mikania 0.3215opqr PH01 1 Pohnpei Papaya 0.3174pqrs DOA16b 1 Brazil Papaya 0.3132qrst GU102 4 Guam Bean 0.3125qrstu PW01 1 Palau Papaya 0.3063rstuv GU92 1 Guam Papaya 0.3028stuv YP01 1 Yap Papaya 0.3014stuv PW57 4 Palau Tomato 0.2993tuvw GU41 4 Guam Eugenia 0.2959tuvwx AS81 1 Samoa Clerodendron 0.2952uvwx SN40 4 Saipan Cucumber 0.2951uvwx PW101 5 Palau Bean 0.2951uvwx

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82 Table 2-5. Continued. Iso. ID PL Location Host Avg GR LSD YP41 2 Yap Saintpaulia 0.2903vwxy FL2920 4 Florida Tomato 0.2889vwxy SN64 5 Saipan Asystasia 0.2829wxyz CLN16 1 Malaysia Rubber 0.2792xyz CSB12 2 Malaysia Rubber 0.2771yz CBPP 1 Malaysia Rubber 0.2736yz YP59 2 Yap SwPotato 0.2688zA FL15 4 Florida Salvia 0.2686zA YP27 2 Yap Cucumber 0.2667zA FL36 2 Florida Catharanthus 0.2521AB PW99 3 Palau Bitter melon 0.2486BC GU109 3 Guam Bauhinia 0.2438BC SN24 4 Saipan Tomato 0.2431BC FL12 4 Florida Tomato 0.2326CD AS80 3 Samoa Basil 0.2313CD GU38 2 Guam Allamanda 0.2312CD SN06 3 Saipan Luffa 0.2250DE RWB321 5 Brazil Coleus 0.2188DE FL09 4 Florida Lantana 0.2097EF AS65 4 Saipan Eggplant 0.2076EF AS50 3 Samoa Tomato 0.1993FG YP42 3 Yap Tomato 0.1938GFH SN59 1 Saipan Lantana 0.1875GHI FL34 1 Florida Tabebouia 0.1763HIJ JMP216a 4 Brazil Lantana 0.1750IJ GU112 3 Guam Bean 0.1709IJK GU120 4 Guam Coleus 0.1688JKL AS49 3 Samoa Tomato 0.1660JKL JMP217 4 Brazil Tomato 0.1549KLM AS117 3 Samoa Papaya 0.1521LM AS119 3 Samoa Papaya 0.1382M PL: Phylogenetic Lineage based on sequence data from 4 loci. Avg GR: Average slope (growth ra te) of three replicate plates. LSD: Average slope (growth rate) values followed by different letters ar e significantly different from one another according to l east significant difference test ( P <0.05).

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83 Figure 2-1. Fifty percent majority rule consen sus tree-phylogram from Bayesian inference analysis of combined data from rDNA ITS, Cc-ga4, Cc-caa5, and Cc-act1 sequences. Numbers above branches indicate maximum parsimony bootstrap > 70% and numbers below branches indicate posterior probability values > 0.90. Pathogenicity profiles on eight crop plants: basil (Ba), bean (Be), cowpea (Co), cucumber (Cu), papaya (Pa), soybean (So), sweet potato (Sw), and tomato (To), and phylogenetic lineage (PL) are indicated.

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84 Figure 2-2. Fifty percent majority rule consen sus tree-phylogram from Bayesian inference analysis of rDNA ITS locus. Numbers above branches indicate maximum parsimony bootstrap > 70% and numbers below branches indicate posterior pr obability values > 0.90. 100,000 maximum parsimony trees were a result of only 3 informative characters within C. cassiicola Phylogenetic lineages (PL) are indicated.

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85 Figure 2-3. Fifty percent majority rule consen sus tree-phylogram from Bayesian inference analysis of the Cc-caa5 locus. Numbers above branches indicate maximum parsimony bootstrap > 70% and numbers below branches indicate posterior probability values > 0.90. Phylogene tic lineages (PL) are indicated.

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86 Figure 2-4. Fifty percent majority rule consen sus tree-phylogram from Bayesian inference analysis of the Cc-ga4 locus. Numbers above branches indicate maximum parsimony bootstrap > 70% and numbers below branches indicate posterior pr obability values > 0.90. Phylogenetic lineages (PL) are indicated.

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87 Figure 2-5. Fifty percent majority rule consen sus tree-phylogram from Bayesian inference analysis of the Cc-act1 locus. Numbers above br anches indicate maximum parsimony bootstrap > 70% and numbers below branches indicate posterior probability values > 0.90. Phylogene tic lineages (PL) are indicated.

PAGE 88

88 Figure 2-6. UPGMA dendrogram of 50 Corynespora cassiicola isolates based on pathogenicity profiles on eight crop plants: basil (Ba), bean (Be), cowpea (Co), cucumber (Cu), papaya (Pa), soybean (So), sweet potato (Sw) tomato (To). Isolates are labeled with their phylogenetic lineag e (PL) designation to demonstrat e that isolates from the same PL cluster together. Statistical s upport for nodes by 1,000 UPGMA Bootstrap repetitions is indicated.

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89 Figure 2-7. Demonstration of the C. cassiicola disease rating system. Symptoms on A) basil, B) bean, C) cowpea, and D) tomato plants se ven days after inoculation with different isolates of Corynespora cassiicola. Plants were rated with the following scale: (0) symptomless; (1) non pathogenic hypers ensitive response, a few to many nonexpanding pinpoint lesions; (2) moderately virulent, many expanding lesions, some coalescing, but not resulting in blight; (3) hi ghly virulent, lesions spreading to form large areas of dead tissue re sulting in a blighting effect.

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90 LIST OF REFERENCES Abul-Hayja ZP, W illiams H, Peterson CE (1978) Inheritance of resistan ce to anthracnose and target leaf spot in cucumbers. Plant Dis Rep 62:43-45 Acharya B, Mishra SK, Acharya A, Mohapatr a KB, and Das, AK (2003) Bioassay of culture metabolites of Corynespora cassiicola ( Berk. Curt. ) Wei on the detached plant parts of betelvine ( Piper betle L.). Orissa J Hortic 31:8-9 Ahmad S (1969) Fungi of West Pa kistan. Biological Society of Pakistan Monograph 5(Sup.1):1110 Alfieri SA Jr, Langdon KR, Wehlburg C, Kimbrough JW (1984) Index of Plant Diseases in Florida. Florida Dept. of Agriculture and C onsumer Sciences, Div. Of Plant Industry. Bull. No. 11 (Revised), pp 389 Alfieri SA Jr, Langdon KR, Kimbrough JW, El-G holl NE, Wehlburg C (1994) Diseases and Disorders of Plants in Florida. Florida Department of Agriculture and Consumer Services, pp 1114 Anderson PJ, Dixon WN (2004) Florida Department of Agriculture and Consumer Services Plant Pathology Section, Ornamentals, Folia ge Plants, Tri-ology, Vol. 43, No. 1 Arnold GRW (1986) Lista de Hongos Fitopatogenos de Cuba. Ministerio de Cultura Editorial Cientifico-Tecnica, 207 pp Atan S, Hamid NH (2003) Differentiating races of Corynespora cassiicola using RAPD and internal transcribed spacer markers. J Rub Res 6:58-64 Barreto RW, Evans HC (1998) Fungal pathogens of Euphorbia heterophylla and E. hirta in Brazil and their potential as weed biocontrol agents Mycopathologia 141:21-36 Barthe P, Pujade-Renauld V, Breton F, Gargani D, Thai R, Roumestand C, de Lamotte F (2007) Structural analysis of cassiicolin, a host-selective protein toxin from Corynespora cassiicola. J Mol Biol 367:89-101 Beaver RG (1981) Guam Agricultural Experiment Station Annual Report, 36 pp Bird J, Krochmal A, Zentmyer G, Adsuar J (1966 ) Fungus diseases of papaya in the U.S. Virgin Islands. J Agric Univ Puer Rico 50:186-200 Blazquez CH (1967) Corynespora leaf spot of cucumber. Fla Agric Exp Stn J Ser No 2858, pp 177-182 Blazquez CH (1968) Corynespora cassiicola on bananas. Phytopath ology 52:1347 (Abstr.) Blazquez CH (1972) Target spot of tomato. Plant Dis Rep 56:243-245

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91 Bliss FA, Onesirosan PT, Arny DC ( 1973) Inheritance of resistance in tomato to target leaf spot. Phytopathology 63:837-840 Boa E, Lenn J (1994) Diseases of Nitrogen Fixing Trees in Developing Countries. An annotated list. Natural Resources Inst., Kent, United Kingdom, pp 82 Boosalis MG, Hamilton RI (1957) Root and stem rot of soybean caused by Corynespora cassiicola Plant Dis Rep 41:8:696-698 Brooks F (2002) List of plant di seases in American Samoa. Land Grant Technical Report No. 38. 50 CABI Databases (2008, July 21). Herb. IMI records for Fungus: Corynespora cassiicola. Retrieved July 21, 2008 from: http://194.203.77.76/herbIMI/DisplayResults .asp? strName=Corynespora+cassiicola Carbone I, Kohn LM (1999) A me thod for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553-556 Casady W (1994) Florida Department of Agricu lture and Consumer Services, P94-5328, Triology, Vol. 33, No.6, Nancy C. Coile Managi ng Editor. Retrieved May 12, 2008 from: http://www.doacs.state.fl .us/pi/enpp/94-11&12all.htm Chase AR (1981) Com parison of Myrothecium sp. and Corynespora cassiicola leaf spots of two cultivars of Aphelandra squarrosa. Proc Fla State Hortic Soc 94:115-116 Chase AR (1982) Corynespora leaf spot of Aeschynanthus pulcher and related plants. Plant Dis 66:739-740 Chase AR (1984) Leaf spot disease of Ficus benjamina caused by Corynespora cassiicola Plant Dis 68:251 Chase AR (1986) Corynespora bract spot of Euphorbia pulcherrima in Florida. Plant Dis 70:1074 Chase AR (1987) Compendium of Ornament al Foliage Plant Diseases. American Phytopathological Society Press, St. Paul, 92 pp Chase AR (1993) Corynespora l eaf spot and stem rot of Salvias. CFREC-Apopka Research Report, RH-93-12 Cheeran A (1968) Leaf and stem bli ght of Japanese Mentha caused by Corynespora cassiicola Agric Res J Karala 6:141

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92 Cho WD, Shin HD (2004) List of Plant Diseases in Korea. Fourth ed. Korean Society of Plant Pathology, 779 pp Coile NC, Dixon WN (1994) Florida Department of Agriculture and Consumer Services Plant Pathology Section, Ornamentals, Folia ge Plants, Tri-ology, Vol. 33, No. 5 Collado J, Platas G, Gonzalez I, and Pelaez F (1999) Geographical and seasonal influences on the distribution of fungal endophytes in Quercus ilex New Phytol 144:525-532 Cutrim FA, Silva GS (2 003) Pathogenicity of Corynespora cassiicola to different plant species. Fitopatol Brasil 28:193-194 Da Silva JL, Soares DJ, Barreto RW (2005) Eye-spot of Rudbeckia laciniata caused by Corynespora cassiicola in Brazil. Br it Soc Plant Pathol, New Dis Rep No. 12 Dade HA (1940) A revised list of Gold Coast fungi and plant diseases. XXIX. Bull. Misc. Inform. Kew 6:205-247 Daughtrey M (2000) Diseases of bleeding heart ( Clerodendrum thomsoniae Balf.). APSnet: Common names of plant diseases. Plant Pathology Online. http://www.apsnet.org/online/ common/names/bleedhrt.asp Delgado-Rodriguez G, Mena-Porta les J (2004) Hifomicetos (hongos anamorficos) de la reserva ecologica "alturas de banao" (Cuba). Bol Soc Micol Madrid 28:115-124 Delgado-Rodriguez G, Mena-Portales J, Cal duch M, Decock C (2002) Hyphomycetes (hongos mitosporicos) del area protegida mil cumbre s, Cuba Occidental. Cryptog Mycol 23:277-293 Dixon WN (1997) Florida Department of Agricu lture and Consumer Services Plant Pathology Section, Ornamentals, Foliage Pl ants, Tri-ology, Vol. 36, No 2 Duarte MLR, Albuquerque FC, Prabhu AS (1978) A new leaf disease of cacao plants ( Theobroma cacao ) caused by the fungus Corynespora cassiicola. Fitopatol Brasil 3:259-265 El-Gholl NE, Schubert TS (1990) Corynespora leaf spot of Tabebuia Fla Dept Agric & Consumer Serv. Division of Plant Industry. Plant Pathol Circ No 328 El-Gholl NE, Schubert TS, Coile NC (1997) Diseases and disorders of plants in Florida. Bulletin No. 14 Supplement No 1. Fla Dept of Agric and Cons Serv, pp 90-91 Ellis MB (1957) Some species of Corynespora. Mycological Papers 65:1-15 Ellis MB, Holliday P (1971) Corynespora cassiicola (Berk. & Curt.) Wei. CMI Descriptions of Fungi and Bacteria No 31, Sheet 303

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93 Fajola AO, Alasoadura SO (1973) Corynespora leaf spot, a new disease of tobacco ( Nicotiana tabacum ). Plant Dis Rep 57:375-378 Farr DF, Rossman AY, Palm ME, McCray EB. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Re trieved May 24, 2008, from http://nt.arsgrin.gov/fungaldatabases/ Felsenstein J (1985) Confidence limit on phylog enies: an approach using the bootstrap. Evolution; International Journa l of Organic Evolution 39:783-791 Fernandes RC, Barreto RW (2003) Corynespora cassiicola causing leaf spots on Coleus barbatus Plant Pathol 52:786 Ferreira FA (1989) Principais Doencas Florestais no Brasil. Patologia Florestal. Vicosa. MG Minas Gerais, 570 pp Florence EJM, Sharma JK (1987) Corynespora cassiicola : a new leaf pathogen for Gmelina arborea in India. J Trop For 3:181-182 Freire FCO (2005) An updated list of plant fungi from Cear state (Brazil) I Hyphomycetes. Revista Cincia Agronmica 36:364-370 Furukawa T, Ushiyama K, and Kishi K (2008) Cor ynespora leaf spot of scarlet sage caused by Corynespora cassiicola J Gen Plant Pathol 74:117-119 Gond SK, Verma VC, Kumar A, Kumar V, Kh arwar RN (2007) Study of endophytic fungal community from different parts of Aegle marmelos Correae ( Rutaceae) from Varanasi (India). World J Microbiol Biotechnol 23:1371-1375 Gowda CLL, Ramakrishna A, Rupela OP, Wa ni SP (2001) Legumes in Rice-Based Cropping Systems in Tropical Asia. Andhra Pradesh, India, pp 11-25 Grand LF (1985) North Carolina Plant Disease I ndex. North Carolina Agric Res Serv Techn Bull 240:1-157 Guo YL (1992) Foliicolous hyphomycetes of G uniujiang in Anhui Province II. Mycosystema 5:109-112 Hasama W, Morita S, Kato T (1991) Cor ynespora leaf spot of Perilla caused by Corynespora cassiicola Annals Phytopathol Soc Jap 57:732-736 Hawaiian Ecosystems at Risk (HEAR). (2008, Ju ly 11). Pathogens of Plants of Hawaii, Corynespora cassiicola Retrieved July 11, 2008, from: http://www.hear.org/pph/pathogens/1065.htm

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94 Holliday P (1980) Fungus Diseases of Tropical Cr ops. Cambridge University Press. Cambridge, UK Hongn S, Ramallo A, Baino O, Ramallo JC (2007) First Report of Target Spot of Vaccinium corymbosum caused by Corynespora cassiicola Plant Dis 91:771 Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogeny. Bioinformatics (Oxford, England) 17:754-755 Hyde KD, Alcorn JL (1993) Some disease-asso ciated microorganisms on plants of Cape York Peninsula and Torres Strait Islands. Australas Plant Pathol 22: 73-83 Hyde KD, McKenzie EHC, Dalisay TU (2001) Saprobic fungi on bamboo culms. Fungal Divers 7:35-48 Isabel N, Beaulieu J, Theriault P, Bousquet J (1999) Direct evid ence for biased gene diversity estimates from dominant random amplified polymorphic DNA (RAPD) fingerprints. Molecular Ecology 8:477-483 Johnston A (1960) A supplement to a host list of plant diseases in Ma laya. Mycol Pap 77:1-30 Jones JP (1961) A leaf spot of cotton caused by Corynespora cassiicola Phytopathology 51:305308 Jones JP, and Jones JB (1984) Target spot of to mato: epidemiology, and control. Proc Fla State Hortic Soc 97:216-218 Jones JB, Jones JP, Stall RE, Zitter TA (1991) Co mpendium of Tomato Diseases. APS Press, St. Paul, MN, 100 pp Khare MN (1991) Lentil diseases with special re ference to seed quality. Indian J Mycol Plant Pathol 21:1-13 Kingsland GC (1985) Pathogenicity and epidemiology of Corynespora cassiicola in the Republic of the Seychelles. Ac ta Hortic (ISHS) 153:229-230 Komaraiah M, Reddy SM (1986) Production of cellulases by Corynespora cassiicola Wei, a seed borne fungus of methi. Acta Botan Ind 14:133-138 Kranz J (1963) Fungi collected in the Republic of Guinea, Colle ctions from the Kindia area in 1962. Sydowia 17:174-185 Kurt S (2004) Host-specific toxin production by the tomato target leaf spot pathogen Corynespora cassiicola Turk J Agric and Fores 28:389-395

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95 Kwon JH, Kang SW, Kim JS, Park CS (2001) First report of Corynespora leaf spot in pepper caused by Corynespora cassiicola in Korea. Plan t Pathol J 17:180-183 Kwon JH, Park CS (2003) Leaf spot of cotton rose caused by Corynespora cassiicola in Korea. Mycobiology 31:57-59 Lakshmanan P, Jeyarajan R, Vidhyasekara n P (1990) A boll rot of cotton caused by Corynespora cassiicola in Tamil Nadu, India. Phytoparasitica 18:171-174 Lee S, Melnik V, Taylor JE, Crous PW ( 2004) Diversity of saprobic hyphomycetes on Proteaceae and Restionaceae from South Africa. Fung Diversity 17:91-114 Leite RS, Barreto RW (2000) Petal s potting of hydrangea flowers caused by Corynespora cassiicola : old pathogen new diseas e. Mycologist 14:80-83 Lenn JM (1990) World List of Fungal Diseases of Tropical Pasture Spec ies. Phytopathol Pap 31:1-162 LSU Ag Center (2008, July 21). Common Diseases of Ornamental Plants. Retrieved July 21, 2008, from: http://www.lsuagcenter.com/NR/rdonlyres/F512A031-FDF9-46C6-AEE2B800409C9FDF/42824/ornam entals1.PDF Lu B, Hyde KD, Ho WH, Tsui KM, Taylor JE, Wong KM, Yanna, Zhou D (2000) Checklist of Hong Kong Fungi. Fungal Divers ity Press, Hong Kong, 207 pp Lumyong P, Photita W, McKenzie EHC, Hyde KD, Lumyong S (2003) Saprobic fungi on dead wild banana. Mycotaxon 85:345-346 Maddison DR, Maddison WP (2005) MacClade 4, version 4.08. Sunderland: Sinuar Associates Mallaiah KV, Vijayalakshmi M, Rao AS (1981) New records of some fo liar diseases. Indian Phytopathology 34: 247 Malvick D (2004) Fungus foliage diseases of soyb eans. University of Illinois Extension. Report on Plant Disease No. 503 Mathiyazhagan S, Kavitha K, Nakkeeran S, Chandrasekar G, Mani an K, Renukadevi P, Krishnamoorthy AS, Fernando WGD (2004) PGPR me diated management of stem blight of Phyllanthus amarus (Schum and Thonn) caused by Corynespora cassiicola (Berk and Curt) Wei. Arch Phytopathol Plant Prot 37:183-199 McDonald BA (2004) Population genetics of plant pathogens. The Plant Health Instructor doi:10.1094/PHI-A-2004-0524-01 McGovern RJ (1994) Target spot of Catharanthus roseus caused by Corynespora cassiicola Plant Dis 78:830

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96 McKenzie EHC, Buchanan PK, Johnston PR (2004) Check-list of fungi on nikau palm ( Rhopalostylis sapida and R. baueri var. cheesemanii ) in New Zealand. N Z J Bot 42:335 355 McKenzie EHC (1990) The fungi, bacteria and pathogenic algae of the Republic of Palau. Technical Paper No. 198. South Pacific Commission, Noumea, New Caledonia, 41 pp McKenzie EHC (1996) Fungi, bacteria and pathog enic algae on plants in American Samoa. Technical Paper No. 206. South Pacific Commission, Noumea, New Caledonia McRitchie JJ, Miller JW (1973) Corynespora leaf spot of zebra plant. Proc Fla State Hort Soc 86:389-390 Mehrotra MD (1987) Corynespora cassiicola leaf spot of Ceiba pentandra and its control in the nursery. Ind For 115:905-909 Mehrotra MD (1997) Diseases of Paulownia and their management. Indian Forester 123:66-72 Mendes MAS, da Silva VL, Dianese JC, Ferreir a MASV, Santos CEN, Gomes Neto E, Urben AF, Castro C (1998) Fungos em Plantas no Brasil. Braslia. Empresa Brasileira de Pesquisa Agropecuria, 555 pp Mercado S (1984) Hifomicetes Demaciaceos de Si erra del Rosario, Cuba. Editorial Academica, Havana, 181 pp Mercado S, Holubov-Jechov V, Mena Portales J (1997) Hifomicetes Demaciceos de Cuba Enteroblsticos. Museo Regionale di Scienze Naturali 23:388 Miller JW (1991) Bureau of Plant Pathology. Tri-ology Techn. Rep. Div. Plant Indust, Florida 30:1-2 Miller JW, Alfieri SA Jr (1973) Leaf spot of Ligustrum sinense caused by Corynespora cassiicola Phytopathology 63:445-446 Minter DW, Rodrguez Hernndez M, Mena Port ales J (2001) Fungi of the Caribbean: an annotated checklist. PDMS Publishing, 946 pp Murali TS, Suryanarayanan TS, Venkatesan G (2007) Fungal endophyte communities in two tropical forests of southern India: diversity and host affiliation. Mycol Progr 6:191-199 Olive LS, Bain DC (1945) A leafspot of cowp ea and soybean caused by an undescribed species of Helminthosporium. Phytopathology 35:822-831 Oluma HOA, Amuta EU (1999) Corynespora cassiicola leaf spot of pawpaw (Carica papaya L.) in Nigeria. Mycopathologia 145:23-27

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97 Onesirosan PT, Arny DC, Durbin RD (1975) Increasing sporulation of Corynespora cassiicola Mycopathologia 55:121-123 Onesirosan PT, Arny DC, Durbin RD (1974) Host specificity of Nigerian and North American isolates of Corynespora cassiicola. Phytopathology 64:1364-1367 Onesirosan PT, Arny DC, Durbin RD (1973) Target Spot of Tomato Incited by Corynespora cassiicola (Berk. & Curt.) Wei. Ph. D. Thesis, University of Wisconsin, Madison, 93 pp Orieux L, Felix S (1968) List of plant dise ases in Mauritius. Phytopathol Pap 7:1-48 Peregrine WTH, Ahmad KB (1982) Brunei: A first annotated list of plant diseases and associated organisms. Phytopathol Pap 27:1-87 Pereira JM, Barreto RW, Ellison CA, Maffia LA (2003) Corynespora cassiicola f. sp. lantanae : a potential biocontrol agent from Brazil for Lantana camara BiolControl 26:21-31 Pernezny K, Simone GW (1993) Target spot of several vegetable crops. PP-39, A series of the Plant Pathology Department, Fla Coop Ext Ser, IFAS, Univ of Fla Pernezny K, Datnoff LE, Mueller T, Collins J (1 996) Losses in fresh-market tomato production in Florida due to target spot and bacterial spot and the benefits of protectant fungicides. Plant Dis 80:559-563 Pernezny K, Datnoff LE, Rutherford B, Carroll A (2000) Relationship of temperature to growth, sporulation, and infection of tomato by the target spot fungus. Florida Tomato Committee Tomato Research Report for 2000, pp 16-19 Pernezny K, Stoffella P, Collins J, Carroll A, Bean ey A (2002) Control of target spot of tomato with fungicides, systemic acquired resistance ac tivators, and a biocontro l agent. Plant Prot Sci 38:81-88 Pernezny KL, Datnoff LE, Smith LJ, Schlub RL (2008) An overview of target spot of tomato caused by Corynespora cassiicola. Acta Hort xxx: Second International Symposium on Tomato Diseases (accepted) Philip S, Ramakrishnan CK, Menon MR (1972) Leaf blight of Coccinia indica (Wight & Arn.) caused by Corynespora cassiicola. Agric Res J Kerala 10:196 Pollack FG, Stevenson JA (1973) A fungal pathogen of Broussonetia papyrifera collected by George Washington Carver. Plant Dis Rep 57:296-298 Poltronieri LS, Duarte MLR, Alfenas AC, Trindade DR, Albuquerque FC (2003) Three new pathogens infecting Antilles Cherry in the state of Para. Fitopatol Brasil 28:424-426

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98 Posada D, Crandall KA (1998) Modeltest: test ing the model of DNA substitution. Bioinformatics 14:817-81 Prakash O, Garg N (2007) A new report of Corynespora casiicola causing black rot of aonla seedlings. J Mycol Plant Pathol 37:120-121 Promputtha I, Lumyong A, Dhanasekaran V, McKenzie EHC, Hyde KD, Jeewon R (2007) A phylogenetic evaluation of whether endophytes become saprotrophs at host senescence. Micro Ecol 53:579-590 Puzari KC, Saikia UN (1981) Amorphophallus campanulatus a new host of Corynespora cassiicola Indian Phytopathol 34:537-538 Quimio RH, Abilay LE (1979) Note: Corynespora di sease of papaya in the Philippines. Philipp Phytopathology 15:158-161 Raabe RD, Conners IL, Martinez AP (1981) Checklist of plant dis eases in Hawaii. College of Tropical Agriculture and Human Resources, Univer sity of Hawaii. Information Text Series No. 22. Hawaii Inst Trop Agric Human Resources, 313 pp Raffel SJ, Kazmar ER, Winberg R, Oplinger ES Handelsman J, Goodman RM, Grau CR (1999) First report of root ro t of soybeans caused by Corynespora cassiicola in Wisconsin. Plant Disease 83:696 Rajak RC, Pandey AK (1985) Fungi from Jabalpu r-II. Indian J Mycol Plant Pathol 15:186-194 Riley EA (1960) A revised list of plant diseases in Tanganyika Territory. Mycol Pap 75:1-42 Romruensukharom P, Tragoonrung S, Vanavichit A, Toojinda T (2005) Genetic variability of Corynespora cassiicola populations in Thai land. J Rub Res 8:38-49 Ronquist F, Huelsenbeck JP (2003) MrBayes3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572-1574 Sadaba RB, Vrijmoed LLP, Jones EBG, Hodgkiss IJ (1995) Observations on vertical distribution of fungi associated w ith standing senescent Acanthus ilicifolius stems at Mai Po Mangrove, Hong Kong. Hydrobiologia 295:119-126 Saikia UN, Sarbhoy AK (1981) Corynespora leaf spot of Eugenia caryophyllata Indian Phytopathol 34:401-402 Sarbhoy AK, Lal G, Varshney JL (1971) Fungi of India. Navyug Traders, New Delhi, 148 pp Sarma YR, Nayudu MV (1970) Corynespora leaf s pot of Brinjal. Proc Indian Acad Sci B(LXXIV):92-97

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99 Schlub RL, Yudin L (2002) Eggplant, pepper, and tomato production guide for Guam. Guam Cooperative Extension Publication, 188 pp Seaman WL, Shoemaker RA, Peters on EA (1965) Pathogenicity of Corynespora cassiicola on soybean. Can J Bot 43:1461-1469 Shaw DE (1984) Microorganisms in Papua New Guinea. Dept. Primary Ind., Res Bull 33:1344 Shivas RG, Alcorn JL (1996) A checklist of plant pathogenic and other microfungi in the rainforests of the wet tropics of northern Queensland. Australas Mycol 25:158-173 Silva WPK, Deverall BJ, Lyon BR (1995) RFLP a nd RAPD analyses in the identification and differentiation of isolates of the leaf spot fungus Corynespora cassiicola Austral J Bot 43:609-618 Silva WPK, Deverall BJ, Lyon BR (1998) Mo lecular, physiological and pathological characterization of Corynespora l eaf spot from rubber plantations in Sri Lanka. Plant Pathol 47:267-277 Silva WPK, Karunanayake EH, Wijesundera RLC, Priyanka UMS (2003) Genetic variation in Corynespora cassiicola : a possible relationship between hos t origin and virulence. Mycol Res 107:567-571 Silva WPK, Wijesundera RLC, Karunanayake EH, Jayasinghe CK, Priyanka UMS (2000) New hosts of Corynespora cassiicola in Sri Lanka. Plant Dis 84:202 Simone GW (2000) Diseases of Cattleya Lindl. spp. APSnet: Common Names of Plant Diseases. http://www.apsnet.org/online/common/names/cattleya.asp Simone GW (2000) Diseases of Pointsettia ( Euphorbia pulcherrina ). APSnet: Common Names of Plant Diseases. http://www.apsnet .org/online/common/names/poinsett.asp Singh KP, Shukla RS, Kumar S, Hussain A (1982) A leaf-spot disease of Dodonaea viscosa caused by Corynespora cassiicola in India. Ind Phytopathol 35:325 Situmorang A, Budiman A (1984) Corynespora cassiicola (Berk. And Curt.) Wei, penyebab penyakit gugur duan pada karet. Kumpulan Ma kalah Lokakarya Karet. PNP/PTP Wilayah 1 dan P4TM, Medan Sivanesan A (1996) Corynesporasca caryote gen. et sp. nov. with a Corynespora anamorph, and the family Corynesporascaceae. Mycol Res 100:783-788 Smith LJ, Datnoff LE, Rollins JA, Pernezny KL Scott JW, Schlub RL (2008a) High genetic diversity within Corynespora cassiicola based on multilocus sequence data, pathogenicity, and growth rate. Acta Hort xxx: Second In ternational Symposium on Tomato Diseases (accepted)

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100 Smith LJ, Datnoff LE, Pernezny KL, Rollins J, Schlub RL (2008b) Phylogenetic analyses of diverse Corynespora cassiicola isolates indicate an evolutionary corr elation with host not geography. 9th European Conference on Fungal Genetics Meeting Abstracts Smith LJ, Datnoff LE, Rollins JA, Pernezny KL, Schlub RL (2007) Phylogenetic analysis of Corynespora isolates from diverse hosts a nd locations. Phytopathology 97:S109 Smith LJ, Datnoff LE, Pernezny KL, Roberts PD, Rollins JA, Schlub RL, Scott JW (2006) Characterization and host-range of the tomato target spot fungus, Corynespora cassiicola and resistance of tomato cultivars. Florida To mato Committee, Tomato Research Report for 2004-2005, pp 14-20 Smith LJ, Schlub RL (2005) Foliar fungi on weeds of Guam and the potential for Corynespora cassiicola as a bioherbicide for Stachytarpheta jamaicensis. Phytopathology 95:S93 Smith LJ, Schlub RL (2004) Host range of Corynespora cassiicola and its occurrence on weeds, ornamentals and crops of Guam. Phytopathology 92:S77 Sobers EK (1966) A leaf spot di sease of azalea and hydrangea caused by Corynespora cassiicola Phytopathology 59:455-457 Spencer JA (1962) Stud y of variations in Corynespora cassiicola (Berk. & Curt.) Wei. M. S. Thesis, University of Arkansas, Fayetteville, 31 pp Spencer JA, Walters HJ (1969) Variations in certain isolates of Corynespora cassiicola Phytopathology 59:58-60 Stone WJ, Jones JP (1960) Corynespora bl ight of sesame. Phytopathology 50:263-266 Strandberg JO (1971) Evaluation of cu cumber varieties for resistance to Corynespora cassiicola. Plant Dis Rep 55:142-144 Suryanarayanan TS, Murali TS, Venkatesan G (2002) Occurrence and distribution of fungal endophytes in tropical forests across a rainfall gradient. Can J Bot 80:818-826 Swofford DL (2002) PAUP*. Phylogenetic anal ysis using parsimony (*and other methods). Version4. Sunderland: Sinauer Associates Taba S, Ooshiro A, Takaesu K (2002) Black stem and root rot of basil Ocimum basilicum L. caused by Corynespora citricola Ann Phytopathol Soc Japan 68:43-45 Tanaka K, Yasuyoshi O, Hatakeyama S, Harada Y, Barr ME (2008) Pleos porales in Japan (5): Pleomassaria, Asteromassaria, and Splanchnonema. Mycoscience 46:248-260

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101 Tsay JG, Kuo CH (1991) The occurrence of Coryne spora blight of cucumber in Taiwan. Plant Prot Bull 33:227-229 Turner GJ (1971) Fungi and Plant Diseas e in Sarawak. Phytopathol Pap 13:1-55 Urtiaga R (1986) Indice de enfe rmedades en plantas de Venezuela y Cuba. Impresos en Impresos Nuevo Siglo. SRL, Ba rquisimeto, Venezuela, 202 pp Urtiaga R (2004) Indice de en fermedades en plantas de Venezuela y Cuba, 2nd Ed, 301 pp Vittal BPR, Dorai M (1995) Studies on litter fung i VIII. Quantitative studies of the mycoflora colonizing Eucalyptus tereticornis Sm. Litter. Kavaka 22/23: 35-41 Volin RB, Pohronezny K (1989) Severe spotting of fresh market tomato fruit incited by Corynespora cassiicola after storm-related inju ry. Plant Dis 73:1018-1019 Vyas SC, Shastry PP, Shukla BN, Varma RK (1985) Two new leaf blight diseases of groundnut. Plant Prot Bull 33:121-122 Wei CT (1950) Notes on Corynespora. Mycol Papers 34:1-10 White TJ, Bruns T, Lee S, Taylor J (1990) Am plification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. P CR Protocols: A Guide to Methods and Applications. Innis MA, Gelfand DH, Sninsky JJ, White TJ (ed.) Academic Press, Inc, New York, NY, ch 38, pp 315-322 Williams TH, Liu PSW (1976) A host list of plant diseases in Sabah, Malaysia. Phytopathol Pap 19:1-67 Yudin L, Schlub RL (1998) Guam Cucurbit Guid e. Guam Cooperative Extension Publication, 64 pp Zhang XG, Ji M (2005) Taxonomic studies of Corynespora from Yunnan, China. Mycotaxon 92:425-429 Zhuang WY (2001) Higher Fungi of Tropica l China. Mycotaxon, Ltd., Ithaca, NY, pp 485

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102 BIOGRAPHICAL SKETCH I was born in Pom pano Beach, FL to Janis Th errell and Kenneth Wayne Smith on October 15, 1977. I have an older sister, Allison, and tw o younger brothers, Scott and Reid. Our family moved to Baltimore, MD when I was nine and I attended Baltimore Friends School, where my father was head of the Middle School. Though I have always loved biology and gardening, my interest in agriculture took off in high school when I attended Maine Coast Semester, a small school for students in their junior year locate d on a coastal farm in Wiscasset, Maine. I received my B. A. at Colorado College in 2000 with a major in Biology, while fostering my interest in farming through summer jobs at nurseries, CSAs, and internships. My sophomore year in college, I traveled abroad to East Africa through The School for Field Studies where I learned the importance of economic value in conservation by focusing on wildlife ranching, national parks, and medicinal plant use as case studies. In 2002, I received my Masters Degree from West Virginia University in Plant Pathology as part of the Organic Farm Project by studying the effect of intercropping on diseases caused by Alternaria solani and Meloidogyne incognita I then spent two years in Micronesia on the island of Guam as a Research Assistant documenting pathogens of agronomically important weeds and working in the diagnostic clinic. It was in Guam where I first became aware of Corynespora cassiicola as an agent of disease. An opportunity pr esented itself to continue the work begun on this pathogen at the University of Florida in the Fall of 2004. At the University of Florida, I became well trained in Phylogenetics and this ha s become my specialty. I plan to continue studying fungal systematics beginning in Septem ber, 2008 as a post-doc with the USDA in Beltsville, MD.







HOST RANGE, PHYLOGENETIC, AND PATHOGENIC DIVERSITY OF Corynespora
cassiicola (Berk. & Curt.) Wei




















By

LINLEY JOY SMITH


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2008


































2008 Linley Joy Smith

































To Peter, for making me laugh.









ACKNOWLEDGMENTS

Funding and support was made possible by the USDA Special Grant Program for Tropical

and Subtropical Agriculture Research, the University of Florida, IFAS, EREC, the Florida

Tomato Committee, the University of Guam, Guam Cooperative Extension, and the USDA IPM

3-D and Hatch funds.

I would like to thank Drs. Ken Pernezny, Pam Roberts, Jeffrey Rollins, and Jay Scott for

their support while serving on my supervisory committee. I would also like to express

appreciation to my major advisor, Dr. Lawrence Datnoff, for his commitment and help

throughout the course of my Ph.D. I would especially like to thank Dr. Robert Schlub for his

willingness to help in every step of the process and for his unwavering support, encouragement,

and friendship. Special thanks to my helpful coworkers in Guam, especially Roger Brown and

Lauren Gutierrez.

Most importantly, my heartfelt appreciation goes to my parents for their unconditional love

and support. Finally, I thank my husband for encouraging me to pursue this opportunity, an

ocean and continent away, for coming to Gainesville for me, and for keeping me smiling

throughout.









TABLE OF CONTENTS



A C K N O W L E D G M E N T S ..............................................................................................................4

LIST OF TABLES ................. ........................ .. ........... ...................................... .. 6

L IST O F F IG U R E S ......................................................................... ................................... . 7

ABSTRACT ....................................................... .......................8

CHAPTER

1 INDEX OF PLANT HOSTS OF Corynespora cassiicola................................................ 10

In tro du ctio n ............................................................................................................ ........ .. 10
M e th o d s ................................................... ..................................................... .................... 12
L literature Survey and H ost Index...................................... ...................... ............... 12
G uam and Florida Surveys .. .................................................................... ............... 13
R esu lts ........................................................................................................... 14
D iscu ssio n .............................................................................................................. ........ .. 16

2 GENETIC AND PATHOGENIC DIVERSITY OF CORYNESPORA CASSIICOLA ........... 48

In tro d u c tio n ............................................................................................................................. 4 8
M eth od s ................ ...... ........ .... .. ...................................................................... . 52
Collection and Solicitation of Fungal Isolates............................................ ................ 52
Primer Development for Random Hypervariable Loci .............................................54
Fungal Cultures and Extraction of Genomic DNA .................................... ................ 55
Phylogenetic Analyses ........................ .. ........... ................ ............... 57
Pathogenicity Analyses .................. ................ ......... ...... ...............59
Growth Rate Analyses ........................... ........... ........................ 60
R esu lts .............................................................................. ................... ... ................... 6 1
Phylogenetic Analyses ........................ ........... .........................61
Pathogenicity Analyses .................. ................ ......... ...... ...............65
Growth Rate Analyses ........................... ........... ........................ 66
D iscu ssio n .............................................................................................................. ........ .. 6 7

L IST O F R EFER EN CE S ............................................................................................. 90

B IO G R A PH IC A L SK E T C H .................................................... ............................................. 102









LIST OF TABLES


Table page

1-1 Taxonomic grouping of Corynespora cassiicola host species. ...................................20

1-2 Occurrence and fungal-host interaction of Corynespora cassiicola identified during
2004-2005 G uam and Florida surveys .......................................................... ................ 21

2-1 Isolate designations, geographic location of isolation, host of isolation, phylogenetic
lineage (PL), type of growth on associated host, and species of Corynespora used in
the phylogenetic analyses. .................... .................................................................. 72

2-2 Summary of sequence data from four loci used to confirm the phylogenetic lineage
of Corynespora cassiicola isolates ...................................... ...................... ................ 76

2-3 Pathogenicity profiles for 50 Corynespora cassiicola isolates....................................77

2-4 Growth rate of Corynespora cassiicola isolates at 230C.............................................79

2-5 Growth rate of Corynespora cassiicola isolates at 330C.............................................81









LIST OF FIGURES


Figure page

1-1 Corynespora cassiicola isolate from Cucumis sativus .................................................45

1-2 Various symptoms caused by Corynespora cassiicola on naturally infected leaves.........46

2-1 Fifty percent majority rule consensus tree-phylogram from Bayesian inference
analysis of combined data from rDNA ITS, Cc-ga4, Cc-caa5, and Cc-act]
sequ en ces. ....................................................................................................... ....... .. 83

2-2 Fifty percent majority rule consensus tree-phylogram from Bayesian inference
analysis of rD N A ITS locus. ............................................. .............. ................ 84

2-3 Fifty percent majority rule consensus tree-phylogram from Bayesian inference
analysis of the C c-caa5 locus ......................................................................... ................ 85

2-4 Fifty percent majority rule consensus tree-phylogram from Bayesian inference
analysis of the C c-ga4 locus ................................................................... ................ 86

2-5 Fifty percent majority rule consensus tree-phylogram from Bayesian inference
analysis of the Cc-act] locus. .......................................... .............. ................ 87

2-6 UPGMA dendrogram of 50 Corynespora cassiicola isolates based on pathogenicity
profi les on eight crop plants:............................................... ......................................... 88

2-7 Demonstration of the C. cassiicola disease rating system...........................................89









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

HOST RANGE, PHYLOGENETIC, AND PATHOGENIC DIVERSITY OF Corynespora
cassiicola (Berk. & Curt). Wei

By

Linley Joy Smith

August 2008

Chair: Lawrence E. Datnoff
Major: Plant Pathology

The fungus Corynespora cassiicola (Berk. & Curt.) Wei is a pathogen, endophyte, and

saprophyte. It can be found growing on at least 530 plant species from 380 genera, primarily in

the tropics. Isolates from diverse hosts were collected or solicited from locations in American

Samoa, Brazil, Malaysia, Micronesia, and Florida, Mississippi, and Tennessee within the United

States. Outgroup taxa including C. citricola, C. melongenea, C. olivaceae, C. proliferate, C.

sesamum, and C. smithii were solicited from culture collections. A multilocus phylogenetic

analysis using 143 isolates was performed to investigate how genetic diversity correlates with

host-specificity, growth rate, and geographic distribution. Phylogenetic trees were congruent

from the rDNA ITS region, two random hypervariable loci (Cs caa5 and Cs ga4), and the actin

encoding locus CC act], indicating asexual propagation. Fifty isolates had different

pathogenicity profiles when tested against eight known C. cassiicola hosts: basil, bean, cowpea,

cucumber, papaya, soybean, sweet potato, and tomato. Phylogenetic lineage correlated with

pathogenicity profiles, host originality, and growth rate, but not with geographic location.

Common fungal genotypes were widely distributed geographically indicating long distance and

global dispersal of clonal lineages. This research reveals an abundance of previously









unrecognized diversity within the species and provides evidence for redefining species

distinctions within Corynespora, which will aid in future disease control strategies.









CHAPTER 1
INDEX OF PLANT HOSTS OF CORYNESPORA CASSIICOLA

Introduction

Corynespora cassiicola (Berk. & Curt.) Wei has been commonly reported as a plant

pathogenic foliar fungus with a wide host range within tropical and subtropical areas (Holliday

1980; Farr et al. 1980; Romruensukharom et al. 2005). In addition to being a pathogen, on some

hosts C. cassiicola is also reported to grow as an endophyte or saprophyte (Collado 1999; Gond

et al. 2007; Promputtha et al. 2007; Suryanarayanan et al. 2002; Kingsland 1985; Hyde et al.

2001; Lee et al. 2004; Lumyong et al. 2003). Though the diseases attributed to C. cassiicola are

mainly foliar, it may also cause fruit, stem, and root diseases (Jones et al. 1991). The

generalization that individual C. cassiicola isolates have a wide host range is not supported by

the literature because host specific isolates, isolates pathogenic to select hosts, and weak

pathogens or secondary invaders of senescent tissue are known to exist (Onesirosan et al. 1974;

Cutrim and Silva et al. 2003; Kingsland 1985; Pereira et al. 2003). Rarely reported outside the

tropics and subtropics, there are occasional reports of the fungus from temperate regions,

particularly on soybean (Boosalis and Hamilton 1957; Malvick 2004; Raffel et al. 1999; Seaman

et al. 1965).

Disease symptoms attributed to C. cassiicola include necrosis, often with a surrounding

yellow halo (Pernezny and Simone 1993) due to the production of a host specific protein toxin,

cassiicolin (Barthe et al. 2007; Kurt 2004). With respect to foliage, young and mature leaves can

be affected, although the pathogen is more commonly associated with older leaves (Pernezny et

al. 2008). Substantial crop losses have been observed in many countries on numerous hosts:

southern United States on ornamentals (Alfieri et al. 1984, 1994; Chase 1981,1982, 1984, 1986,

1987, 1993; El-Gholl and Schubert 1990; El-Gholl et al. 1997; Miller and Alfieri 1973;









McRitchie and Miller 1973; Simone 2000, 2000), cucumber (Abul-Hayja et al. 1978; Blazquez

1967; Strandburg 1971), and tomato (Bliss et al. 1973; Blazquez 1972; Jones and Jones 1984;

Pernezny et al. 1996, 2002; Smith et al. 2006, Smith et al. 2008b); Midwestern United States on

soybean (Boosalis and Hamilton 1957), cowpea (Olive and Bain 1945) and sesame (Stone and

Jones 1960); India on ornamentals (Cheeran 1968; Mallaiah et al. 1981; Mehrotra 1987, 1997;

Silva et al. 2000; Singh et al. 1982), Hevea rubber trees (Atan and Hamid 2003; Silva et al.

1998), cotton (Lakshmanan et al. 1990), and weeds (Philip et al. 1972); Brazil on ornamentals

(Da Silva et al. 2005; Leite and Barreto 2000; Pohltronieri 2003), and weeds (Pereira et al.

2003); Philippines, Nigeria, and U.S. Virgin Islands on papaya (Quimio and Abilay 1979; Oluma

and Amuta 1999; Bird et al. 1966); and Micronesia and Asia on ornamentals (Florence and

Sharma 1987; Hasama et al. 1991), cucurbits (Yudin and Schlub 1998; Tsay and Kuo 1991),

tomato (Schlub and Yudin 2002), and pepper (Kwon et al. 2001).

Most regions report C. cassiicola diseases on only a few host species, despite the broad

host range of the fungus, prompting questions pertaining to isolate host specificity and

distribution. Addressing such questions will have implications for disease control and

quarantine. The host -specificity and severity of the fungus on Lantana camera in Brazil led to

the discovery of a new forma specialist, C. cassiicola f. sp. lantanae, and the use of the isolate as

a bioherbicide (Pereira et al. 2003). Based on the vast number of weeds that serve as hosts, and

past demonstration of host-specificity in some isolates, there is great potential for the discovery

of additional isolates useful for biological control. Further information on the fungal-host

interaction and host range of individual isolates will be useful in the study of disease epidemics.

The objective of this study was to compile a list of C. cassiicola hosts into a single

document, thereby aiding further research on the host range of individual isolates. Prior to this









study, the most complete host listing is in the fungal database of the ARS/USDA Systematic

Mycology and Microbiology Laboratory, which included 257 plant host species (Farr et al.

2008). This study will provide a more complete index for use by those engaged in phylogenetic

analysis of Corynespora spp. and in disease management. Awareness of the potential host range

of the fungal species is vital to the determination of the host-specificity of individual isolates.

The host range of individual isolates has direct implications for disease management, including

the identification of potential inoculum sources, recommendations for intercropping and crop

rotation, weed management, biological control candidacy, and isolate choice for resistance

breeding.

In order to obtain an estimate of the completeness of the list of hosts known to harbor C.

cassiicola, surveys were conducted to identify hosts in Guam and Florida. Guam is an ideal

location to discover new hosts due to its tropical climate, wet and dry seasons, and lack

heretofore of a Corynespora host survey (Schlub and Yudin 2002). Florida was included

because outbreaks of target spot on tomato caused by C. cassiicola are common and it represents

a subtropical environment located an ocean and a continent away from Guam.

Methods

Literature Survey and Host Index

An index of plant hosts of C. cassiicola was compiled from a search of world literature for

any reference regarding its presence on plant tissue. All plant-fungus associations were included

such as pathogenic, endophytic, and saprophytic. Resources included articles in refereed

journals, graduate student theses, books, and web-based resources such as annual reports,

production guides, and plant clinic lists. The final list of susceptible hosts of C. cassiicola was

compiled from the literature and personal observation from surveys in Florida and Guam.









All plant species, genera, and families were named and classified according to the USDA

Germplasm Resources Information Network (GRIN) taxonomy, which follows the APGII

system. In some cases, the host name given in the original citation was changed to be consistent

with GRIN taxonomy. In a few cases, neither the species cited nor a proper synonym was

identified using GRIN taxonomy and the species name was kept as originally cited. Only one

reference was provided per host, with emphasis on citing the first known report of that host. For

some hosts, the only reference that could be found was a website, and in those cases the website

is listed. The number of plant host species was conservatively determined by counting only

unique species within each genus. Genera with unidentified species (e.g. Crossandra spp.) were

counted only once when no other named species were present within that genus.

Guam and Florida Surveys

Surveys for the presence of C. cassiicola were conducted throughout Guam and Florida.

The Guam survey was conducted for one year beginning in January of 2004 and the Florida

survey was conducted for one year beginning in January of 2005. Survey areas focused on

roadsides, nurseries, and farms. During the course of the survey, leaves from plants with

characteristic C. cassiicola foliage disease symptoms were collected and placed in individual

plastic bags. Known hosts of C. cassiicola were sampled more intensely through the additional

collection of old and young asymptomatic leaves. An effort was made to sample from an equal

number of individual plants and unique plant species in Florida and Guam.

To induce sporulation, leaf tissue was placed abaxial side up in the moisture chamber for

10 days. Moisture chambers were created on the lab bench by placing 10 ml of sterilized

distilled water on a paper towel in a 150 mm petri plate. A plant species was identified as a host

of C. cassiicola if characteristic structures of the fungus developed within 10 days. An isolate

was labeled a pathogen if conidiophores arose from a necrotic spot and an endophyte if









conidiophores arose from healthy, green tissue. Petri plates were inspected under a dissecting

microscope daily for spores and conidiophores of C. cassiicola. Structures were confirmed

based on microscopic morphological features such as percurrent proliferation of the

conidiophores and pseudoseptation. Single spore isolates were obtained for long-term storage by

needle transfer of spores to antibiotic V8 agar agar slants (340 ml V8 juice, 660 ml water, 3 g

CaCO3, 17 g agar, 100 [tg/ml ampicillin or kanamycin). Slants were left at room temperature

until colonies reached at least 5 cm in diameter, covered with autoclaved mineral oil, and stored

at 5 C until further study.

Results

Over 900 individual plants were surveyed in both Guam and Florida from 320 unique plant

species in Guam and 289 unique plant species in Florida. Compilation of Corynespora

cassiicola hosts from the literature and surveys conducted in Guam and Florida resulted in an

index of 530 plant species from 380 genera. The majority of index host species for C. cassiicola

are herbaceous Eudicotyledonae, but 52 Monocotyledonae, eight Magnoliids, five Filicopsida

(ferns), and one cycad are also represented. No hosts were found within the Anthocerotophyta

(hornworts), Bryophyta (mosses), Equisetopsida (horsetails, sphenophytes), Lycopsida

(lycophytes), or Marchantiomorpha (liverworts) (Table 1-1).

Hosts were found in two plant divisions: Filicopsida and Spermatopsida. The five hosts in

the Filicopsida include Arachniodes aristata (Davalliaceae), Athyrium niponicum

(Dryopteridaceae), Adiantum cuneatum (Pteridaceae), Davallia repens (Davalliaceae), and

Platycerium spp. (Pteridaceae). The plant division Spermatophyta (Cycadales, Magnoliidae,

Monocotolydonae, and Tricolpates) contains 99% of the host species (Table 1-1). There are

eight species from the Magnoliidae. Three species are from the Piperaceae (Piper betle, P.









hispidinervum, and Perperomia obtusifolia). Three species are from the Magnoliales in the

family Annonaceae (Annona reticulata, A. squamosa and Asimina triloba). Two species are

from the Laurales in the Hernandiaceae (Hernandia ovigera) and the Lauraceae (Ocotea

leucoxylon) (Table 1-2).

The 52 host species from the Monocotolydonae are from 16 families: Araceae (13

species), Poaceae (9 species), Arecaceae (7 species), Dioscoreaceae (5 species, all from the

genus Dioscorea), Orchidaceae (4 species), Agavaceae (3 species), Musaceae (2 species),

Alismataceae (1 species), Asparagaceae (1 species), Bromeliaceae (1 species), Commelinaceae

(1 species), Heliconiaceae (1 species), Hemerocallidaceae (1 species), Marantaceae (1 species),

Restonaceae (1 species), and Strelitziaceae (1 species), in decreasing order of host species

numbers.

The remaining 464 host species are Eudicots. Families that contain the largest number of

hosts include Fabaceae (70 species), Lamiaceae (33 species), Malvaceae (32 species),

Asteraceae (26 species), Apocynaceae (21 species), Acanthaceae (20 species), Euphorbiaceae

(20 species), Verbenaceae (17 species), Convolvulaceae (14 species), Cucurbitaceae (13

species), and Solanaceae (13 species), in decreasing order of host species numbers.

Between the two surveys, 91 new hosts species were identified, 87 of which were found in

the survey conducted on Guam. New hosts were found in 32 families, of which three families

had never been reported to harbor the fungus: Hernandiaceae, Moringaceae, and Mutingiaceae.

Ten new host species were found to harbor the fungus in the survey conducted in Florida

(Cerinthe major, Corchorus aestuans, Fatshedera lizei, Hibiscus rosa-sinensis, Jatropha spp.,

Salviafarinacea, Salvia microphylla, Salcia officinalis, Sida spinosa, and Stachytarpheta









jamaicensis). Six new hosts were found in both Guam and Florida (Corchorus aestuans, Salvia

farinacea, S. microphylla, S. officinalis, Sida spinosa, and Stachytarpheta jamaicensis).

From the Guam and Florida surveys, C. cassiicola was more often identified as a pathogen

than as an endophyte on 191 and 121 plant species, respectively. On 48 hosts, the fungus was

identified as both a pathogen and an endophyte. Endophytic isolates of C. cassiicola were most

likely recovered from young leaves and pathogenic isolates from older leaves.

Discussion

The index produced here contains 530 C. cassiicola host plant species. Four hundred

thirty nine species were identified from the literature and 91 new species were identified from

the field surveys conducted in Guam and Florida. The number of new hosts found to harbor the

fungus in Guam was 87 and in Florida was 10, with six new hosts found in both Guam and

Florida. This suggests that there are many additional host species remaining to be discovered.

Although most of the literature on C. cassiicola relates to the diseases it causes, in this

study the fungus was often isolated from asymptomatic tissue, indicative of endophytic growth.

There are likely many additional endophytic hosts that remain to be discovered considering only

healthy leaves from previously reported hosts were sampled. The extent to which C. cassiicola

was occurring as an endophyte was not appreciated prior to this survey. During the course of the

Guam survey, C. cassiicola often sporulated from healthy tissue when placed in a moisture

chamber instead of necrotic tissue. In these cases, C. cassiicola was likely not the cause of the

necrosis because other fungi were often found to sporulate in those areas.

There seems to be no clear demarcation as to the presence of C. cassiicola on a particular

host and its ability to grow endophytically or pathogenically. Publications on C. cassiicola are

usually restricted to a description of symptoms on a particular host or as part of a list of fungi

from an endophyte study. Koch's postulates are rarely completed, and when they are, often the









fungus is not pathogenic on the host it was isolated from without wounding (Kingsland 1985;

Pernezny et al. 1996). This study recorded 48 cases from the Guam and Florida surveys where

plants were found to harbor pathogenic isolates of C. cassiicola and in other locations harbor

endophytic isolates. It may be that the fungus has the ability to delay symptoms by growing

initially as an endophyte. Pathogenic isolates were often found on older leaves indicating that

endophytic isolates may become pathogens as the host tissue ages or begins senescence. Despite

the symptomless nature of an endophytic relationship with the host, it is likely that the potential

exists for the fungus to switch to an opportunistic pathogen and/or a saprophyte on the same host

because individual hosts were found to harbor both pathogenic and endophytic isolates.

The likelihood of finding the fungus as an endophyte or as a pathogen may depend on the

plant family. In this study, plant families more likely found harboring the fungus growing as an

endophyte were Araceae, Bignoniaceae, Convolvulaceae, Crassulaceae, Elaeocarpaceae,

Hernandaceae, Magnoliaceae, Meliaceae, and Moraceae. Magnolia liliifera (Magnoliaceae)

was recently reported as hosting a Corynespora spp. endophyte with ribosomal DNA (ITS 1-

5.8S-ITS2) sequence homology to C. cassiicola (Promputtha et al. 2007) and was therefore

included in our list. In the Guam survey, Hernandia sp. (Magnoliaceae) was also found to

support endophytic growth of C. cassiicola. Families that were likely to support pathogenic

growth of the fungus in these surveys were Acanthaceae, Amaranthaceae, Apocynaceae,

Asteraceae, Begoniaceae, Boragniaceae, Gesnariaceae, Lamiaceae, and Verbenaceae.

Throughout the survey, it was difficult to determine whether the Corynespora species

observed were in fact C. cassiicola. At least one hundred and thirteen species of Corynespora

are currently described, but a monograph is needed, including molecular analyses, in order to

assess the validity of these species (Sivanesan 1996). Most species have been named according









to host identity, and only a few species have been described in culture. In addition, single

isolates exhibit considerable morphological plasticity that depends on humidity, light,

temperature, and substrate; therefore, morphological differences need to be compared with

molecular differences. Although the hosts included in this index are restricted to those reported

for C. cassiicola, some may actually be hosts of other Corynespora species due to

misidentification. Likewise, there may be hosts reported to harbor other species of Corynespora

that may, in fact, be harboring C. cassiicola because the morphological distinctions between

species are based on overlapping, variable, morphological characters. Phylogenetic analyses of

the isolates should help to clarify these issues.

Despite these complications, this is the first step taken to consolidate our knowledge of the

potential host range of C. cassiicola, which is vital for further studies of the biology of individual

isolates and ultimately in future studies of Corynespora species evolution. Although there is no

teleomorphic stage currently known for C. cassiicola, the Ascomycete species Corynesporasca

caryote and Pleomassaria swidae have unknown Corynespora species anamorphs (Sivanesan

1996; Tanaka et al. 2008). There is no evidence to suggest that C. cassiicola is reproducing

other than by asexual spores. However, evidence for sexual recombination needs to be tested

between isolates within and among host species. Insight into the evolutionary potential of the

fungus will lead to a better understanding of how to control its diseases (McDonald 2004).

The literature search and surveys elucidated several characteristics of C. cassiicola that

warrant further investigation: (1) the inability of some isolates recovered from symptomatic

tissue to re-infect the original hosts; (2) the ability to be endophytic, pathogenic, and saprophytic

on individual hosts; (3) the wide host range of the fungal species, yet restricted host ranges of

individual isolates; (4) the ability to grow on some members of a plant taxonomic group and not









others; (5) a lack of understanding of the diversity within the fungal species and how it relates to

host range; (6) the taxonomic validity of the 113 species of Corynespora considering the high

morphological plasticity of individual isolates. Future research should attempt to address these

issues and the organization of the plant hosts in a single publication will facilitate this.












Table 1-1. Taxonomic grouping of Corynespora cassiicola host species.
Number of Number of
Number of Host Species Host Species
Host Species Sampled in Sampled in
Plant Group in the Index Guam Florida
Anthocerotphyta (hornworts) 0 2 3
Bryophyta (mosses) 0 5 2
Filicopsida (ferns) 5 14 21
Spermatopsida (seed plants) 525 299 263
Conifers 0 3 6


Cycads
Gnetales
Angiosperms
Magnoliids
Monocotyledons
Eudicots


1
0
524
8
52
464


4
2
290
6
61
223


5
1
251
4
38
209










Table 1-2. Occurrence and fungal-host interaction of Corynespora cassiicola identified during 2004-2005 Guam and Florida surveys.
Host Fungal-Host Interaction Location Reference


Acanthaceae Juss. dicott)
Acanthus ilicifolius L.
Aphelandra squarrosa Nees
Asystasia spp. Blume
Asystasia gungcticu (L.) T. Anders.
Crossandra spp. Salisb.
Eranthemum pulchellum Andrews
Fittonia spp. Coem.
Fittonia albivenis (Lindl. ex hort. Veitch) Brummitt
Hygrophila spp. R. Br.
Justicia spp. L.
Justicia brandegeeana Wasshausen & L.B. Sm.
Justicia carnea Lindl.
Justicia ventricosa Wall. ex Hook.
Meisosperma oppositifolium
Pachystachys coccinea (Aubl.) Nees
Pachystachys lutea Nees
Peristrophe spp. Nees
Pseuderanthemum spp. Radlk.
Pseuderanthemum carruthersii (Seem.) Guillaumin
Ruellia humboldtiana (Nees) Lindau
Strobilanthes dyerianus M.T. Mast.
Thunbergia fragrans Roxb.
Warpuria clandestine Stapf.
Actinidiaceae Gilg & Werderm. dicott)
Actinidia chinensis Planch.
Adoxaceae E. Mey. dicott)
Viburnum spp. L.
Viburnum odoratissimum Ker Gawl.


endophytic
pathogenic

pathogenic
pathogenic
pathogenic
pathogenic
endophytic, pathogenic



pathogenic
pathogenic

endophytic

pathogenic



pathogenic
endophytic, pathogenic
pathogenic

pathogenic


endophvtic


GU Sadaba et al. 1995
FL, GU Chase 1982
Alfieri et al. 1984
GU Alfieri et al. 1984
FL Alfieri et al. 1994
FL Alfieri et al. 1994
FL Chase 1982
FL, GU Chase 1982
FL Alfieri et al. 1994
Ellis 1957
FL, GU Alfieri et al. 1994
GU Ellis 1957
Zhuang 2001
GU Smith et al. 2007
Urtiaga 1986
FL, GU Alfieri et al. 1994
Alfieri et al. 1994
El-Gholl et al. 1997
GU El-Gholl et al. 1997
FL Urtiaga 2004
GU Coile and Dixon 1994
Zhuang 2001
GU Ellis 1957

Peregrine and Ahmad 1982

Alfieri et al. 1994
FL. GU Alfieri et al. 1994










Table 1-2. Continued
Host Fungal-Host Interaction Location Reference


Agavaceae Dumort. monocott)
Agave sisalana Perrine
Cordylinefruticosa (L.) Chev.
Dracaena spp. Vand. ex L.
Dracaena reflexa Lam.
Alismataceae Vent. monocott)
Echinodorus spp. Rich. ex Engelm.
Amaranthaceae Juss. dicott)
Achyranthes aspera L.
Alternantheraficoidea (L.) P. Beauv.
Amaranthus spp. L.
Amaranthus spinosus L.
Amaranthus tricolor L.
S Celosia argentea L. var. cristata (L.) Kuntze
Digera muricata (L.) Mart.
Anacardiaceae R. Br. dicott)
Lannea coromandelica (Houtt.) Merr.
Mangifera indica L.
Schinus spp. L.
Spondias purpurea L.
Vernicia montana Lour.
Annonaceae Juss. dicott)
Annona reticulata L.
Annona squamosa L.
Asimina triloba (L.) Dunal
Apiaceae Lindl. dicott)
Foeniculum vulgare Mill.


endophytic


endophytic, pathogenic FL


pathogenic

pathogenic

pathogenic





endophytic, pathogenic
pathogenic
endophytic


endophytic


Ellis 1957
Situmorang and Budimen 1984
Alfieri et al. 1984
Alfieri et al. 1994


Alfieri et al. 1994


CABI, Herb. IMI 191361
GU first report
Alfieri et al. 1994
FL, GU Alfieri et al. 1994
Peregrine and Ahmad 1982
GU first report
Sarma and Nayudu 1970


CABI, Herb. IMI 266196
Rajak and Pandey 1985
Alfieri et al. 1984
Freire 2005
Ellis 1957

Peregrine and Ahmad 1982
first report
CABI, Herb. IMI 364250

Peregrine and Ahmad 1982










Table 1-2. Continued
Host Fungal-Host Interaction Location Reference


Apocynaceae Juss. dicott)
Adenium obesum (Forssk.) Roem. & Schult.
Allamanda spp. L.
Allamanda cathartica L.
Alstonia scholars (L.) R. Br.
Calotropis procera (Aiton) W. T. Aiton
Carissa spp. L.
Catharanthus roseus (L.) G. Don
Conopharyngia i, ,.-itii a (Benth.) Stapf
Cryptolepis buchananii Schult.
Funastrum clausum (Jacq.) Schltr.
Hoya spp. R. Br.
Mandevilla spp. Lindl.
Mandevilla splendens (Hook. f.) Woodson
Nerium oleander L.
Plumeria rubra L. forma acutifolia (Poir.) Woodson
Rauvolfia serpentina (L.) Benth. ex Kurz
Tabernaemontana divaricata (L.) R. Br. ex Roem. & Schult.
Tabernaemontana sananho Ruiz & Pav.
Tacazzea spp. Decne.
Telosma cordata (Burm. f.) Merr.
Thevetia peruviana (Pers.) K. Schum.
Trachelospermumjasminoides (Lindl.) Lem.
Vinca spp. L.
Aquifoliaceae Bercht. & J. Presl dicott)
Ilex vomitoria Sol. ex Aiton
Araceae Juss. monocott)
Aglaonema spp. Schott
Alocasia macrorrhizos (L.) G. Don
Amorphophallus paeoniifolius (Dennst.) Nicolson


endophytic
pathogenic
endophytic, pathogenic

pathogenic
pathogenic




pathogenic

pathogenic
pathogenic
endophytic, pathogenic





endophytic, pathogenic

pathogenic



endophytic

pathogenic
endophytic, pathogenic


El-Gholl 1997
FL Alfieri et al. 1984
GU Alfieri et al. 1994
FL Suryanarayanan et al. 2002
CABI, Herb. IMI 173980
FL Alfieri et al. 1994
FL, GU McGovern 1994
Kranz 1963
CABI, Herb. IMI 221003
Urtiaga 2004
FL Alfieri et al. 1994


FL, GU
FL
GU





GU

FL



FL

FL
GU


Alfieri et al. 1984
Alfieri et al. 1994
Alfieri et al. 1994
Ellis 1957
CABI, Herb. IMI 122395
CABI, Herb. IMI 209321
Urtiaga 2004
Ellis 1957
first report
CABI, Herb. IMI 231448
Alfieri et al. 1984
Alfieri et al. 1994

Alfieri et al. 1994

Alfieri et al. 1994
Mercado et al. 1997
Puzari and Saikia 1981










Table 1-2. Continued
Host
Anthurium spp. Schott
Anthurium andraeanum Linden ex Andre
Anubias afzelii Schott
Caladium bicolor (Aiton) Vent.
Colocasia esculenta (L.) Schott
Dieffenbachia spp. Schott
Epipremnum pinnatum (L.) Engl.
Philodendron bipinnatifidum Schott ex Endl.
Syngonium podophyllum Schott
Xanthosoma ,.,,udfil,,,n (L.) Schott
Zantedeschia spp. Spreng.
Zantedeschia aethiopica (L.) Spreng.
Araliaceae Juss. dicott)
Fatshedera spp. Guillaumin
0 Fatshedera lizei (hort. ex Cochet) Guillaumin
Polyscias balfouriana L.H.Bailey
Polysciasfruticosa (L.) Harms
Polyscias scutellaria (Burm. f.) Fosberg
Arecaceae Bercht. & J. Presl monocott)
Attalea butyracea (Mutis ex L. f.) Wess. Boer
Calyptronoma plumeriana (Mart.) Lourteig
Cocos nucifera L.
Dypsis lutescens (H. Wendl.) Beentje & J. Dransf.
Elaeis guineensis Jacq.
Licuala ramsayi (Mueler) Domin.
Rhopalostylis sapida H. Wendl and Drude
Asparagaceae Juss. monocott)
Asparagus officinalis L.
Asteraceae Bercht. & J. Presl dicott)
Ageratum conyzoides L.


Fungal-Host Interaction Location Reference
pathogenic Alfieri et al. 1994
pathogenic GU Alfieri et al. 1994
El-Gholl 1997
endophytic, pathogenic GU first report
endophytic GU Onesirosan et al. 1974
endophytic FL Alfieri et al. 1994
pathogenic FL Alfieri et al. 1984
endophytic GU first report
pathogenic GU Coile and Dixon 1994
endophytic GU Ellis 1957
Raabe et al. 1981
Raabe et al. 1981

Alfieri et al. 1984
endophytic FL first report
Alfieri et al. 1984
pathogenic FL Alfieri et al. 1994
pathogenic GU first report

Urtiaga 2004
Delgado-Rodriguez and Mena-Portales 2004
CABI, Herb. IMI 317357
endophytic, pathogenic FL Alfieri et al. 1994
Ellis 1957
Shivas and Alcorn 1996
McKenzie et al. 2004

Urtiaga 2004


pathogenic GU


Smith and Schlub 2004










Table 1-2. Continued
Host
Aspilia africana (Pers.) C. D. Adams
Bidens spp. L.
Bidens alba (L.) DC.
Calyptocarpus vialis Less.
Chromolaena odorata (L.) R. M. King & H. Rob.
hC hy,,t, lii mm spp. L.
C hl, 11111h 1,1,n indicum L.
Elephantopus mollis Kunth
Elephantopus scaber L.
Elephantopus tomentosus L.
Emilia sonchifolia (L.) DC
Gaillardia aristata Pursh
Lactuca sativa L.
Liatris spp. Gaertn. ex Schreb.
0 Melanthera biflora (L.) Wild
Mikania micrantha Kunth
Pseudelephantopus spicatus (B. Juss. ex Aubl.) C. F. Baker
Pseudogynoxys chenopodioides (Kunth) Cabrera
Sp hagneticolu trilobata (L.) Pruski
Symphyotrichum novi-belgii (L.) G. L. Nesom
Synedrella nodiflora (L.) Gaertn.
Tithonia rotundifolia (Mill.) S. F. Blake
Tridax procumbens L.
Verbesina turbacensis Kunth
Vernonia cinerea (L.) Less.
Zinnia violacea Cav.
Balsaminaceae A. Rich. dicott)
Impatiens balsamina L.
Impatiens noli-tangere L.
Impatiens sultanii Hook. f.


Fungal-Host Interaction
pathogenic

pathogenic
pathogenic
pathogenic
endophytic

endophytic



pathogenic
pathogenic
pathogenic
endophytic, pathogenic

pathogenic
endophytic
endophytic, pathogenic
endophytic

pathogenic

pathogenic

pathogenic


pathogenic
pathogenic


Location Reference


FL, GU
GU
GU
FL

GU



GU
GU
GU
FL

GU
GU
FL
GU

GU

GU

GU



GU
FL


Onesirosan et al. 1974
Alfieri et al. 1984
Alfieri et al. 1984
Smith and Schlub 2004
CABI, Herb. IMI 147913
Turner 1971
Peregrine and Ahmad 1982
first report
CABI, Herb. IMI 199985
Zhuang 2001
McKenzie 1990
Ellis 1957
Ellis 1957
Alfieri et al. 1994
Ellis 1957
Smith et al. 2007
first report
Alfieri et al. 1994
Alfieri et al. 1994
Dixon 1997
Onesirosan et al. 1974
Wei 1950
first report
Urtiaga 2004
Cutrim and Silva 2003
Urtiaga 2004

Wei, 1950
CABI, Herb. IMI 124564
Urtiaga 2004










Table 1-2. Continued
Host
Impatiens walleriana Hook. f.
Begoniaceae C. Agardh dicott)
Begonia spp. L.
Begonia coccinea Hook.
Begonia cucullata Willd.
Bignoniaceae Juss. dicott)
Bignonia spp. L.
Crescentia cujete L.
Handroanthus serratifolius (Vahl) S. Grose
Newbouldia laevis (P. Beauv.) Seem. ex Bureau
Radermachera sinica (Hance) Hemsl.
Radermachera xylocarpa (Roxb.) K. Schum.
Stereospermum colais (Buch.-Ham. ex Dillwyn) Mabb.
Tabebuia spp. Gomes ex DC.
Tabebuia aurea (Silva Manso) Benth. & Hook. f. ex S. Moore
Tabebuia heterophylla (DC.) Britton
Tabebuiapallida (Lindl.) Miers
Tabebuia odontodiscus (Bureau & K. Schum.) Toledo
Tecoma capensis (Thunb.) Lindl.
Boraginaceae Juss. dicott)
Cerinthe major L.
Cordia collococca L.
Cordia curassavica (Jacq.) Roem. & Schult.
Cordia obliqua Willd.
Cordia wallichii G. Don.
Cordia subcordata Lam.
Tournefortia argentea L. f.
Brassicaceae Burnett dicott)
Brassica rapa L.


Fungal-Host Interaction Location


pathogenic
pathogenic



pathogenic

endophytic, pathogenic
endophytic
endophytic
endophytic

pathogenic
endophytic
pathogenic




pathogenic





pathogenic
pathogenic


Reference
Alfieri et al. 1994

Chase 1982
Chase 1982
first report

Orieux and Felix 1968
Alfieri et al. 1994
Mendes et al. 1998
Ellis 1957
Alfieri et al. 1994
Suryanarayanan et al. 2002
Murali et al. 2007
Mendes et al. 1998
Alfieri et al. 1984
Alfieri et al. 1994
Alfieri et al. 1994
Mendes et al. 1998
Urtiaga 2004

first report
Urtiaga 2004
Urtiaga 2004
Murali et al. 2007
Murali et al. 2007
first report
first report

Peregrine and Ahmad 1982










Table 1-2. Continued
Host
Bromeliaceae Juss. monocott)
Ananas comosus (L.) Merr.
Burseraceae Kunth dicott)
Bursera simaruba (L.) Sarg.
Canarium album (Lour.) Raeusch.
Cannabaceae Martinov dicott)
Trema micrantha (L.) Blume
Trema orientalis (L.) Blume
Capparaceae Juss. dicott)
Capparis spp. L.
Caprifoliaceae Juss. dicott)
Lonicera japonica Thunb.
Lonicera sempervirens L.
S Caricaceae Dumort. dicott)
Carica papaya L.
Vasconcellea cauliflora (Jacq.) A. DC.
Vasconcellea pubescens A. DC.
Celastraceae R. dicott)
Celastrus paniculatus Willd.
Elaeodendron glaucum (Rottb.) Pers.
Euonymus spp. L.
Salacia senegalensis (Lam.) DC.
Combretaceae R. Br. dicott)
Anogeissus latifolia (Roxb. ex DC.) Wall. ex Guill. & Perr.
Terminalia arjuna (Roxb. ex DC.) Wight & Am.
Terminalia catappa L.
Terminalia crenulata Roth.
Terminalia elliptica Willd.


Fungal-Host Interaction Location


Reference


Blazquez 1968


endophytic, pathogenic FL


Alfieri et al. 1994
Zhang and Ji 2005


Arnold 1986
CABI, Herb. IMI 256125

CABI, Herb. IMI 259297


endophytic



pathogenic













endophytic


Alfieri et al. 1984
Alfieri et al. 1994


FL, GU Beaver 1981
Urtiaga 2004
Johnston 1960

CABI, Herb. IMI 302698
Murali et al. 2007
Alfieri et al. 1994
Ellis 1957


Suryanarayanan et al. 2002
CABI, Herb. IMI 302839
first report
Murali et al. 2007
Suryanarayanan et al. 2002










Table 1-2. Continued


Host
Commelinaceae Mirb. monocott)
Commelina benghalensis L.
Convolvulaceae Juss. dicott)
Evolvulus glomeratus Nees & Mart.
Ipomoea alba L.
Ipomoea aquatica Forssk.
Ipomoea batatas (L.) Lam.
Ipomoea indica (Burm.) Merr.
Ipomoea littoralis (L.) Blume
Ipomoea obscura (L.) Ker Gawl.
Ipomoeapes-caprae (L.) R. Br.
Ipomoea triloba L.


Lepistemon spp. Blume
Merremia ci,, *. p/vi (L.) Urb.
Merremia peltata (L.) Merr.
00 Operculina turpethum (L.) Silva Manso
Stictocardia tiliifolia (Desr.) Hallier f.
Cornaceae Bercht. & J. Presl dicott)
Alangium chinense (Lour.) Harms
Cornus florida L.
Crassulaceae J. St.-Hil. dicott)
Crassula ovata (Mill.) Druce
Kalanchoe spp. Adans.
Kalanchoe pinnata (Lam.) Pers.
Kalanchoe ;h.y, iji 1 Harv.
Sedum spp. L.
Cucurbitaceae Juss. dicott)
Citrullus lanatus (Thunb.) Matsum. & Nakai
Coccinia grandis (L.) Voigt
Cucumis anguria L.


Fungal-Host Interaction Location Reference


pathogenic

endophytic
endophytic, pathogenic
endophytic
endophytic, pathogenic
endophytic, pathogenic
endophytic, pathogenic
endophytic, pathogenic
endophytic
endophytic, pathogenic

endophytic, pathogenic
endophytic, pathogenic

endophytic


GU

GU
GU
GU
FL, GU
GU
GU
GU
GU
GU

GU
GU
GU
GU


Cutrim and Silva 2003

Alfieri et al.1994
McKenzie 1990
McKenzie 1990
Silva et al. 2003
first report
first report
Smith and Schlub 2004
Hawaiian Ecosystems at Risk (HEAR) 2008
Smith and Schlub 2004
Onesirosan et al. 1974
first report
first report
first report
first report


Guo 1992
Alfieri et al. 1994


endophytic
endophytic
endophytic, pathogenic



pathogenic
endophytic, pathogenic
endophytic


Alfieri et al. 1994
Alfieri et al. 1994
first report
first report
Chase 1982

Sobers 1966
Philip et al. 1972
Cutrim and Silva 2003










Table 1-2. Continued
Host
Cucumis melo L.
Cucumis sativus L.
Cucurbita spp. L.
Cucurbita maxima Duchesne
Cucurbita moschata Duchesne
Cucurbita pepo L.
Lagenaria siceraria (Molina) Standl.
Luffa acutangula (L.) Roxb.
Luffa i, .,, -y,/ i Mill.
Momordica charantia L.
Sechium edule (Jacq.) Sw.
Davalliaceae M. R. Schomb. dicott)
Arachniodes aristata (G. Forst.) Tindale
Davallia spp. Sm.
0 Davallia repens (L. f.) Kuhn
Dioscoreaceae R. Br. monocott)
Dioscorea alata L.
Dioscorea bulbifera L.
Dioscorea cayenensis Lam.
Dioscorea esculenta (Lour.) Burkill
Dioscorea pentaphylla L.
Dryopteridaceae Herter (fern)
Athyrium niponicum (Mett.) Hance
Ebenaceae Guirke dicott)
Diospyros montana Roxb.
Elaeocarpaceae Juss. ex DC. dicott)
Elaeocarpus joga Merr.
Elaeocarpus tuberculatus Roxb.
Ahunmtin.ia calabura L.


Fungal-Host Interaction
endophytic, pathogenic
pathogenic




pathogenic
endophytic, pathogenic
endophytic, pathogenic

pathogenic
endophytic, pathogenic

endophytic, pathogenic

pathogenic


endophytic, pathogenic

endophytic, pathogenic



endophytic


Location Reference
GU Ellis and Holliday 1971
FL, GU Wei 1950
Grand 1985
Williams and Liu 1976
Minter et al. 2001
GU Cutrim and Silva 2003
GU Ellis 1957
GU Onesirosan et al. 1974
Onesirosan et al. 1974
GU Alfieri et al. 1994
FL, GU Alfieri et al. 1984


Anderson and Dixon 2004
Alfieri et al. 1994
Alfieri et al. 1994

CABI, IMI 229871
Onesirosan et al. 1974
CABI IMI 83832
Onesirosan et al. 1974
Peregrine and Ahmad 1982

El-Gholl 1997


Murali et al. 2007


endophytic

endophytic


first report
Suryanarayanan et al. 2002
first report










Table 1-2. Continued
Host
Ericaceae Juss. dicott)
Oxydendrum arboreum (L.) DC.
Rhododendron spp. L.
Rhododendron canescens (Michx.) Sweet
Rhododendron obtusum (Lindl.) Planch.
Vaccinium corymbosum L.
Erythroxylaceae Kunth dicott)
Erythroxylum monogynum Roxb.
Euphorbiaceae Juss. dicott)
Acalypha macrostachya Jacq.
Bridelia ferruginea Benth.
Chamaesyce hirta (L.) Millsp.
Codiaeum variegatum (L.) A. Juss.
Cnidoscolus aconitifolius (Mill.) I. M. Johnst.
0 Croton bonplandianus Baill.
Croton fragrans Kunth.
Drypetes alba Poit.
Euphorbia spp. L.
Euphorbia cyathophora Murray
Euphorbia pulcherrima Willd. ex Klotzsch
Euphorbia milii Des Moulins
Givotia rottleriformis Griff.
Hevea brasiliensis (Willd. ex A. Juss.) Muill. Arg.
Hura crepitans L.
Jatropha spp. L.
Jatropha gossypiifolia L.
Manihot spp. Mill.
Manihot cu,, dth,e; n, a,, (Jacq.) Mtill. Arg.
Manihot esculenta Crantz


Fungal-Host Interaction Location


Reference


Alfieri et al. 1994
Alfieri et al. 1984


endophytic, pathogenic FL
pathogenic FL


Ellis and Holliday 1971
Hongn et al. 2007


Murali et al. 2007


pathogenic
endophytic, pathogenic

endophytic, pathogenic




endophytic, pathogenic
pathogenic
pathogenic




pathogenic
pathogenic



endophvtic, pathogenic


Urtiaga 2004
Ellis 1957
GU first report
GU CABI, IMI 179212
Peregrine and Ahmad 1982
FL, GU Sarma and Nayudu 1970
Urtiaga 2004
Mercado 1984
Ellis 1957
GU Barreto and Evans 1998
FL Chase 1986
GU Smith et al. 2007
Murali et al. 2007
Silva et al. 1995
Urtiaga 1986
FL first report
GU Smith et al. 2007
Malvick 2004
Onesirosan et al. 1974
GU Ellis 1957










Table 1-2. Continued
Host
Phyllanthus amarus Schumach. & Thonn.
Phyllanthus emblica L.
Tragia spp. L.
Fabaceae Lindl. dicott)
Acacia spp. Mill.
Acacia auriculiformis A. Cunn. ex Benth.
Afzelia africana Sm. ex Pers.
Albizia lebbeck (L.) Benth.
Albizia _ygia (DC.) J. F. Macbr.
Alysicarpus vaginalis (L.) DC.
Arachis hypogaea L.
Bauhinia spp. L.
Bauhinia galpinii N. E. Br.
Bauhinia purpurea L.
Bauhinia racemosa Lam.
Butea monosperma (Lam.) Taub.
Caesalpinia granadillo Pittier
Cajanus cajan (L.) Millsp.
Calopogonium mucunoides Desv.
Cassia fistula L.
Clitoria ternatea L.
Crotalaria goreensis Guill. & Perr.
Crotalaria juncea L.
Crotalaria micans Link
Crotalaria pallida Aiton
Crotalaria retusa L.
Crotalaria spectabilis Roth
Cyamopsis tetragonoloba (L.) Taub.
Dalberuia spp. L. f.


Fungal-Host Interaction Loc
endophytic, pathogenic GU





endophytic, pathogenic GU


endophytic

endophytic



pathogenic
pathogenic


pathogenic
endophytic
pathogenic


GU


endophytic, pathogenic GU


action Reference
Mathiyazhagan et al. 2004
Prakash and Garg 2007
Ellis 1957

Situmorang and Budimen 1984
first report
Dade 1940
first report
Ellis 1957
first report
Vyas et al. 1985
Alfieri et al. 1994
Smith and Schlub 2004
GU Ellis 1957
Suryanarayanan et al. 2002
Murali et al. 2007
Urtiaga 2004
Lenne 1990
Onesirosan et al. 1974
Suryanarayanan et al. 2002
first report
Hyde and Alcorn 1993
Wei 1950
Shaw 1984
Turner 1971
first report
Malvick 2004
Spencer 1962
Ellis 1957










Table 1-2. Continued
Host
Dalbergia latifolia Roxb.
Dalbergia lanceolaria L. f
Delonix regia (Bojer ex Hook.) Raf.
Desmodium spp. Desv.
Desmodium incanum DC.
Desmodium tortuosum (Sw.) DC.
Desmodium triflorum (L.) DC.
Erythrina spp. L.
Gliricidia sepium (Jacq.) Kunth ex Walp.
Glycine max (L.) Merr.
Glycine soja Siebold & Zucc.
Hymenaea courbaril L.
Lens culinaris Medik.
Lupinus albus L.
Lupinus i ,i,,r,, fi ,i L.
Lupinus luteus L.
Lupinus pilosus L.
Macrolobium spp. Schreb.
Macroptilium atropurpureum (Moc. & Sess6 ex DC.) Urban
Macroptilium lathyroides (L.) Urban
Mimosa diplotricha C. Wright
Mimosa pudica L.
Mucunapruriens (L.) DC.
Phaseolus lunatus L.
Phaseolus vulgaris L.
Pisum sativum L.
Pithecellobium dulce (Roxb.) Benth.
Psophocarpus tetragonolobus (L.) DC.
Pterocarpus indicus Willd.


Fungal-Host Interaction Location


endophytic




pathogenic
pathogenic
pathogenic



pathogenic


pathogenic
pathogenic

endophytic, pathogenic



pathogenic
pathogenic
pathogenic


Reference
Suryanarayanan et al. 2002
Murali et al. 2007
CABI, Herb. IMI 314022


Lenn6, 1990
GU Smith and Schlub 2004
GU Smith and Schlub 2004
GU Smith and Schlub 2004
Delgado-Rodriguez et al. 2002
Boa and Lenn6 1994
FL, GU Olive et al. 1945
Lenn6 1990
Urtiaga 2004
Khare 1991
Sobers 1966
Sobers 1966
Sobers 1966
Malvick 2004
Kranz 1963
GU first report
GU Smith and Schlub 2004
Silva 1995
GU Smith and Schlub 2004
Sobers 1966
Malvick 2004
GU Wei 1950
GU first report
GU first report
Ellis 1957
Situmorans and Budimen 1984










Table 1-2. Continued
Host Fungal-Host Interaction Location Reference


Pueraria montana (Lour.) Merr.
Ricinus communis L.
Saraca indica L.
Senna alata (L.) Roxb.
Senna occidentalis (L.) Link
Senna surattensis (Burm. f.) H. S. Irwin & Bameby
Senna tora (L.) Roxb.
Sesamum indicum L.
Spathodea campanulata P. Beauv.
Teramnus labialis (L. f.) Spreng.
Trifolium repens L.
Trigonella foenum-graecum L.
Tylosema esculentum (Burch.) A. Schreib.
Vicia spp. L.
Vigna mungo (L.) Hepper
Vigna radiata (L.) R. Wilczek
Vigna unguiculata (L.) Walp. subsp. sesquipedalis (L.) Verdc.
Vigna umbellata (Thunb.) Ohwi & H. Ohashi
Wisteria sinensis (Sims) DC.
Fagaceae Dumort. dicott)
Quercus ilex L.
Gesneriaceae Rich. & Juss. dicott)
Aeschynanthus longicaulis Wall. ex R. Br.
Aeschynanthus radicans Jack
Columnea spp. L.
Episcia cupreata (Hook.) Hanst.
Gloxinia perennis (L.) Fritsch
Nematanthus spp. Schrad.
Saintpaulia ionantha H. Wendl.


pathogenic
pathogenic

endophytic
pathogenic
pathogenic


pathogenic

endophytic





pathogenic

pathogenic


pathogenic GU


Peregrine and Ahmad 1982
Spencer and Walters 1968
CABI, Herb. IMI 210811
Wei 1950
first report
first report
Situmorang and Budimen 1984
Wei 1950
Smith et al. 2007
Smith et al. 2007
Cho and Shin 2004
Komaraiah and Reddy 1986
Alfieri et al. 1994
Alfieri et al. 1984
Gowda et al. 2001
Malvick 2004
Seaman et al. 1965
Peregrine and Ahmad 1982
Alfieri et al. 1984

Collado et al. 1999

Chase 1982
Chase 1982
Chase 1982
Alfieri et al. 1994
Brooks 2002
Chase 1982
Smith et al. 2007










Table 1-2. Continued
Host
Sinningia speciosa (Lodd. et al.) Hiern
Streptocarpus spp. Lindl.
Streptocarpus rexii (Bowie ex Hook.) Lindl.
Heliconiaceae Nakai monocott)
Heliconia caribaea Lam.
Hemerocallidaceae R. Br. monocott)
Hemerocallis spp. L.
Hernandiaceae Blume dicott)
Hernandia spp. L.
Hernandia ovigera L.
Hydrangeaceae Dumort. dicott)
Hydrangea spp. L.
Hydrangea macrophylla (Thunb.) Ser.
Lamiaceae Martinov dicott)
Ajuga spp. L.
Ajuga reptans L.
Anisochilus carnosus (L. f.) Wall. ex Benth.
Coleus barbatus (Andrews) Benth.
Congea tomentosa Roxb.
Clerodendrum inerme (L.) Gaertn.
Clerodendrum infortunatum L.
Clerodendrum speciosissimum Van Geert ex C. Morren
Hyptis suaveolens (L.) Poit.
Leucas aspera (Willd.) Link
Mentha arvensis L.
Mentha xpiperita L.
Moluccella spp. L.
Moluccella laevis L.
Monarda punctata L.


Fungal-Host Interaction
pathogenic

pathogenic


endophytic
endophytic


pathogenic FL



pathogenic FL

pathogenic FL,





endophytic GU

endophytic, pathogenic GU


Location Reference
FL Alfieri et al. 1994
Alfieri et al. 1994
FL, GU Alfieri et al. 1994

Urtiaga 2004

Peregrine and Ahmad 1982


first report
first report

Alfieri et al. 1984
Sobers 1966


Alfieri et al. 1984
Alfieri et al. 1984
CABI, Herb. IMI 151008
GU Fernandes and Barreto 2003
Peregrine and Ahmad 1982
Ahmad 1969
CABI, Herb. IMI 112265
Urtiaga 1986
Smith et al. 2007
Sarma and Nayudu 1970
Cheeran 1968
Williams and Liu 1976
Alfieri et al. 1984
Alfieri et al. 1984
Alfieri et al. 1994










Table 1-2. Continued
Host
Ocimum basilicum L.
Ocimum tenuiflorum L.
Origanum vulgare L.
Perillafrutescens (L.) Britton
Plectranthus amboinicus (Lour.) Spreng.
Plectranthus barbatus Andrews
Plectranthus parviflorus Willd.
Premna serratifolia L.
Premna tomentosa Willd.
Rosmarinus officinalis L.
Salvia spp. L.
Salvia farinacea Benth.
Salvia leucantha Cav.
Salvia microphylla Kunth
Salvia officinalis L.
Salvia splendens Sellow ex Schult.
Solenostemon scutellarioides (L.) Codd
Stachysfloridana Shuttlew. ex Benth.
Thymus vulgaris L.
Tectona grandis L. f.
Teucrium canadense L.
Lauraceae Juss. dicott)
Ocotea leucoxylon (Sw.) Laness.
Lecythidaceae A. Rich. dicott)
Careya arborea Roxb.
Lecythis ollaria Loefl.
Loganiaceae R. Br. ex Mart. dicott)
Buddleja asiatica Lour.
Strychnos potatorum L. f.


Fungal-Host Interaction
endophytic, pathogenic


pathogenic
pathogenic
pathogenic

pathogenic
pathogenic




pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic

pathogenic


pathogenic


Location Reference
GU Taba et al. 2002
Sarma and Nayudu 1970
FL, GU
GU Hasama et al. 1991
GU Miller 1991
Smith et al. 2007
FL Alfieri et al. 1994
GU first report
Murali et al. 2007
Alfieri et al. 1994
Peregrine and Ahmad 1982
FL, GU first report
FL Riley 1960
FL, GU first report
FL, GU first report
FL Chase 1982
FL, GU Alfieri et al. 1994
Alfieri et al. 1994
FL Silva 1995
Murali et al. 2007
El-Gholl 1997


Delgado-Rodriguez et al. 2002

Murali et al. 2007
Urtiaga 2004


Smith and Schlub 2004
Murali et al. 2007










Table 1-2. Continued
Host
Lythraceae J. St.-Hil. dicott)
Lagerstroemia indica L.
Lagerstroemia microcarpa Wight
Lagerstroemia parviflora Roxb.
Pemphis acidula Forst. & Forst.
Magnoliaceae Juss. dicott)
Magnolia champaca (L.) Baill. ex Pierre
Magnolia liliifera (L.) Baill.
Malpighiaceae Juss. dicott)
Malpighia glabra L.
Malvaceae Juss. dicott)
Abelmoschus esculentus (L.) Moench
Abutilon theophrasti Medik.
Ceiba pentandra (L.) Gaertn.
Ceiba speciosa (A. St.-Hil.) Ravenna
Corchorus aestuans L.
Corchorus capsularis L.
Corchorus olitorius L.
Desplatsia spp. Bocq.
Durio zibethinus L.
Gossypium barbadense L.
Gossypium hirsutum L.
Grewia tiliifolia Vahl
Helicteres isora L.
Hibiscus spp. L.
Hibiscus cannabinus L.
Hibiscus mutabilis L.
Hibiscus rosa-sinensis L.
Hibiscus sabdariffa L.


Fungal-Host Interaction Location Reference


pathogenic


endophytic



endophytic




pathogenic
endophytic, pathogenic
endophytic

pathogenic
pathogenic
endophytic, pathogenic



endophytic, pathogenic








endophytic
endophytic


GU



FL




GU
GU
GU

FL, GU
GU
GU



GU








FL
GU


Alfieri et al. 1994
Murali et al. 2007
Murali et al. 2007
first report

CABI, Herb. IMI 254407
Promputtha et al. 2007

Poltronieri et al. 2003

Wei 1950
Spencer and Walters 1969
Mehrotra 1989
Ferreira 1989
Smith and Schlub 2004
Wei 1950
Ellis 1957
Ellis 1957
Williams and Liu 1976
Jones 1961
Jones 1961
Suryanarayanan et al. 2002
Murali et al. 2007
Urtiaga 2004
Shaw 1984
Kwon and Park 2003
first report
Wei 1950










Table 1-2. Continued
Host
Kydia calycina Roxb.
Pavonia spp. Cav.
Pseudobombax septenatum (Jacq.) Dugand
Sida acuta Burm. f.
Sida glomerata Cav.
Sida rhombifolia L.
Sida spinosa L.
Sida urens L.
Sterculia apetala (Jacq.) H. Karst.
Talipariti tiliaceum (L.) Fryxell
Theobroma cacao L.
Thespesia populnea (L.) Soland. ex Correa
Triumfetta rhomboidea Jacq.
Waltheria indica L.
Urena lobata L.
Marantaceae R. Br. monocott)
Maranta leuconeura E. Morren
Marcgraviaceae Bercht. & J. Presl dicott)
Norantea guianensis Aubl.
Meliaceae Juss. dicott)
Chukrasia velutina M. Roem.
Guarea guidonia (L.) Sleumer
Melia azedarach L.
Moraceae Gaudich. dicott)
Artocarpus altilis (Parkinson) Fosberg
Broussonetia spp. L'Her. ex Vent.
Broussonetiapapyrifera (L.) L'Her. ex Vent.
Ficus spp. L.
Ficus benjamina L.


Fungal-Host Interaction


pathogenic

pathogenic
pathogenic



endophytic

endophytic pathogenic
endophytic
endophytic
pathogenic


pathogenic

endophytic

endophytic

endophytic


endophytic

endophytic


Location Reference
CABI, Herb. IMI 264454
Urtiaga 2004
Urtiaga 2004
GU Smith and Schlub 2004
Urtiaga 2004
GU CABI, Herb. IMI 180198
FL, GU first report
Ellis 1957
Urtiaga 2004
GU first report
Duarte et al. 1978
GU first report
GU Onesirosan et al. 1974
GU CABI, Herb. IMI 123575
GU first report


Alfieri et al. 1994

Wei 1950

first report
Urtiaga 2004
first report

CABI, Herb IMI 351978
Pollack and Stevenson 1973
Alfieri et al. 1994
Ellis 1957
Chase 1984










Table 1-2. Continued
Host
Ficus elastica Roxb. ex Homem.
Ficus exasperata Vahl
Ficus hispida L. f.
Ficus lyrata Warb.
Ficus racemosa L.
Ficus religiosa L.
Moringaceae Martinov dicott)
Moringa oleifera Lam.
Muntingiaceae C. Bayer et al. dicott)
Mhuningiu calabura L.
Musaceae Juss. monocott)
Musa xsapientum L.
Musa acuminata Colla
Myrsinaceae R. Br. dicott)
00 Ardisia foetida Willd.
Myrtaceae Juss. dicott)
Eucalyptus spp. L'Her.
Eucalyptus grandis W. Hill ex Maiden
Eucalyptus tereticornis Sm.
Eugenia uniflora L.
Psidium guajava L.
Syivgiimn aromaticum (L.) Merr. & L. M. Perry
Syvzygiin cumin (L.) Skeels
Sy'zgium jambos (L.) Alston
Nyctaginaceae Juss. dicott)
Bougainvillea spectabilis Willd.
Mirabilis jalapa L.
Nymphaeaceae Salisb. dicott)
Nvmphaea ampla (Salisb.) DC.


Fungal-Host Interaction
endophytic



endophytic


endophytic

pathogenic


pathogenic



pathogenic
pathogenic

endophytic


Location Reference
GU Chase 1987
Onesirosan et al. 1974
CABI, Herb IMI 311137
FL Alfieri et al. 1994
Gilson 2002
CABI, Herb. IMI 217075


Smith et al. 2007

first report

Blazquez 1968
Lumyong et al. 2003

Urtiaga 2004



C.M.I. No. 303
Vittal and Dorai 1994
CABI, Herb IMI 99533
Alfieri et al. 1984
Saikia and Sarbhoy 1981
Sarbhoy et al. 1971
Smith et al. 2007

first report
CABI, Herb IMI 259283


Urtiaga 2004










Table 1-2. Continued
Host
Nyssaceae Juss. ex Dumort. dicott)
Nyssa spp. L.
Oleaceae Hoffmanns. & Link dicott)
Chionanthus retusus Lindl. & Paxton
Jasminum spp. L.
Jasminum laurifolium Roxb. forma nitidum (Skan) P. S. Green
Jasminum multiflorum (Burm. f.) Andrews
Jasminum sambac (L.) Aiton
Jasminum simplicifolium G. Forst.
Ligustrum lucidum W. T. Aiton
Ligustrum japonicum Thunb.
Ligustrum sinense Lour.
Orchidaceae Juss. monocott)
S Cattleya spp. Lindl.
Dendrobium spp. Sw.
Phalaenopsis spp. Blume
Vanilla planifolia Andrews
Passifloraceae Juss. ex Roussel dicott)
Passiflora spp. L.
Passiflora edulis Sims
Passiflora foetida L.
Passiflora suberosa L.
Pedaliaceae R. Br. dicott)
Josephinia imperatricis Vent.
Martynia annua L.
Sesamum indicum L.
Piperaceae Giseke dicott)
Piper betle L.


Fungal-Host Interaction Location


Reference


Alfieri et al. 1994


Alfieri et al. 1994
Alfieri et al. 1984
Alfieri et al. 1994
Alfieri et al. 1994
CABI Herb. IMI 111858
Alfieri et al. 1994
Alfieri et al. 1994
Alfieri et al. 1994
Alfieri et al. 1994


pathogenic



endophytic


Simone 2000
Alfieri et al. 1994
Alfieri et al. 1994
Urtiaga 2004


endophytic
pathogenic
endophytic


Pemezny and Simone 1993
Alfieri et al. 1994
Smith et al. 2007
first report

Hyde and Alcorn 1993
CABI, Herb IMI 264260
Riley 1960


endophytic, pathogenic GU


Acharva et al. 2003










Table 1-2. Continued
Host
Piper hispidinervum C. DC.
Peperomia obtusifolia (L.) A. Dietr.
Poaceae Barnhart monocott)
Arundinariapygmaea (Miq.) Asch. & Graebn.
Bambusa vulgaris Schrad. ex J. C. Wendl.
Dendrocalamus spp. Nees
Oryza sativa L.
Ottochloa nodosa (Kunth) Dandy
Panicum repens L.
Pennisetum glaucum (L.) R. Br.
3A ,, ih, ,i, maximus (Jacq.) B. K. Simon & S. W. L. Jacobs
Sorghum bicolor (L.) Moench
Polypodiaceae Bercht. & J. Presl (fern)
Platycerium spp. Desv.
0 Polygonaceae Juss. dicott)
Coccoloba fallax Lindau
Pteridaceae E. D. M. Kirchn. (fern)
Adiantum spp. L.
Adiantum tenerum Sw.
Restionaceae R. Br. monocott)
Ischyrolepis subverticillata Steud.
Rhamnaceae Juss. dicott)
Colubrina retusa (Pittier) Cowan
Ziziphus cyclocardia S.F. Blake
Ziziphus mauritiana Lam.
Ziziphus xylopyrus (Retz.) Willd.
Rosaceae Juss. dicott)
Malus pumila Mill.
Pvrus communis L.


Fungal-Host Interaction Location Reference


pathogenic



endophytic


Poltronieri et al. 2003
Chase 1982

LSU Ag Center 2008
first report
Lu et al. 2000
CABI, Herb IMI 280017
Situmorang and Budimen 1984
Situmorang and Budimen 1984
Lenne 1990
Smith and Schlub 2004
Mendes et al. 1998

Alfieri et al. 1994


endophytic



pathogenic


Urtiaga 2004


Situmorang and Budimen 1984
Alfieri et al. 1984


pathogenic


Lee et al. 2004

Urtiaga 2004
Urtiaga 2004
first report
Murali et al. 2007


pathogenic
pathogenic


CABI, Herb IMI 284207
Alfieri et al. 1984










Table 1-2. Continued
Host Fungal-Host Interaction Location Reference


Rubiaceae Juss. dicott)
Guettarda speciosa L.
Ixora coccinea L.
Ixora nigricans R. Br. ex Wt. & Am.
Morinda citrifolia L.
Nauclea diderrichii (De Wild.) Merr.
Pentas lanceolata (Forssk.) Deflers
Spermacoce spp. L.
Rutaceae Juss. dicott)
Aegle marmelos
Naringi crenulata (Roxb.) Nicolson
Salicaceae Mirb. dicott)
Casearia decandra Jacq.
Sapindaceae Juss. dicott)
Acer negundo L.
Acer rubrum L.
Cupaniopsis anacardioides (A. Rich.) Radlk.
Dodonaea viscosa Jacq.
Litchi chinensis Sonn.
Matayba scrobiculata (Kunth) Radlk.
Saxifragaceae Juss. dicott)
, \ if ,., stolonifera Curtis
Tolmiea spp. Torr. & A. Gray
Tolmiea menziesii (Pursh) Torr. & Gray
Scrophulariaceae Juss. dicott)
Alectra %. iylmjo, (Vahl) Kuntze
Antirrhinum majus L.
Buchnera americana L.
Digitalis SDD. L.


endophytic


endophytic

pathogenic


pathogenic


pathogenic


first report
CABI, Herb. IMI 129296
Murali et al. 2007
first report
CABI, Herb. IMI 126192
first report
Situmorang and Budimen 1984

Gond et al. 2007
Murali et al. 2007

Urtiaga 2004

El-Gholl 1997
Alfieri et al. 1994
Alfieri et al. 1994
Singh et al. 1982

Urtiaga 2004

El-Gholl 1997
Alfieri et al. 1984
Alfieri et al. 1984

Urtiaga 2004
Alfieri et al. 1994
Smith and Schlub 2004
Alfieri et al. 1994










Table 1-2. Continued
Host
Paulownia spp. Siebold & Zucc.
Paulownia tomentosa (Thunb.) Steud.
Russelia equisetiformis Schltdl. & Cham.
Simaroubaceae DC. dicott)
Ailanthus excelsa Roxb.
Solanaceae Juss. dicott)
Capsicum annuum L.
Capsicum frutescens L.
Nicotiana glutinosa L.
Nicotiana tabacum L.
Petunia xhybrida hort. ex E. Vilm.
Petunia integrifolia (Hook.) Schinz & Thell.
Solanum erianthum D. Don
Solanum lycopersicum L.
Solanum melongena L.
Solanum nigrum L.
Solanum torvum Sw.
Solanum tuberosum L.
Solanum viarum Dunal
Strelitziaceae Hutch. monocott)
Strelitzia spp. Aiton
Strelitzia reginae Aiton
Theaceae Mirb. dicott)
Camellia sinensis (L.) Kuntze
Turneraceae Kunth ex DC. dicott)
Turnera ulmifolia L.
Urticaceae Juss. dicott)
Boehmeria nivea (L.) Gaudich.
Cecropia peltata L.


Fungal-Host Interaction

endophytic
endophytic, pathogenic


endophytic



pathogenic
pathogenic



pathogenic
endophytic
endophytic
endophytic





pathogenic

endophytic


Location Reference
Mehrotra 1997
GU Mehrotra 1997
FL, GU Alfieri et al. 1984

CABI, Herb IMI 337615


FL, GU
GU
FL, GU
GU


Kwon et al. 2001
Pemezny and Simone 1993
Tsay and Kuo 1991
Fajola and Alasoadura 1973
Alfieri et al. 1994
Peregrine and Ahmad 1982
Shaw 1984
Wei 1950
Onesirosan et al. 1974
Sarma and Nayudu 1971
Onesirosan et al. 1974
Peregrine and Ahmad 1982
Casady 1994


Alfieri et al. 1994
FL, GU Alfieri et al. 1994


El-Gholl et al. 1997


Urtiaga 2004


Minter et al. 2001










Table 1-2. Continued
Host
Cecropia schreberiana Miq.
Laportea aestuans (L.) Chew
Pilea spp. Lindl.
Pilea cadiereJ Gagnep. & Guillaumin
Pilea microphylla (L.) Liebm.
Pilea nummularilfolia (Sw.) Weddell
Verbenaceae J. St.-Hil. dicott)
Callicarpa americana L.
Citharexylum spinosum L.
Clerodendrum buchananii (Roxb.) Walp.
Clerodendrum paniculatum L.
Clerodendrum quadriloculare (Blanco) Merr.
Clerodendrum thomsoniae Balf.
Gmelina arborea Roxb.
Lantana camera L.
Petrea spp. L.
Stachytarpheta ,i, ..',ih '" (Mill.) Vahl.
Stachytarpheta cayennensis (Rich.) Vahl
Stachytarphetajamaicensis (L.) Vahl
Vitex agnus-castus L.
Vitex negundo L.
Vitex parviflora Juss.
Vitex pinnata L.
Vitex trifolia L.
Vitaceae Juss. dicott)
Cissus spp. L.
Cissus alata Jacq.
Tetra.tingma voinierianum (Baltet) Pierre ex Gagnep.
Vitis sDD. L.


Fungal-Host Interaction Location


pathogenic
pathogenic
pathogenic


pathogenic
pathogenic
pathogenic
pathogenic
endophytic
pathogenic

pathogenic
pathogenic
pathogenic



pathogenic

pathogenic


GU
GU
FL, GU


GU
FL
GU
FL
GU
FL, GU

GU
GU
FL, GU


Reference
Minter et al. 2001
Alfieri et al. 1994
Chase 1982
Alfieri et al. 1994
Smith and Schlub 2004
Alfieri et al. 1994

Alfieri et al. 1994
El-Gholl 1997
first report
Ellis 1957
first report
Daughtrey 2000
Florence and Sharma 1987
Pereira et al. 2003
Ellis 1957
Ellis 1957
McKenzi 1990
Smith and Schlub 2004
Alfieri et al. 1994
CABI, Herb. IMI 244917
Smith and Schlub 2004
Ellis 1957
McKenzie 1996


Alfieri et al. 1994
Alfieri et al. 1994
Alfieri et al. 1994
Alfieri et al. 1994










Table 1-2. Continued
Host Fungal-Host Interaction Location Reference
Zamiaceae Horan. gymnospermm)
Encephalartos spp. Lehm. Alfieri et al. 1994

Host plants are listed alphabetically by family (in bold). Each species is followed by the first known reported reference. Fungal-host
interaction refers to the endophytic or pathogenic nature of the fungus and was only reported for hosts that were collected during the
Guam (GU) and Florida (FL) surveys. Location refers to whether the plant species was found as a host of C. cassiicola in FL or GU.
Forty of the hosts were found on the CABI online database website
(http://194.203.77.76/herblMI/DisplayResults.asp? strName=Corynespora+cassiicola
CABI Databases: Herb. IMI records for Fungus: Corynespora cassiicola).











S


B)


Figure 1-1. Corynespora cassiicola isolate from Cucumis sativus A) sporulating on naturally
infected leaf tissue after 24 hours in the moisture chamber, B) germinating spore on
water agar, and C) growing on V8 agar after single spore isolation (images are not
shown to scale).

























D) E) F)













G) H) )

Figure 1-2. Various symptoms caused by Corynespora cassiicola on naturally infected leaves of
A) Vaccinium corymbosum, B) Carica papaya, C) Ageratum conyzoides, D)
Allamanda spp., E) Macroptilium lathyroides, F) Abutilon theophrasti, G) Bidens
alba, H) Euphorbia cyathophora, I) Chromolaena odorata Continued. J) Corchorus
aestuans, K) Passiflorafoetida, L) Ipomoeapes-caprae, M) Ipomoea obscura, N)
Lantana camera, 0) Merremia peltata, P) Bauhinia galpinii, Q) Catharanthus
roseus, R) Phyllanthus amarus, S) Hydrangea macrophylla, and T) Salviafarinacea.
Images are not to scale.













JVT


R) -'S)
Figure 1-2. Continued.










CHAPTER 2
GENETIC AND PATHOGENIC DIVERSITY OF CORYNESPORA CASSIICOLA

Introduction

Target spot, caused by the fungal pathogen Corynespora cassiicola (Berk. & Curt.) Wei, is

common in the tropics, subtropics, and greenhouses (Chase 1987). C. cassiicola is reported to

infect 530 plant species from 380 genera, including monocots, dicots, ferns, and one cycad

(Chapter 1, this dissertation). Isolate characterization is needed to determine which hosts might

serve as sources of inoculum for target spot of tomato species and other hosts since there is much

variability concerning the host range of individual isolates. Some isolates show pathogenicity to

a wide range of hosts, whereas others exhibit host specificity, and some are only pathogenic

when associated with wounding (Chase 1982; Cutrim and Silva 2003; Kingsland 1985;

Onesirosan et al. 1973, 1974; Pereira et al. 2003; Poltronieri et al. 2003; Seaman et al. 1965;

Smith and Schlub 2004; Smith and Schlub 2005; Spencer and Walters 1969; Volin and

Pohronezny 1989). At least two races of the fungus have been distinguished based on their

differential pathogenicity response on soybean and cowpea (Olive and Bain 1945; Spencer and

Walters 1969). However, isolates from soybean, sesame, cowpea and cotton in Mississippi were

alike in pathogenicity (Jones 1961). A more extensive study found eight different pathogenicity

profiles among 28 isolates from soybean in Mexico, cucumber in Florida, and diverse hosts in

Nigeria (Onesirosan et al. 1974). Furukawa et al. (2008) found that an isolate from Salvia

splendens was not pathogenic to cucumber, green pepper or hydrangea; however isolates from

these hosts were pathogenic to Salvia splendens. Furukawa et al. (2008), therefore,

demonstrated that isolates with different pathogenicity profiles can be found on the same host.

Since the 1960's, a leaf and fruit spot disease of tomato caused by C. cassiicola has

become increasingly serious in tropical countries worldwide (Jones and Jones 1984). It was first









reported in Florida in 1972 and has since become one of state's most damaging foliage and fruit

diseases (Blazquez 1972; Pernezny et al. 1993, 1996, 2000, 2002). Under warm, humid,

conditions the disease leads to heavy defoliation and significant losses in yield (Volin and

Pohronezny 1989). Currently, there are no resistant tomato cultivars available, although

resistance found in PI 120265 (Lycopersicon esculentum) and PI 11215 (L. pimpinellifolium) and

was controlled by a single recessive gene (Bliss et al. 1973). Understanding the genetic and

pathogenic diversity of the pathogen and its distribution is vital to isolate selection for resistance

screening.

Kingsland (1985) compared three isolates from tomato, cucumber and papaya debris and

found that tomato and cucumber were susceptible to all isolates, but the isolate from papaya

debris was not pathogenic on papaya, indicating that it was possibly growing as a saprophyte. In

many studies, isolates were found to be non-pathogenic on the hosts from which they were

isolated, further indicating that C. cassiicola can grow as a saprophyte (Chase 1982; Kingsland

1985; Onesirosan et al. 1974; Hyde et al. 2001; Lee et al. 2004). Other studies show that isolates

are only secondary invaders, or invaders of senescent tissue. Isolates from the ornamental hosts

A eh. /1i un/ni1 pulcher (lipstick vine), Aphelandra squarrosa (zebra plant), azalea and

hydrangea were pathogenic on all hosts in cross-pathogenicity trials when wounded; however,

only A. pulcher was susceptible without wounding (Chase 1982).

Silva et al. (1998) compared pathogenicity of 16 isolates from rubber trees in Sri Lanka

and five isolates from diverse hosts in Australia. Papaya isolates from Australia were pathogenic

to tomato and rubber, but not cowpea and eggplant. Mimosa and thyme isolates from Australia

were pathogenic to eggplant, rubber, and tomato, but not cowpea. Isolates from Sri Lanka









collected from different rubber clones were either pathogenic to all hosts cowpeaa, eggplant,

rubber, and tomato), or pathogenic to all hosts but eggplant.

The host specificity and severity of the fungus on Lantana camera in Brazil has led to the

discovery that C. cassiicola may be useful as a bioherbicide (Pereira et al. 2003). Based on the

vast number of weeds that serve as hosts of the fungus, there is great potential for the discovery

of several more isolates useful for biological control of weeds. Considering the wide variation in

isolate pathogenicity that has been previously reported, additional studies are needed to further

understand the host range of individual isolates from different hosts and locations.

Prior research on the genetic characterization of C. cassiicola is limited to restriction

fragment length polymorphism (RFLP) of ITS rDNA and random amplified polymorphic DNA

(RAPD) studies. No variation between five isolates of C. cassiicola collected from mimosa,

papaya, and thyme in Australia was found based on RFLP of ITS (Silva et al. 1995). Silva et al.

(1995) concluded that RFLP of the ITS regions of rDNA can be used to distinguish between

Corynespora and the morphologically similar genus Helminthosporium, but not different isolates

of C. cassiicola. However, the three isolates from papaya had identical RAPD patterns, growth

rate, isolate color, and pathogenicity profiles, which were different from the isolates from

mimosa and thyme, indicating an ongoing process of host specialization on papaya (Silva et al.

1995).

RAPD analyses from 27 isolates collected from Hevea brasiliensis, in Sri Lanka revealed

correlations between host location, host genotype, isolate morphology, and isolate pathogenicity

(Silva et al. 1998). Silva et al. (1998) concluded that a progenitor strain may have been spread in

India by distribution of live plant material. Prior outbreaks of the disease on the susceptible









rubber clone RRIC 103 in other countries, and the sudden appearance and severity of target spot

on the same clone in Sri Lanka in 1985, is evidence for such dissemination.

Silva et al. (2003) characterized 42 isolates from bitter gourd, cocoa, manihot, papaya,

rubber, sweet potato, tomato, and wing-bean from various regions in India based on RAPD

analyses. RAPD groups did not correlate with geographic origin, but isolates obtained from

rubber clone RRIC 103 grouped together. This strain might be responsible for several recent

outbreaks on this clone. In addition, all but one of the isolates from rubber clone RRIC 110

clustered in 2 RAPD groups, which may identify the strain that caused the outbreak on this clone

in 1995. Silva et al. (2003) concluded that correlation of RAPD groups with pathogenicity was

needed to help develop resistant clones against all pathogenic isolates.

Atan and Hamid (2003) characterized nine C. cassiicola isolates from Hevea brasiliensis

in Malaysia using RAPD of genomic DNA and RFLP of amplified ITS regions. RFLP analyses

with three restriction enzymes yielded monomorphic patterns. However, isolate OPEN 1 from

clone RRIM 2020 had a distinct RFLP pattern from the other eight isolates after digestion with

HaeIII. RAPD results indicated the presence of at least two genetically distinct races that infect

rubber. Seven isolates pathogenic to clones RRIM 600, RRIM 2009, and two unidentified

rubber clones were molecularly similar and identified as Race 1. The remaining two isolates,

both pathogenic on clone RRIM 2020, had identical banding patterns and were considered Race

2.

Unfortunately, the majority of the diversity assessments are limited to rubber isolates from

Malaysia and Sri Lanka and are based on RAPD techniques, which is problematic with respect to

repeatability and homology assessment (Isabel et al. 1999). In addition, all the RFLP studies

used the ITS rDNA region which has minimal variation among isolates (Silva et al. 1995, 1998).









Investigations into the genetic variation among C. cassiicola isolates using more reliable

molecular methods and more diverse isolates are needed.

In this study, we collected and solicited 143 isolates from diverse hosts and locations. To

test whether C. cassiicola is panmictic throughout its range, allelic genealogies were constructed

from four loci including the rDNA ITS region, two random hypervariable loci, Cc caa5 and Cc

ga4, and the single copy actin-encoding nuclear gene, Cc act]. Fifty of these isolates were spray

inoculated on seedlings of eight crop plants to test pathogenicity profiles. Correlations among an

isolate's pathogenicity profile, its host of origin, and genotype were investigated. The purpose of

this research is to gain knowledge of the diversity within the species C. cassiicola because of its

implications for resistance breeding and disease management of target spot of basil, bean,

cowpea, cucumber, papaya, soybean, sweet potato, tomato, and potentially other crops.

Methods

Collection and Solicitation of Fungal Isolates

C. cassiicola isolates were collected from diverse plant hosts during 5-day collecting trips

to locations in the Pacific: American Samoa (AS), Hawaii (HI), Palau (PW), Pohnepei (PH),

Saipan (SN), and Yap (YP) in the summer of 2005. More extensive surveys were conducted to

collect the fungus in Florida (FL) and Guam (GU) between 2004-2006 (see Chapter 1).

Farms, nurseries, and roadsides were surveyed for plants with target spot symptoms. First,

second, and third priority was given to crops, weeds, and naturalized or indigenous hosts of C.

cassiicola, respectively. Symptomatic leaves were put into individual plastic bags in the field

and later placed abaxial side up in petri dishes with moistened paper towels in a laboratory.

After 24 hours in the moisture chamber, petri plates were placed under the dissecting microscope

and suspected spores and conidiophores of C. cassiicola were confirmed microscopically.









Single spores were captured at the end of a teasing needle and transferred to antibiotic V8

agar (340 ml V8 juice, 660 ml water, 3g CaCO3, 17g agar, 100 [tg/ml Ampicillin or Kanamycin)

slants, left at room temperature until the colony reached at least 5 cm in diameter, whereby it was

covered with autoclaved mineral oil, and stored at 5 C until further study. Sporulation from

non-symptomatic leaf material was noted, possibly indicating non-pathogenic growth.

To obtain globally diverse isolates, individual researchers in Brazil (BZ), Malaysia (MY),

Mississippi (MS), and Tennessee (TN) were solicited for additional C. cassiicola cultures.

Isolates from BZ on lantana (JMP216), papaya (DOA16b), soybean (RWB321) and tomato

(JMP217) came from Alvaro Almeida, EMBRAPA. Isolates CBPP, CLN 16 and CSB1 2 were

received from MY off of rubber from Dr. Safiah Atan, Malaysian Rubber Board. Isolate TN13-3

was received from Nashville, TN on greenhouse African violet from Justin S. Clark, University

of Tennessee. Isolate MS01 was received from MS on greenhouse tomato leaves from David

Ingram, Central MS Research and Extension Center.

Isolates of different species were also solicited from culture collections to serve as

outgroups. Cultures from Commonwealth Agricultural Bureaux International (CABI) in the

United Kingdom included C. smithii IMI 5649b and C. citricola IMI 211585. Cultures from

National Institute of Agrobiological Sciences (NIAS) in Japan included C. citricola MAFF No.

425231, C. melongenea MAFF No. 712045, and C. sesamum MAFF No. 305095. Cultures from

Centraalbureau voor Schimmelcultures (CBS) in the Netherlands included C. proliferate CBS

112393, C. citricola CBS 169.77, and C. olivaceae CBS 291.74. Cultures were single-spored

after they were received. A complete list of isolates used in these studies, along with the plant

host, geographic location and the type of association with the host plant (endophytic or

pathogenic growth) can be found in Table 2-1.










Primer Development for Random Hypervariable Loci

Three C. cassiicola isolates from long-term storage (FL31, GUI 12, and PW56) were

chosen based on unique host and location. A small piece of mycelium from the monosporic

cultures was extracted with tweezers and placed onto a V8 agar plate. The isolates were grown

under constant fluorescent light for 7 days. Aerial mycelium was scraped from the agar surface,

placed in 1.5 ml microcentrifuge tubes, lyophilized overnight, and then frozen in liquid nitrogen.

Genomic DNA was purified using the DNeasy plant Mini Kit (Quiagen, Inc.) according to the

manufacturer's specifications.

Genomic DNA combined from all three isolates was digested with the Sau 3AI restriction

enzyme (7.2 [tl of DNA from each of the three isolates; 2.5 [tl 10X buffer; 1.0 [tl 10 U/dtl Sau 3A

I enzyme; incubated at 37C for 2 hours). The digested genomic DNA was fractionated to

remove fragments less than 400 bp using a Chroma Spin column (Chroma Spin + TE 400,

Clonetech Laboratories, Inc.) according to the manufacturer's specifications. The digested

fractionated DNA was quantified and ligated to Sau 3AI linkers and incubated at 16C overnight.

Excess linkers were removed using the same Chroma Spin column as above. The linker-

ligated fragments were PCR amplified using SauL-A primers and a program consisting of initial

denaturation for 3 min at 94C, followed by 25 cycles of 94C for 1 min, 68C for 1 min, and

72C for 2 min, and a final amplification at 72C for 10 min.

The amplified genomic PCR library (composed of 400-1500 bp fragments) was enriched

for fragments containing two different microsatellite repeats, (CAA)n and (GA)n. The denatured

genomic PCR library was hybridized to the following biotinylated oligoprobes:

[5'(CAA)i5TATAAGATA-Biotin] and [5'(GA)i5TATAAGATA-Biotin] (Tepnel Lifecodes

Corporation) and incubated at 48C overnight. The PCR fragments that hybridized to the repeat









probes were captured and eluted using two VECTREX Avidin D matrix columns (cat. No. A-

2020, Vector Laboratories, Burlingame, CA) according to the manufacturers specifications. The

two mixtures containing genomic fragments enriched for the (CAA)n tri-repeat and the (GA)n di-

repeat were PCR amplified using SauL-A primers following the same PCR conditions as above.

PCR products from the amplification of the enriched microsatellite library were ligated

into a plasmid vector (pCR 2.1-TOPO vector; Invitrogen, Inc.) and transformed into E. coli

(One ShotTM TOP 10 Cells, Invitrogen, Inc.) using the TOPO TA Cloning Kit (Invitrogen, Inc.)

according to the manufacturers instructions. Transformed colonies were lifted and crosslinked

onto nylon membranes in an UV chamber (GS Gene LinkerTM, Bio-Rad Laboratories, Inc.,

Hercules, CA) using the "optimal crosslink" program.

Nylon membranes were hybridized with alkaline phosphatase-labeled repeat probes

((CAA)n and (GA)n) and the Quick-LightTM hybridization Kit (Tepnel Lifecodes Corp.)

according to the manufacturers recommendation. Colonies containing plasmids that tested

positive for inserts with repeats were sequenced in one direction. Primers were designed to

amplify 300-500 base pair fragments flanking low repeat number (<10) sequences using Primer3

(v. 0.4.0). Sequences with repeats of less than 10 were likely to be non-variable microsatellite

loci, but may contain polymorphic flanking sequences. Sequences were screened for

polymorphisms using five isolates from different hosts and locations. Primers that amplified the

Cc ga4 and Cc caa5 loci were chosen for further study because they amplified sequences with

relatively high levels of polymorphism (>5%).

Fungal Cultures and Extraction of Genomic DNA

Genomic DNA from 143 isolates (Table 2-2) in long-term storage was purified and

amplified using Extract-N-AmpT (Sigma-Aldrich) according to the manufacturer's

specifications.









The following primers were used for PCR amplification: ITS1 and ITS4 (White et al.

1990) for the internal transcribed spacer region, including the 5.8 rRNA coding region; ACT-

512F and ACT-783R (Carbone and Kohn 1999) for the single copy nuclear actin locus Cc actl;

GA4-F (5'-CCT GCT CCG ACT TTG TTG AG-3') and GA4-R (5'-GTC TGG GAG CAG CAA

AGA CT-3') for the random hypervariable Cc ga4 locus; CAA5-F (5'-GTC CAC AAG TGG

AAC CTC GT-3') and CAA5-R (5'-CCT CGT CTG CCA GTT CTT CT-3') for the random

hypervariable Cc caa5 locus. "Hot-start" PCR was performed with a MyCyclerTM thermocycler

(BioRad) with a program consisting of initial denaturation for 3 min at 94C, followed by 30

cycles of 30 sec at 94C, 30 sec at 58C, and 30 sec at 72C, and a final cycle of 5 min at 72C

for the ITS, Cc ga4, and Cc caa5 loci. For the Cc act] locus, the program was identical except

for an annealing temperature of 61C. PCR products were purified using the QIA quick PCR

purification Kit (QIAGEN Inc.) according to the manufacturer's instructions. The purified

products were then quantified on 1% ethidium bromide-stained agarose gels. Sequencing of the

DNA samples was done at the University of Florida DNA Sequencing Core Laboratory using

ABI Prism BigDye Terminator cycle sequencing protocols (part number 4303153) developed by

Applied Biosystems (Perkin-Elmer Corp., Foster City, CA). The excess dye-labeled terminators

were removed using MultiScreen 96-well filtration system (Millipore, Bedford, MA, USA).

The purified extension products were dried in SpeedVac (ThermoSavant, Holbrook, NY, USA)

and then suspended in Hi-di formamide. Sequencing reactions were performed using POP-7

sieving matrix on 50-cm capillaries in an ABI Prism 3130 Genetic Analyzer (Applied

Biosystems, Foster City, CA, USA) and were analyzed by ABI Sequencing Analysis software v.

5.2 and KB Basecaller.









Phylogenetic Analyses

Four loci (rDNA ITS, Cc caa5, Cc ga4, and Cc act]) from 143 isolates were sequenced.

Forward and reverse sequences from each PCR product were concatenated in SequencherTM 4.8

and trimmed to include only bases sequenced in both directions. Samples with ambiguities were

sent for re-sequencing. Multiple alignments from each locus were executed separately with

Clustal X (1.83.1) and the alignments were inspected and adjusted manually using MacClade

4.08 OS X (Maddison and Maddison 2005). Data from ITS rDNA, Cc ga4, Cc caa5, and Cc

act] loci were partitioned to facilitate different permutations of combined analysis. A partition-

homogeneity test incongruencee length-difference test or ILD) was implemented to evaluate the

homogeneity of different data partition subsets using PAUP* v4.0bl0 (Swafford 2002). The test

implemented 1,000 replicates (heuristic search; random simple sequence additions; TBR; max-

trees = 1,000). Comparisons were evaluated using a threshold ofp < 0.001 and were made

between all data partitions.

With the ILD test indicating the combinability of all molecular data, neighbor joining (NJ)

and maximum parsimony (MP) analyses were conducted for each data partition and the

combined data set using PAUP* (Swafford 2002). C. smithii IMI 5649b, C. citricola IMI

211585, C.proliferata CBS 112393, C. citricola CBS 169.77, and C. olivaceae CBS 291.74

were defined as outgroups. Cultures from National Institute of Agrobiological Sciences (NIAS)

in Japan (C. citricola MAFF No. 425231, C. melongenea MAFF No. 712045, and C. sesamum

MAFF No. 305095) were not included as outgroups because they grouped with C. cassiicola

isolates in phylogenetic analyses (see Results below). For the NJ analyses, default settings were

used except ties were broken randomly by initial seed.

Due to long computational time, MP analyses were conducted in the following manner.

An initial heuristic search was conducted with one random addition replicate, TBR (tree-









bisection-reconnection) branch swapping, and the MulTrees option (saving all optimal trees) in

effect. A second heuristic search was conducted using 1000 random addition replicates with the

above settings and saving no more than 10 trees with a score greater than or equal to the best tree

score from the first replicate in the previous analysis. In all analyses, gaps were treated as

missing data. Strict consensus trees were generated from analyses with multiple equally

parsimonious trees. For all MP analyses, statistical support for nodes was estimated using

maximum parsimony bootstrap (BS) replicates (Felsenstein 1985). For the combined data set,

BS estimates were obtained using 1,000 replicates, each with 100 random taxon addition

replicates and saving no more than 1,500 trees per bootstrap replicate, TBR branch swapping and

the MulTrees option in effect.

All data were also analyzed by Bayesian inference (BI) methods with MrBayes v3.1.2

(Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). An appropriate model of

evolution (under the AIC criterion) was selected for each data partition using the program

Modeltest v3.4 (Posada and Crandall 1998). All Bayesian analyses (individual loci and

combined data) were conducted while retaining the appropriate model for each data partition.

Markov Chain Monte Carlo was implemented with four heated chains and trees were sampled

every 1,000th generation for one million generations. The first 25 percent of the total number of

generations was discarded as bum-in. A 50 percent majority rule consensus tree was generated

from the remaining trees, in which the percentage of nodes recovered represented their posterior

probability (PP).

Congruent nodes resulting from the NJ, MP, and BI analyses of the combined molecular

data was used to assign isolates to a phylogenetic lineage (PL). Only isolates that fell within

clades of high support (BS value >70 and PP value > 95) were assigned to a PL.









Pathogenicity Analyses

Fifty out of the 143 Corynespora isolates were used for pathogenicity profiling (Table 2-3)

on eight crop plants. Isolates originally isolated from crop plants and from all phylogenetic

lineages were chosen. Each isolate was spray-inoculated onto four replicate plants of eight-

week-old: basil 'Italian Large Leaf (Ba), bean 'Bush Kentucky Wonder' (Be), cowpea

'California black-eye' (Co), cucumber 'Straight 8' (Cu), soybean 'AG00901' (So), and tomato

'Rutgers' (To) seedlings; 8-week-old sweet potato 'Beauregard' (Sw) cuttings; and 12-week-old

papaya 'HI Sunrise' (Pa) seedlings. Cultivars were chosen based on their known susceptibility

in the survey regions.

To increase colony sporulation for inoculum preparation, aerial mycelium from 10-day-old

V8 agar plates was gently scraped with a glass cover slip to flatten mycelium and then placed

under constant cool-white fluorescent light (Onesirosan et al. 1975). Three days later, the

surface of the agar was scraped with a glass cover slip and the resulting mycelia was blended in

200 ml sterile distilled water for two seconds and filtered through three layers of cheesecloth.

Spores were counted under a hemacytometer and the concentration was adjusted to 20,000

spores/ml. One drop of Tween 20 per 100 ml was added to the inoculum. Plants were sprayed

with the spore suspension until leaf run off (about 500 ml), making sure that both leaf surfaces

were fully covered. Plants were kept on a mist bench to maintain constant leaf moisture 3 days

prior to inoculation and for the remainder of the experiment. Plants were rated 7 days after

inoculation using the rating system developed by Onesirosan et al. (1973): (0) symptomless, no

lesions on leaves or stems; (1) non pathogenic hypersensitive response, a few to many non-

expanding pinpoint lesions; (2) moderately virulent, many expanding lesions, some coalescing,

but not resulting in blight; (3) highly virulent, lesions spreading to form large areas of dead tissue

resulting in a blighting effect. Incidence (I), defined as the number of plants showing symptoms









(with ratings of 1, 2, or 3), and severity (S), defined as the average rating for all symptomatic

plants, were recorded. The experiment was repeated.

Each isolate was assigned a pathogenicity profile (PP), which is a list of susceptible hosts.

Hosts were considered susceptible if at least one of the replicates from the two experiments (total

of eight plants) received a rating of 2 or 3. PPs were converted to a binary character matrix so

that each isolate received a zero (non-pathogenic, all reps with ratings of 0 or 1) or a one

(pathogenic, at least one rep with a rating of 2 or 3) for each host. Unweighted pair group

method with arithmetic mean (UPGMA) trees were constructed from the binary matrix and

internal support for nodes was estimated using bootstrap analyses with 1,000 reps and a UPGMA

algorithm. The tree topology was visually compared to the PL designation of each isolate tested

(Figure 2-6). PPs were also visually mapped on the four-locus combined BI phylogenetic tree

(Figure 2-1).

Growth Rate Analyses

Seventy-seven isolates were tested for growth rate at two temperatures (23 C and 33 C). A

small piece of aerial mycelium was extracted from the monosporic cultures in long-term storage

with tweezers and placed onto a V8 agar plate. After 5 days, the 77 colonies had grown beyond

the mineral oil and six 4 mm agar plugs were cut from actively growing mycelium at the colony

edge. A single plug was placed in the center of six V8 agar plates. Three replicate plates of each

isolate were immediately placed in growth chambers at 23 C and 33 C under 12 hours of

alternating fluorescent light (ca. 25 lux) and dark.

The average of two colony diameters at 90 degrees from each other was recorded at 48, 72,

96, 120, 144, and 166 hours. Average colony diameter was plotted against time and a line of

best fit was generated for each replicate. The slope of the line of best fit (R2>0.98) was used to

compare variation within reps to variation between isolates in SAS Statistical Software









(Version 8, 1999). The experiment was repeated with five isolates with no statistically

significant variation (data not shown). A correlation between isolate growth rate and

phylogenetic lineage was tested using SAS statistical software.

Results

Phylogenetic Analyses

The General Time Reversible model (GTR + I + F) was selected by Modeltest for each of

the four gene partitions. The corresponding model for each locus was applied to all BI analyses

and the combined dataset was partitioned. The final combined dataset contains 2,136 aligned

characters used for analyses. Tree topologies resulting from NJ, MP, and BI analyses recovered

essentially the same well-supported nodes. The analyses reveal four major phylogenetic lineages

(PL) with high statistical support (BS value >70 and PP value > 95) (Figure 2-1).

All major PLs contain isolates from diverse locations, indicating their global dispersal.

PL1 contains a distinct clade with high statistical support (designated PL1.1) containing only

isolates collected from papaya from around the world indicating specialization on this host.

PL1.2 contains two isolates from Stachytarphetajamaicensis, collected from Guam and Palau,

indicating potential specialization on this host. This supports pathogenicity studies showing

isolate specificity to this host (Smith and Schlub 2005). Isolates from diverse hosts are present

in PL1 including crops (basil, bitter melon, eggplant, cowpea, cucumber, oregano, pumpkin,

rubber, soybean, sweet potato, watermelon), ornamentals (Buddleja, Catharanthus, Codiaeum,

Coleus, Episcia, and Tabebouia), and weeds (Bidens, Buchnera, Clerodendrum, Commelina,

Lantana, Macroptilium, Meisosperma, Vitex). Tomato isolates are missing from PL1, indicating

that isolates in this lineage may not be pathogenic to tomato.

Isolates in PL2 are also globally distributed and include crops (cucumber, rubber, sweet

potato), ornamentals (African violet, Allamanda, Catharanthus, Pilea), and weeds (Piper, Pilea).









There is also a lack of tomato isolates in PL2 indicating that isolates from this lineage may be

nonpathogenic on this host. Though PL2 was highly supported (BS and PP values of 100), its

sister relationship to PL1, PL3, and PL4 remains unresolved.

Globally distributed isolates from PL3 include crops (basil, bitter melon, cucumber,

pumpkin, soybean, tomato), ornamentals (Bauhinia, Moringa, Pachystachys, Plectranthus,

Saintpaulia), and weeds (Acanthus, Asystasia, Calopogonium, Coccinia, Euphorbia, Luffa,

Passiflora, Teramnus). These are hosts that may harbor isolates pathogenic to tomato. PL5 and

PL6 group with PL3 with low support (MPBS value of 60). PL5 contain C. cassiicola isolates

from African violet in Guam and Tennessee that are very similar in sequence, especially at the

Cc-caa5 locus, indicating specialization on this host. African violet isolates from Saipan and

Yap are found in PL3. PL6 is highly supported and contains isolates from Brazil on Coleus,

Palau on cowpea, and Saipan on Asystasia.

The majority of tomato isolates group in PL4 from diverse locations including American

Samoa, Brazil, Florida, Guam, Mississippi, Palau, and Saipan. These twelve tomato isolates also

group with isolates from crops (bean, cassava, cucumber, sweet potato), ornamentals (Bauhinia,

Cassia, Coleus, Eugenia, Ficus, Jatropha, Salvia, Syzygium), and common weeds

(Calopogonium, Calyptocarpus, Chromolaena, Euphorbia, Hyptus, Lantana, Mikania,

Spathodea), which are likely inoculum sources for the initiation of disease on tomato.

The rDNA ITS region (Figure 2-2) is composed of 1,013 characters, 400 of which are an

insertion in the outgroup taxa C. smithii. Of the 612 remaining characters, 141 are variable and

107 are informative. The rDNA ITS sequences reveal the misidentification of three outgroup

taxa from the NIAS culture collection. C. sesamum 305095, C. citricola 425231, and C.

melongenea 712045 should be reclassified as C. cassiicola based on rDNA ITS sequences.









When the outgroup taxa C. citricola, C. olivaceae, C. proliferata, and C. smithii are removed

from the analyses, the rDNA ITS sequences of C. cassiicola contain only three informative

characters out of 584 bases. Two of these characters separate the isolates into three distinct

phylogenetic lineages that correlate with the PLs in the combined analysis. These two characters

are base pair 158 (C or T), and base pair 497 (A or G) of the C. cassiicola rDNA ITS alignment.

Three haplotypes are represented by these two characters: CA, CG, and TG (no haplotype TA);

all isolates with haplotype CA group in PL4, isolates with haplotype TG group in PL1, isolates

with haplotype CG group in PL2, PL3, PL5 and PL6. CG is also the ancestral haplotype, present

in all outgroups except for C. proliferata (haplotype CA). The third informative character in the

rDNA ITS sequences of C. cassiicola is base pair 123, which is a T in the majority of isolates,

but a C in isolates PW101 (PL5), RWB321 (PL5), SN64 (PL5.1), and TN13-3 (PL6). It is this

character (bp 123) that caused the polymorphic band pattern observed by Atan and Hamid (2003)

in their RFLP analysis of the rDNA ITS region of rubber isolates using HaelII (recognition

sequence GGCC).

The sister relationships between the phylogenetic lineages remain unresolved in the

analyses of the individual loci and in the combined analyses. In addition, the ITS rDNA region

was the only locus that showed good support for C. citricola, C. olivaceae, C. proliferata, and C.

smithii as sister taxa to the ingroup of C. cassiicola isolates. The phylogenetic placement of the

outgroup taxa was not well supported in the combined analyses, or the GA4 locus. The CAA5

locus showed support for C. olivaceae, C. proliferata, and C. smithii as basal to PL1, PL2, PL3,

PL5, and PL6, but PL4 and C. citricola fell basal to that group. The apparent paraphyly of C.

cassiicola at the Cc-caa5 locus may be a result of character variation that occurred at this locus









before the species evolved. Additional loci containing characters that reveal the sister

relationships of the different PLs are needed in future analyses.

The Cc-caa5 locus (Figure 2-3) reveals similar tree topologies to the combined analyses

with high support for the four major PLs. Differences at this locus in PL1 include the lack of

PL1.1 that distinguishes papaya isolates from other isolates in PL1 in the combined analyses. In

addition, PL1.2, which includes two rubber isolates from Malaysia, has low support. GU70 and

SN59, isolates basal to PL1 in the combined analyses, groups with other isolates in PL1 at this

locus. The Cc-caa5 locus shows strong support for PL2 with the same nine isolates as in the

combined analysis. The Cc-caa5 locus does not resolve PL3.1 or PL3.3 as distinct from PL3,

although the five isolates in PL3.2 group together with strong support. This locus does not

distinguish isolates FL2920, GU120, GU136 as distinct from other isolates in PL4. PL5 and

PL5.1 isolates are group basal to PL3, but with low support. PL6, which includes African violet

isolates from Guam and Tennessee, group with isolate NIAS 712045 with high support. Isolates

FL50 (Hydrangea macrophylla) and FL51 (Vaccinium corymbosum) are unresolved at this locus

as well as in combined analyses.

The Cc-ga4 locus (Figure 2-4) highly supports PL1, PL2, PL4, and PL6, although the

sister relationships between the PLs are unresolved. Isolates in PL3 form a clade with low

support. The Cc-ga4 locus did reveal a shared haplotype between papaya isolates with a point

mutation from an A to a G at base 74. C. cassiicola isolates are not monophyletic at this locus

because the outgroups C. proliferate, C. olivaceae, and C. smithii fall basal to PL1, PL2, and

PL6 with low support. This may be a result of character variation before speciation, a high

incidence of homoplasious characters, or the convergent evolution of specific adaptations.









The Cc-act] locus could not be amplified in the outgroup taxa C. citricola, C. proliferate,

and C. smithii, perhaps due to mutations in the primer annealing site. The locus did amplify in

C. olivaceae and shows high variation from C. cassiicola isolates (Figure 2-5). There is good

support for PL3, PL2, and PL4 at this locus, although only marginal support for PL1 (BS value

of 68). Again, the sister relationships between the PLs are unresolved. The Cc-act] locus also

reveals a shared haplotype between papaya isolates with a point mutation from an A to a G at

base 229.

Pathogenicity Analyses

As a result of screening fifty isolates for pathogenicity on eight index hosts, 16 unique

pathogenicity profiles (PP) were developed (Table 2-3). The most common PP was CuTo,

followed by Pa and CuSwTo. Cucumber was the most susceptible host, with all isolates

producing symptoms and an average severity rating of 2.3. Tomato was also highly susceptible

with 49 out of 50 isolates showing symptoms with an average severity rating of 1.8. Even

though only eight isolates were pathogenic on papaya, the average severity rating was 2.1

indicating that pathogenic isolates were highly virulent. Isolates pathogenic to basil, bean,

cowpea, soybean and sweet potato were less virulent on these hosts with average severity ratings

less than 1.5.

There was a strong correlation between PP and PL (Figure 2-6). Seven out often isolates

with PP CuTo were from PL4 and all isolates with PP CuSwTo and BeCuSwTo were from PL4.

In PL4, all isolates but SN37 were highly virulent on tomato (average severity ratings ranging

from 2.5 to 3) and all isolates but GU28 were pathogenic to cucumber (average severity ratings

ranging from 1.3 to 3). In addition, the only isolates pathogenic to bean were from PL4,

although these five isolates were weakly virulent (average severity ratings ranging from 1.3 to

1.9).









In PL3, all isolates were strongly pathogenic to cucumber (average severity ratings ranging

from 2.3 to 2.8) and six out of seven isolates were strongly pathogenic to tomato (average

severity ratings ranging from 2.5 to 3). Four out of the six isolates also were pathogenic to basil

and all isolates with PP BaCuTo were from PL3.

Pathogenicity profile CuSw was unique to isolates from PL2 and all isolates tested from

PL2 had this profile. In addition, isolates collected in the field from papaya in PL1.1 were

specific to papaya in pathogenicity studies, although all isolates were weakly virulent on

cucumber with average severity ratings of 1.3 or less. All isolates from PL1 were pathogenic to

cucumber with average severity ratings ranging from 1.3 to 3. Nine out of the 13 isolates from

PL1 were pathogenic to cowpea and seven were pathogenic to basil. The only other host

susceptible to isolates from PL1 was soybean, which was only weakly susceptible when

inoculated with isolate PW87.

Growth Rate Analyses

The null hypotheses of no growth rate differences among isolates, phylogenetic lineage,

and temperatures were rejected (P<0.0001), while the null hypotheses of no growth rate

differences among repetitions was accepted with a probability of 0.7546. The 77 isolates tested

all grew faster at 23 C than 33 C. At 23 C, average isolate growth rate (average of three

repetitions) was between 0.1479 and 0.474 with an overall mean of 0.3855 (Table 2-4). At 33 C,

average isolate growth rate was between 0.1382 and 0.4153, with an overall mean of 0.2958

(Table 2-5). At 23 C, there were 29 significantly different growth rates and at 33 C there were

39 significantly different growth rates. Among the fastest growing isolates at both temperatures

were FL37, GU90, GU99, AS67, and HI01. The slowest growing isolates were very different at

the two temperatures. Slow growing isolates at 23 C were PH01, JMP216a, GU120, FL15, and









DOA16b. Slow growing isolates at 33 C were AS119, AS117, JMP217, AS49, GU120, and

GU 112.

Though there were not enough replicates to test for interactions among effects, growth rate

alone correlated with location, phylogenetic lineage, and with host. Isolates from Oahu and

Palau grew the fastest at both temperatures. Isolates from Brazil, Florida and Malaysa tended to

have slower growth rates at both temperatures. Surprisingly, American Samoan isolates grew

proportionately much faster at 23 C than 33 C, despite its tropical climate. Isolates from PL6

and PL1 grew the fastest at both temperatures. Isolates from PL2 and PL4 grew the slowest at

23 C and isolates from PL5 and PL3 grew the slowest at 33 C. All isolates from Clerodendrum,

Commelina, Ficus, Macroptilium, pumpkin, and Stachytarpheta were fast growing at both

temperatures. In addition, isolates from Allamanda, Coleus, eggplant, Lantana, and tomato

isolates had slower growth rates at both temperatures.

Discussion

The current study presents the first robust, global phylogeny of the species Corynespora

cassiicola. Based on sequence data from four unique loci, there is evidence for high genetic

diversity within the species. The highly clonal nature of C. cassiicola is demonstrated in the

congruence of the phylogenetic trees from distinct loci. All loci distinguish four major clonal

lineages within C. cassiicola. The low level of sequence variation at the rDNA ITS region

within the species relative to other Corynespora species suggests that these lineages are in fact

clonal populations, rather than taxonomically distinct species.

As reported previously, the pattern of distribution of the diversity within the species

correlates with the host (Smith et al. 2008a). Identical haplotypes are widely distributed

geographically. The lack of correlation between phylogenetic data and location provides

evidence for the recent global dispersal of isolates from all four phylogenetic lineages. In









addition, geographically diverse isolates from the same host plant shared identical haplotypes,

potentially indicating host specialization. For example, isolates collected from tomato in Brazil,

Florida, Guam, Mississippi, Palau, and Saipan had identical haplotypes at all four loci. Isolates

collected from Lantana in Florida and Brazil were also identical at all four loci. Isolates

collected from African violet in Guam and Tennessee were unique from all other isolates and

nearly identical to each other. Perhaps the most compelling evidence for host specialization is

the shared identical sequences of all isolates collected from papaya from very diverse locations.

Tomato isolates from diverse locations including North and South America and the Pacific

Islands are found in only two of the five major phylogenetic lineages (PL3 and PL4). Isolates

from other hosts that fall into these same PLs are likely pathogenic to tomato and may serve as

source hosts or alternative hosts for target spot of tomato. Tomato isolates are genetically similar

to isolates from common crops (basil, bean, bitter melon, cassava, cucumber, papaya, pumpkin,

soybean, sweet potato), weeds (Acanthus, Calopogonium, Calyptocarpus, Chromolaena,

Coccinia, Euphorbia, Lantana, Macroptilium, Mikania, Momordica, Passiflora, and Teramnus),

and ornamentals (Asystasia, Bauhinia, Cassia, Coleus, Eugenia, Euphorbia, Ficus, Hyptus,

Jatropha, Luffa, Moringa, Pachystachys, Plectranthus, Saintpaulia, Salvia, Spathodea, and

Syzygium). Based strictly on these data, control of target spot should involve isolation of tomato

fields from these plant species, when possible.

Pathogenicity testing, in addition to phylogenetics, should be used to determine which

hosts might serve as sources of inoculum for the initiation of target spot of tomato. There are at

least sixteen unique pathogenicity profiles within C. cassiicola on the eight crop plants that were

tested. Isolates from the same lineages show similar but not identical profiles (Figure 2-1 and

Figure 2-6). For example, all but two isolates in PL3 and PL4 are pathogenic to tomato, and









isolates from all other lineages are nonpathogenic to tomato. All isolates pathogenic to basil are

from PL1 and PL3, but not all isolates in these clades are pathogenic to basil. Interestingly, the

majority of isolates, excluding the isolates collected from papaya, were pathogenic on cucumber.

Though there were no isolates collected from tomato that grouped in PL1 and PL2, isolates from

these lineages produced a hypersensitive response on tomato, showing pinpoint lesions that were

given a disease rating of one.

These data are similar to pathogenicity tests using 18 C. cassiicola isolates from Nigeria,

the Southern U.S., and Mexico (Onesirosan et al. 1973) in that both studies found isolates

specific to papaya and cucumber. Likewise, both studies found that isolates pathogenic to

tomato also were likely to be pathogenic on several other hosts. The number of isolates screened

compared to the number of unique pathogenicity profiles in both studies indicates that gains and

losses of pathogenicity are common.

Growth rate at different temperatures has provided evidence for isolates adapted to tropical

and temperate environments. Using an isolate collected from tomato in Florida, Pernezny et al.

(2000) found the best colony growth occurred at 32C, whereas Sobers (1966) reported an

optimum growth rate at 24C for Florida isolates collected from hydrangea and azalea. Jones and

Jones (1984) report higher disease severity on tomato inoculated and maintained at temperatures

between 20-23 C. In this study, two temperature extremes (23 C and 33 C) were chosen in

attempt to discern between isolates adapted to temperate and tropical climates. Though the

majority of isolates were collected from tropical climates, all isolates grew faster at 23 C than 33

C. Growth rate also strongly correlated with phylogenetic lineage. Isolates from PL2 and PL4

may be more adapted to warmer temperatures, and isolates from PL5 and PL3 might be more

adapted to cooler temperatures. Such physiological traits, including growth rate and









pathogenicity profile, correlate with phylogenetic data and may be useful for isolate

classification.

These studies have shown that the rDNA ITS sequence will be useful for the initial

screening of isolates and for isolate selection for resistance breeding. The rDNA ITS region was

useful for the grouping of isolates into three groups (PL1 (haplotype TG), PL4 (haplotype CA),

and PLs 2, 3, 5, and 6 (haplotype CG)) that correlate with phylogenetic data from the combined

four locus data set. For example, isolates from PL1 (rDNA ITS haplotype TG) should be used to

screen for resistance to target spot in papaya. In contrast, isolates from PL2 and PL4 (rDNA ITS

haplotypes CA and CG) should be used to screen for resistance to target spot in tomato. In

addition, genotyping by restriction digest of the amplified ITS region is possible now that

specific polymorphisms have been identified and mapped. For example, use of the enzyme

HpyCH4V (recognition sequence TGCA) will cut in two positions in haplotypes CA and CG, but

only one position in haplotype TG. Additionally, this research found isolates with the same

unique genotype found in Atan and Hamid's (2003) RFLP analysis of the rDNA ITS region

using HaeIII. However, only four of the 143 isolates we sequenced shared this polymorphism at

base pair 123, rendering RFLP analysis of the rDNA ITS region using HaelII ineffective for

distinguishing among the majority of isolates.

Despite evidence for host specificity (on African violet, Lantana, papaya, and

Stachytarpheta, for example), the combined pathogenicity and phylogenetic data indicate that

there are many hosts with the potential to harbor C. cassiicola isolates pathogenic to susceptible

crops such as basil, cucumber, and tomato. Studies that incorporate many isolates from the same

host across diverse locations, the sequencing of additional loci, and subsequent pathogenicity

screening, will no doubt reveal additional genetic diversity and host specificities.









It is hoped that this research will aid others in unraveling the many complexities that

remain to be discovered with respect to C. cassiicola and its disease development in the field.

For example, more studies are needed to explain why C. cassiicola is rare in Hawaii on all

cultivated crops except basil, if there are isolates adapted to tropical and temperate climates, and

how isolate genotype and pathogenicity profiles are correlated using more diverse isolates and

hosts.












Table 2-1. Isolate designations, geographic location of isolation, host of isolation, phylogenetic
lineage (PL), type of growth on associated host, and species of Corynespora used in
the phylogenetic analyses.


Isolate ID
CABI 211585
CBS 169.77
NIAS 425231
NIAS 712045
CBS 291.74
CBS 112393
NIAS 305095
CABI 5649b
AS49
AS50
AS54
AS58
AS65
AS67
AS71
AS78
AS80
AS81
AS92
AS98
AS117
AS119
DOA16b
JMP216a
JMP217
JMP218
RWB321
FL09
FL11
FL12
FL15
FL21
FL34
FL36
FL37
FL50
FL51
FL62
FL757
FL2920
MS31
TN3-3
GU01


Location
New Zealand
New Zealand
Japan
Japan
Netherlands
Italy
Japan
England
Amer. Samoa
Amer. Samoa
Amer. Samoa
Amer. Samoa
Amer. Samoa
Amer. Samoa
Amer. Samoa
Amer. Samoa
Amer. Samoa
Amer. Samoa
Amer. Samoa
Amer. Samoa
Amer. Samoa
Amer. Samoa
Brazil
Brazil
Brazil
Brazil
Brazil
FL, USA
FL, USA
FL, USA
FL, USA
FL, USA
FL, USA
FL, USA
FL, USA
FL, USA
FL, USA
FL, USA
FL, USA
FL, USA
MS, USA
TN, USA
Guam


Host
Poncirus trifoliatus
Poncirus trifoliatus
Ocimum basilicum
Solanum melongenea
Tilia spp.
Fagus sylvatica
Sesamum indicum
Fagus sylvatica
Solanum lycopersicum
Solanum lycopersicum
Vigna unguiculata
Vigna unguiculata
Solanum melongenea
Commelina benghalensis
Cucurbita pepo
Ocimum basilicum
Ocimum basilicum
Clerodendrum quadriloculare
Cucumis sativus
Cucumis sativus
Carica papaya fruit
Cucurbita pepo
Carica papaya
Lantana camera
Solanum lycopersicum
Glycine max
Coleus barbatus
Lantana camera
Carica papaya
Solanum lycopersicum
Salvia farinacea
Bauhinia galpinii
Tabebouia pallida
Catharanthus roseus
Clerodendrum paniculatum
Hydrangea macrophylla
Vaccinium corymbosum
Coleus barbatus
Origanum vulgare
Solanum lycopersicum
Solanum lycopersicum
Saintpaulia ionantha
Cassia fistula


Growth
endophytic
endophytic
pathogen
pathogenic
saprophyte
endophytic
pathogenic
saprophyte
pathogenic
pathogenic
saprophyte
saprophyte
saprophyte
pathogenic
saprophyte
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
saprophyte
saprophyte
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
saprophvte


Species
C. citricola
C. citricola
C. citricola
C. melongenae
C. olivacea
C. proliferata
C. sesamum
C. smithii
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola










Table 2-1. Continued.
Isolate ID Location
GU06 Guam
GU08 Guam
GU10 Guam
GU11 Guam
GU12 Guam
GU14 Guam
GU16 Guam
GU21 Guam
GU23 Guam
GU25 Guam
GU28 Guam
GU32 Guam
GU38 Guam
GU41 Guam
GU42 Guam
GU44 Guam
GU49 Guam
GU51 Guam
GU55 Guam
GU65 Guam
GU68 Guam
GU70 Guam
GU79 Guam
GU83 Guam
GU90 Guam
GU92 Guam
GU93 Guam
GU98 Guam
GU99 Guam
GU101 Guam
GU102 Guam
GU103 Guam
GU104 Guam
GU107 Guam
GU109 Guam
GU110 Guam
GU 11 Guam
GU112 Guam
GU 114 Guam
GU115 Guam
GU120 Guam
GU128 Guam
GU136 Guam
HI01 Oahu, Hawaii
CBPP Malaysia
CLN16 Malaysia


Host
Hyptus suarelens
Lantana camera
Codiaeum variegatum
Citrullus vulgaris
Calopogonium mucunoides
Calyptocarpus vialis
Asystasia gangeticai
Buddleja asiatica
Ipomoea batatas
Buchnera floridana
Solanum lycopersicum
Euphorbia heterophylla
Allamanda cathartica
Eugenia uniflora
Bidens alba
Jatropha curcas
S)-n*gin jambos
Meisosperma oppositifolium
Calopogonium mucunoides
Puc%,II ,a' foetida
Moringa oleifera
Solanum melongenea
Acanthus ilicifolius
Euphorbia heterophylla
Stachytarpheta jamaicensis
Carica papaya
Capsicum annum
Spathodea campanulata
Saintpaulia ionantha
Euphorbia milii
Phaseolus vulgaris
Pilea nummulariifolia
Macroptilium atropurpureum
Mikania micrantha
Bauhinia galpinii
Plectranthus ambionicus
Manihot esculenta
Glycine max
Teramnus labialis
Vitex ]pi, ,ijlI, ia
Coleus barbatus
Solanum lycopersicum
Ficus benjamani
Ocimum basilicum
Hevea brasiliensis clone unk.
Hevea brasiliensis RRIM 2020


Growth
endophytic
pathogenic
endophytic
saprophyte
pathogenic
pathogenic
pathogenic
pathogenic
endophytic
pathogenic
pathogenic
endophytic
pathogenic
endophytic
pathogenic
endophytic
endophytic
endophytic
pathogenic
endophytic
endophytic
endophytic
endophytic
endophytic
pathogenic
pathogenic
endophytic
pathogenic
pathogenic
saprophyte
saprophyte
endophytic
pathogenic
pathogenic
pathogenic
pathogenic
endophytic
endophytic
endophytic
pathogenic
pathogenic
pathogenic
endophytic
pathogenic
pathogenic
pathogenic


Species
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola










Table 2-1. Continued.
Isolate ID Location
CSB12 Malaysia
GU136 Guam
HI01 Oahu, Hawaii
CBPP Malaysia
CLN16 Malaysia
CSB12 Malaysia
PH01 Pohnpei
PW01 Palau
PW12 Palau
PW17 Palau
PW20 Palau
PW25 Palau
PW27 Palau
PW34 Palau
PW37 Palau
PW38 Palau
PW43 Palau
PW48 Palau
PW53 Palau
PW56 Palau
PW57 Palau
PW63 Palau
PW69 Palau
PW79 Palau
PW80 Palau
PW83 Palau
PW87 Palau
PW89 Palau
PW91 Palau
PW92 Palau
PW94 Palau
PW99 Palau
PW101 Palau
SN03 Saipan
SN05 Saipan
SN06 Saipan
SN07 Saipan
SN18 Saipan
SN24 Saipan
SN27 Saipan
SN30 Saipan
SN37 Saipan
SN40 Saipan
SN43 Saipan
SN48 Saipan
SN53 Saipan


Host PL Growth Species
Hevea brasiliensis RRIM 725 2 pathogenic C. cassiicola
Ficus benjamani 6.1 endophytic C. cassiicola
Ocimum basilicum 1 pathogenic C. cassiicola
Hevea brasiliensis clone unk. 1.2 pathogenic C. cassiicola
Hevea brasiliensis RRIM 2020 1.2 pathogenic C. cassiicola
Hevea brasiliensis RRIM 725 2 pathogenic C. cassiicola
Carica papaya 1.1 endophytic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Solanum lycopersicum 6 pathogenic C. cassiicola
Solanum lycopersicum 6 pathogenic C. cassiicola
Piper betle 2 endophytic C. cassiicola
Pilea microphylla 2 pathogenic C. cassiicola
Saintpaulia ionantha 1 pathogenic C. cassiicola
Saintpaulia ionantha 1 pathogenic C. cassiicola
Cucumis sativus 1 pathogenic C. cassiicola
Chromolaena odorata 6 endophytic C. cassiicola
Luffa acutangula 1 endophytic C. cassiicola
Catharanthus roseus 1 pathogenic C. cassiicola
Stachytarpheta jamaicensis 1 pathogenic C. cassiicola
Momordica charantia 3 pathogenic C. cassiicola
Vigna unguiculata 4 saprophyte C. cassiicola
Momordica charantia 1 pathogenic C. cassiicola
Ipomoea batatas 1 pathogenic C. cassiicola
Luffa acutangula 3.1 endophytic C. cassiicola
Carica papaya 1.1 endophytic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola
Solanum lycopersicum 6 pathogenic C. cassiicola
Solanum lycopersicum 6 pathogenic C. cassiicola
Solanum lycopersicum 6 pathogenic C. cassiicola
Vigna unguiculata 6 saprophyte C. cassiicola
Cucumis sativus 6 pathogenic C. cassiicola
Saintpaulia ionantha 3 pathogenic C. cassiicola
Coccinia grandis 3 endophytic C. cassiicola
Carica papaya 1.1 pathogenic C. cassiicola










Table 2-1. Continued.
Isolate ID Location
SN59 Saipan
SN64 Saipan
SN69 Saipan
YP01 Yap
YPO8 Yap
YP17 Yap
YP26 Yap
YP27 Yap
YP29 Yap
YP41 Yap
YP42 Yap
YP51 Yap
YP59 Yap


Host
Lantana camera
Asystasia gangetich
Pachystachys lutea
Carica papaya
Carica papaya
Carica papaya
Cucumis sativus
Cucumis sativus
Cucumis sativus
Saintpaulia ionantha
Solanum lycopersicum
Vigna unguiculata
Ipomoea batatas


Growth
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
pathogenic
saprophyte
endophytic


Species
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola
C. cassiicola


Information on Corynespora cassiicola isolates used in this study including location, original host,
phylogenetic lineage (PL), type of growth in association with the host (endophytic or pathogenic), and the
Corynespora species. The first eight isolates were solicited from culture collections as outgroups (0).
Three isolates from the NIAS culture collection (305095, 425231, and 712045) are likely misidentified
because they grouped with C. cassiicola isolates according to sequence data. They are labeled here
according to the original culture collection designations, though they should be re-classified as C.
cassiicola. The remaining isolates were collected as part of this study or solicited from other researchers
and are listed according to geographic location.











Table 2-2. Summary of sequence data from four loci used to confirm the phylogenetic lineage of
Corynespora cassiicola isolates.


Locus Total Variable Informative Tree Score No. MP Trees


Combined a
rDNA ITS
rDNA ITSb
Cc-ga4
Cc-g_-4
Cc-caa5
Cc-caa5b
Cc-act]
Cc-actlb


2136
1013
584
414
414
366
366
343
343


a Combined loci: rDNA ITS, Cc-ga4,


174
100


330
158


15 17
Cc-caa5, and Cc-actl.


7430
9990
1
9530
9560
40
12
11
4


b Locus analyzed with only C. cassiicola taxa represented (no outgroups).










Table 2-3. Pathogenicity profiles for 50 Corynespora cassiicola isolates.
Path Pro PL Isolate Host Ba Be Co Cu I


BaCoCu
BaCoCu
BaCoCu
BaCoCu
BaCoCuSo
BaCu
BaCu
BaCuTo
BaCuTo
BaCuTo
BaCuTo
BeCoCuSw
BeCuSwTo
BeCuSwTo
BeCuSwTo
BeCuTo
CoCu
CoCu
CoCu
CoCu
Cu
Cu
CuPa
CuPa
CuPa
CuSo
CuSw
CuSw
CuSw
CuSwTo
CuSwTo
CuSwTo


1 AS78
1 AS58
1 YP29
1 AS71
1 PW87
1 HI01
1 SN05
3 AS50
3 YP42
3.1 AS80
3.1 AS117
4 SN37
4 JMP217
4 GU102
4 SN40
4 PW57
1 AS98
1 YP26
1 JMP218
1 GU08
1 AS54
1 YP51
1.1 DOA16b
1.1 FL11
1.1 PH01
3 GU112
2 YP27
2 YP59
2 SN59
4 PW63
4 SN24
4 SN27


I S I
Ba 5 2.6 0
Co 6 2.3 0
Cu 7 2.1 0
Pu 8 2.1 0
Cu 5 2.2 0
Ba 4 2.5 0
Sw 7 1.9 1
To 5 2.2 0
To 6 2.3 0
Ba 5 1.8 0
Sap 7 1.9 0
Co 7 1 5
To 5 1 7
Be 0 7
Cu 0 6
To 0 6
Cu 0 5
Cu 0 7
So 0 0
La 0 0
Co 0 1
Co 0 0
Pa 7 1 0
Pa 0 5
Pa 1 1 1
So 0 0
Cu 1 1 0
Sw 2 1 0
La 1 1 5
To 4 1 0
To 0 0
To 5 1 0


S I S I S I
- 4 1.8 6 2.3 0
- 3 2.3 6 3 0
- 3 1.7 8 3 0
- 2 2 7 1.3 0
- 5 1.6 8 2.5 0
- 0 0 7 3 0
1 0 0 6 2.7 0
- 2 1 6 2.7 0
- 0 7 2.6 0
- 0 8 2.5 0
- 2 1 8 2.8 0
1.6 5 1.4 8 2.8 0
1.9 0 8 2.9 0
1.3 0 8 2.6 0
1.3 0 8 2.5 0
1.7 0 8 2.1 0
1 6 1.8 8 2.6 0
1 4 1.5 7 2.4 0
- 7 2.1 8 3 0
- 3 1.7 7 3 0
1 0 8 3 0
0 8 3 0
0 8 1.1 8
1 0 8 1.3 7
1 0 7 1.1 6
5 1 8 2.8 0
2 1 6 2.8 0
1 1 7 2.6 0
1 0 8 2.8 0
7 1 8 2.6 0
0 8 2.5 0
0 8 3 0


Pa So
S I S I
0 1
0 0
0 0
1 1 0
2 1.5 0
0 1
0 0
1 1 2
0 0
1 1 0
2 1 8
2 1 3
0 1
0 2
0 1
2 1 0
0 0
0 8
0 7
0 0
1 1 8
0 7
2.3 0 8
2.6 0 0
1.8 0 0
1 2 1
0 5
0 6
0 7
0 1
0 2
0 5


Sw To
S I S
1 2 1
7 1
0
2 1
7 1
1 8 1
2 1
1 8 3
8 2.6
8 2.8
1 7 2.9
1.3 8 1
2 8 2.5
1.5 8 2.5
2 8 2.6
8 3
7 1
1 8 1
1 7 1
8 1
1 1 1
1 1 1
1 1 1
8 1
8 1
1 2 1
1.4 2 1
1.4 7 1
1.3 7 1
2 8 3
1.5 8 2.6
1.2 8 2.9











Path Pro PL Isolate

CuSwTo 4 GU23
CuSwTo 4 FL09
CuTo 3 AS49
CuTo 3.1 AS119
CuTo 4 FL12
CuTo 4 FL2920
CuTo 4 MS31
CuTo 4 GU128
CuTo 4 SN30
CuTo 4 AS92
CuTo 4 JMP216a
CuTo 5 PW101
Pa 1.1 GU92
Pa 1.1 PW01
Pa 1.1 PW12
Pa 1.1 SN03
Pa 1.1 YP01
To 4 GU28


Host Ba Be Co Cu Pa
I S I S I S I S I S
Sw 0 0 7 1 8 2.4 0 -
La 0 1 1 0 8 1.4 0 -
To 0 0 0 4 2.8 0 -
Pu 0 0 0 8 2.3 0 -
To 0 5 1 5 1 8 3 0 -
To 4 1 0 0 8 2 0 -
To 0 7 1 8 1 8 2.8 0 -
To 0 1 1 0 8 2.9 0 -
To 0 0 0 8 3 0 -
Cu 2 1 1 1 0 8 2.8 0 -
La 7 1 0 0 7 1.3 0 -
Co 0 0 4 1 7 2.9 0 -
Pa 2 1 0 0 1 1 7 2.1
Pa 0 0 0 8 1 8 2.4
Pa 0 0 1 1 1 1 4 1.4
Pa 1 1 0 0 8 1 6 2.3
Pa 0 7 1 0 1 1 8 1.9
To 0 0 0 8 1 0 -


So
I S I
0 5
2 1 3
0 1
1 1 7
2 1 1
0 0
0 1
2 1 1
2 1 2
0 1
0 2
0 0
0 1
0 0
2 1 0
0 1
0 0
0 1


Sw

1[
1


To
S I S
.8 8 2.9
.3 8 2.8
1 7 2.6
1 8 2.5
1 8 3
8 2.6
1 8 2.3
1 8 2.5
1 8 3
1 8 2.9
1 8 3
7 1.3
1 7 1
8 1
1 1
1 1 1
8 1
1 8 3


Path Pro (Pathogenicity Profile): A list of susceptible hosts, or plants with an average disease rating greater than 1.
PL: Phylogenetic lineage designation based on combined sequence analysis of ITS rDNA, CAA5, GA4, and ACT.
Isolate: Corynespora cassiicola isolate code.
Host: Original host the isolate was collected from. Ba (Ocimum basilicum), Be (Phaseolus vulgarus), Co (Vigna unquiculata), Cu (Cucumis
sativus), La (Lantana camera), Pa (Carica papaya), Pu (Cucurbita pepo), Sw (Ipomoea batatas), To (Solanum lycopersicum).
I (Incidence): Number of plants (out of 8 reps) that showed symptoms seven days after inoculation with 20,000 C. cassiicola spores per ml.
S (Severity): Average rating of symptomatic plants (these rated 1, 2, or 3). Plants were rated with the following scale: (0) symptomless; (1) non
pathogenic hypersensitive response, a few to many non-expanding pinpoint lesions; (2) moderately virulent, many expanding lesions, some
coalescing, but not resulting in blight; (3) highly virulent, lesions spreading to form large areas of dead tissue resulting in a blighting effect.


Sw










Table 2-4. Growth rate of Corynespora cassiicola isolates at 230C.
Iso. ID PL Location Host Avg GR LSD
GU99 6 Guam Saintpaulia 0.4743 a
AS81 1 Samoa Clerodendron 0.4639 ab
GU90 1 Guam Stachytarpheta 0.4528 abc
HI01 1 Oahu Basil 0.4521 abcd
PW94 1 Palau Stachytarpheta 0.4521 abcd
FL37 1 Florida Clerodendron 0.4514 abcd
GU104 1 Guam Macroptilium 0.4507 abcde
AS67 1 Samoa Commelina 0.4479 bcde
AS54 1 Samoa Bean 0.4444 bcdef
GU08 1 Guam Lantana 0.4438 bcdef
AS71 1 Samoa Pumpkin 0.4410 bcdef
SN03 1 Saipan Bitter melon 0.4389 cdef
YP26 1 Yap Cucumber 0.4375 cdef
GU136 4 Guam Ficus 0.4375 cdef
PW80 1 Palau Saintpaulia 0.4375 cdef
SN05 1 Saipan SwPotato 0.4375 cdef
AS58 1 Samoa Bean 0.4368 cdef
YP29 1 Yap Cucumber 0.4361 cdef
YP51 1 Yap Bean 0.4326 cdef
SN37 4 Saipan Bean 0.4313 cdef
GUI15 1 Guam Vitex 0.4278 defg
PW92 1 Palau Catharanthus 0.4264 efg
AS78 1 Samoa Basil 0.4229 fgh
GU21 1 Guam Buddleja 0.4215 fgh
AS80 3 Samoa Basil 0.4202 fgh
PW91 1 Palau Luffa 0.4202 fgh
AS50 3 Samoa Tomato 0.4063 ghi
FL34 1 Florida Tabebouia 0.4055 ghi
YPO8 1 Yap Papaya 0.4000 hij
PW79 2 Palau Pilea 0.3951 ijk
SN59 1 Saipan Lantana 0.3951 ijk
RWB321 5 Brazil Coleus 0.3945 ijk
JMP218 1 Brazil Soybean 0.3924 ijkl
CSB12 2 Malaysia Rubber 0.3917 ijklm
SN06 3 Saipan Luffa 0.3903 ijklmn
PW37 1 Palau Papaya 0.3896 ijklmn
FL2920 4 Florida Tomato 0.3882 ijklmn
SN07 1 Saipan Papaya 0.3868 ijklmno
PW101 5 Palau Bean 0.3833 ijklmno
PW99 3 Palau Bitter melon 0.3833 ijklmno
SN64 5 Saipan Asystasia 0.3822 ijklmno
GU109 3 Guam Bauhinia 0.3820 ijklmno
AS98 1 Samoa Cucumber 0.3819 ijklmno
CLN16 1 Malaysia Rubber 0.3778 jklmnop
PW89 4 Palau Chromolaena 0.3778 jklmnop
YP59 2 Yap SwPotato 0.3778 iklmnop










Table 2-4. Continued.
Iso. ID PL Location Host Avg GR LSD
AS49 3 Samoa Tomato 0.3771 jklmnopq
GU107 4 Guam Mikania 0.3729 klmnopqr
CBPP 1 Malaysia Rubber 0.3695 Imnopqrs
AS119 3 Samoa Papaya 0.3687 Imnopqrst
YP42 3 Yap Tomato 0.3674 mnopqrstu
YP01 1 Yap Papaya 0.3667 nopqrstu
AS92 4 Samoa Cucumber 0.3632 opqrstu
GU112 3 Guam Bean 0.3632 opqrstu
FL12 4 Florida Tomato 0.3556 pqrstuv
GU98 4 Guam Spathodea 0.3549 pqrstuv
GU92 1 Guam Papaya 0.3529 qrstuvw
FL09 4 Florida Lantana 0.3521 rstuvw
YP17 1 Yap Papaya 0.3521 rstuvw
GU102 4 Guam Bean 0.3507 rstuvwx
GU38 2 Guam Allamanda 0.3507 rstuvwx
PW57 4 Palau Tomato 0.3500 rstuvwx
YP41 2 Yap Saintpaulia 0.3480 stuvwxy
AS117 3 Samoa Papaya 0.3458 stuvwxy
SN40 4 Saipan Cucumber 0.3444 tuvwxy
AS65 4 Saipan Eggplant 0.3437 uvwxy
PW01 1 Palau Papaya 0.3368 vwxyz
GU41 4 Guam Eugenia 0.3361 vwxyz
YP27 2 Yap Cucumber 0.3340 vwxyz
FL36 2 Florida Catharanthus 0.3312 vwxyz
JMP217 4 Brazil Tomato 0.3299 wxyz
SN24 4 Saipan Tomato 0.3264 xyz
DOA16b 1 Brazil Papaya 0.3250 yz
FL15 4 Florida Salvia 0.3146 z
GU120 4 Guam Coleus 0.2792 A
JMP216a 4 Brazil Lantana 0.2535 B
PH01 1 Pohnpei Papaya 0.1479 C
PL: Phylogenetic Lineage based on sequence data from 4 loci.
Avg GR: Average slope (growth rate) of three replicate plates.
LSD: Average slope (growth rate) values followed by different letters are significantly different
from one another according to least significant difference test (P<0.05).










Table 2-5. Growth rate of Corynespora cassiicola isolates at 330C.


Iso. ID
AS71
FL37
AS78
SN37
PW92
SN03
GU90
GU99
AS67
PW79
PW80
HI01
GU136
YP51
SN05
GU115
GU21
YP26
YP29
AS54
AS58
GU104
PW91
GU08
AS98
SN07
YP08
AS92
PW94
JMP218
GU98
PW89
YP17
PW37
GU107
PH01
DOA16b
GU102
PW01
GU92
YP01
PW57
GU41
AS81
SN40
PW101


PL Location
1 Samoa
1 Florida
1 Samoa
4 Saipan
1 Palau
1 Saipan
1 Guam
6 Guam
1 Samoa
2 Palau
1 Palau
1 Oahu
4 Guam
1 Yap
1 Saipan
1 Guam
1 Guam
1 Yap
1 Yap
1 Samoa
1 Samoa
1 Guam
1 Palau
1 Guam
1 Samoa
1 Saipan
1 Yap
4 Samoa
1 Palau
1 Brazil
4 Guam
4 Palau
1 Yap
1 Palau
4 Guam
1 Pohnpei
1 Brazil
4 Guam
1 Palau
1 Guam
1 Yap
4 Palau
4 Guam
1 Samoa
4 Saipan
5 Palau


Host
Pumpkin
Clerodendron
Basil
Bean
Catharanthus
Bitter melon
Stachytarpheta
Saintpaulia
Commelina
Pilea
Saintpaulia
Basil
Ficus
Bean
SwPotato
Vitex
Buddleja
Cucumber
Cucumber
Bean
Bean
Macroptilium
Luffa
Lantana
Cucumber
Papaya
Papaya
Cucumber
Stachytarpheta
Soybean
Spathodea
Chromolaena
Papaya
Papaya
Mikania
Papaya
Papaya
Bean
Papaya
Papaya
Papaya
Tomato
Eugenia
Clerodendron
Cucumber
Bean


Avg GR LSD
0.4153 a
0.3972 b
0.3965 b
0.3917 bc
0.3813 bcd
0.3799 bcd
0.3778 cd
0.3771 cde
0.3764 cde
0.3722 def
0.3715 defg
0.3680 defgh
0.3674 defghi
0.3653 defghi
0.3597 efghij
0.3577 fghij
0.3576 fghij
0.3569 fghij
0.3548 fghij
0.3542 ghijk
0.3542 ghijk
0.3542 ghijk
0.3514 hijkl
0.3500 ijklm
0.3451 jklmno
0.3368 klmno
0.3354 Imno
0.3327 mnop
0.3292 nopq
0.3285 nopq
0.3278 nopq
0.3278 nopq
0.3236 opqr
0.3224 opqr
0.3215 opqr
0.3174 pqrs
0.3132 qrst
0.3125 qrstu
0.3063 rstuv
0.3028 stuv
0.3014 stuv
0.2993 tuvw
0.2959 tuvwx
0.2952 uvwx
0.2951 uvwx
0.2951 uvwx










Table 2-5. Continued.
Iso. ID PL Location Host Avg GR LSD
YP41 2 Yap Saintpaulia 0.2903 vwxy
FL2920 4 Florida Tomato 0.2889 vwxy
SN64 5 Saipan Asystasia 0.2829 wxyz
CLN16 1 Malaysia Rubber 0.2792 xyz
CSB12 2 Malaysia Rubber 0.2771 yz
CBPP 1 Malaysia Rubber 0.2736 yz
YP59 2 Yap SwPotato 0.2688 zA
FL15 4 Florida Salvia 0.2686 zA
YP27 2 Yap Cucumber 0.2667 zA
FL36 2 Florida Catharanthus 0.2521 AB
PW99 3 Palau Bitter melon 0.2486 BC
GU109 3 Guam Bauhinia 0.2438 BC
SN24 4 Saipan Tomato 0.2431 BC
FL12 4 Florida Tomato 0.2326 CD
AS80 3 Samoa Basil 0.2313 CD
GU38 2 Guam Allamanda 0.2312 CD
SN06 3 Saipan Luffa 0.2250 DE
RWB321 5 Brazil Coleus 0.2188 DE
FL09 4 Florida Lantana 0.2097 EF
AS65 4 Saipan Eggplant 0.2076 EF
AS50 3 Samoa Tomato 0.1993 FG
YP42 3 Yap Tomato 0.1938 GFH
SN59 1 Saipan Lantana 0.1875 GHI
FL34 1 Florida Tabebouia 0.1763 HIJ
JMP216a 4 Brazil Lantana 0.1750 IJ
GU112 3 Guam Bean 0.1709 IJK
GU120 4 Guam Coleus 0.1688 JKL
AS49 3 Samoa Tomato 0.1660 JKL
JMP217 4 Brazil Tomato 0.1549 KLM
AS117 3 Samoa Papaya 0.1521 LM
AS119 3 Samoa Papaya 0.1382 M
PL: Phylogenetic Lineage based on sequence data from 4 loci.
Avg GR: Average slope (growth rate) of three replicate plates.
LSD: Average slope (growth rate) values followed by different letters are significantly different
from one another according to least significant difference test (P<0.05).


















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Figure 2-1. Fifty percent majority rule consensus tree-phylogram from Bayesian inference
analysis of combined data from rDNA ITS, Cc-ga4, Cc-caa5, and Cc-act] sequences.
Numbers above branches indicate maximum parsimony bootstrap > 70% and
numbers below branches indicate posterior probability values > 0.90. Pathogenicity
profiles on eight crop plants: basil (Ba), bean (Be), cowpea (Co), cucumber (Cu),
papaya (Pa), soybean (So), sweet potato (Sw), and tomato (To), and phylogenetic
lineage (PL) are indicated.

















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Figure 2-2. Fifty percent majority rule consensus tree-phylogram from Bayesian inference

analysis of rDNA ITS locus. Numbers above branches indicate maximum parsimony

bootstrap > 7000 and numbers below branches indicate posterior probability values >

0.90. 100,000 maximum parsimony trees were a result of only 3 informative

characters within C. cassiicola. Phylogenetic lineages (PL) are indicated.















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analysis of the Cc-caa5 locus. Numbers above branches indicate maximum


parsimony bootstrap > 70% and numbers below branches indicate posterior

probability values > 0.90. Phylogenetic lineages (PL) are indicated.


















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Figure 2-5. Fifty percent majority rule consensus tree-phylogram from Bayesian inference

analysis of the Cc-act] locus. Numbers above branches indicate maximum

parsimony bootstrap > 70% and numbers below branches indicate posterior

probability values > 0.90. Phylogenetic lineages (PL) are indicated.






87


L_










UPGMA
SN30 PL4
JMP216a PL4
AS92 PL4
AS49 PL3
8 CuTo FL12 PL4
GU128 PL4
MS31 PL4
R0 FL2920 PL4
AS119 PL3.1
0 BeCuTo PW101 PL5
PW57 PL4
TO GU28 PL4
0 PW63 PL4
25 CuSwTo FL09 PL4
SN27 PL4
GU23 PL4
0 SN24 PL4
S41 BeCuS o JMP217 PL4
o41 BeCuSwTo Gu102PL4
GU102 PLA
SN40 PL4
37 CuSw YP27 PL2
SN59 PL2
0 YP59 PL2
0 0 11Cu AS54 PL1
I YP51 PL1
GUI 12 PL3
AS50 PL3
46 BaCuTo AS80 PL3.1
AS1l7 PL3.1
21 YP42 PL3
32 BaCuM HI01 PL1
..I SNO5 PL1
AS78 PL1
o BaCoCu AS71 PL1
0AS58 PL1
YP29 PL1
o BaCoCuSo PW87 PL1
YP26 PL1
38 CoCu GU08 PL1
JMP218 PLI
BeCoCuSw AS98 PLI
SN37 PL4
GU92 PL1.1
57 Pa PW12 PLI.1
PW01 PL1.1
YP01 PLI.1
52- SN03 PL1.1
DOA16b PL1
44 FLll PL1.1
PH01 PL1.1
0.01 changes
Figure 2-6. UPGMA dendrogram of 50 Corynespora cassiicola isolates based on pathogenicity
profiles on eight crop plants: basil (Ba), bean (Be), cowpea (Co), cucumber (Cu),
papaya (Pa), soybean (So), sweet potato (Sw), tomato (To). Isolates are labeled with
their phylogenetic lineage (PL) designation to demonstrate that isolates from the same
PL cluster together. Statistical support for nodes by 1,000 UPGMA Bootstrap
repetitions is indicated.

















A) 1 J


B) 1


C) 1 W









D) 2 3
Figure 2-7. Demonstration of the C. cassiicola disease rating system. Symptoms on A) basil, B)
bean, C) cowpea, and D) tomato plants seven days after inoculation with different
isolates of Corynespora cassiicola. Plants were rated with the following scale: (0)
symptomless; (1) non pathogenic hypersensitive response, a few to many non-
expanding pinpoint lesions; (2) moderately virulent, many expanding lesions, some
coalescing, but not resulting in blight; (3) highly virulent, lesions spreading to form
large areas of dead tissue resulting in a blighting effect.









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BIOGRAPHICAL SKETCH

I was born in Pompano Beach, FL to Janis Therrell and Kenneth Wayne Smith on October

15, 1977. I have an older sister, Allison, and two younger brothers, Scott and Reid. Our family

moved to Baltimore, MD when I was nine and I attended Baltimore Friends School, where my

father was head of the Middle School. Though I have always loved biology and gardening, my

interest in agriculture took off in high school when I attended Maine Coast Semester, a small

school for students in their junior year located on a coastal farm in Wiscasset, Maine.

I received my B. A. at Colorado College in 2000 with a major in Biology, while fostering

my interest in farming through summer jobs at nurseries, CSA's, and internships. My

sophomore year in college, I traveled abroad to East Africa through The School for Field Studies

where I learned the importance of economic value in conservation by focusing on wildlife

ranching, national parks, and medicinal plant use as case studies.

In 2002, I received my Masters Degree from West Virginia University in Plant Pathology

as part of the Organic Farm Project by studying the effect of intercropping on diseases caused by

Alternaria solani and Meloidogyne incognita. I then spent two years in Micronesia on the island

of Guam as a Research Assistant documenting pathogens of agronomically important weeds and

working in the diagnostic clinic. It was in Guam where I first became aware of Corynespora

cassiicola as an agent of disease. An opportunity presented itself to continue the work begun on

this pathogen at the University of Florida in the Fall of 2004. At the University of Florida, I

became well trained in Phylogenetics and this has become my specialty. I plan to continue

studying fungal systematics beginning in September, 2008 as a post-doc with the USDA in

Beltsville, MD.





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1 HOST RANGE, PHYLOGENETIC, AND PATHOGENIC DIVERSITY OF Corynespora cassiicola (Berk. & Curt.) Wei By LINLEY JOY SMITH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Linley Joy Smith

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3 To Peter, for making me laugh.

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4 ACKNOWLEDGMENTS Funding and support was m ade possible by the USDA Special Grant Program for Tropical and Subtropical Agriculture Research, the Univ ersity of Florida, IFAS, EREC, the Florida Tomato Committee, the University of Guam, Guam Cooperative Extens ion, and the USDA IPM 3-D and Hatch funds. I would like to thank Drs. Ken Pernezny, Pam Roberts, Jeffrey Rollins, and Jay Scott for their support while serving on my supervisory committee. I would also like to express appreciation to my major a dvisor, Dr. Lawrence Datnoff, for his commitment and help throughout the course of my Ph.D. I would especially like to thank Dr. Robert Schlub for his willingness to help in every step of the process and for his unwavering support, encouragement, and friendship. Special thanks to my helpful coworkers in Guam, especially Roger Brown and Lauren Gutierrez. Most importantly, my heartfelt appreciation goe s to my parents for their unconditional love and support. Finally, I thank my husband for en couraging me to pursue this opportunity, an ocean and continent away, for coming to Gainesville for me, and for keeping me smiling throughout.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........6 LIST OF FIGURES.........................................................................................................................7 ABSTRACT.....................................................................................................................................8 CHAP TER 1 INDEX OF PLANT HOSTS OF Corynespora cassiicola .....................................................10 Introduction................................................................................................................... ..........10 Methods..................................................................................................................................12 Literature Survey and Host Index.................................................................................... 12 Guam and Florida Surveys..............................................................................................13 Results.....................................................................................................................................14 Discussion...............................................................................................................................16 2 GENETIC AND PATHOGENIC DIVERSITY OF CORYNESPORA CASSIICOLA ...........48 Introduction................................................................................................................... ..........48 Methods..................................................................................................................................52 Collection and Solicitation of Fungal Isolates................................................................. 52 Primer Development for Ra ndom Hypervariable Loci................................................... 54 Fungal Cultures and Extraction of Genomic DNA......................................................... 55 Phylogenetic Analyses.....................................................................................................57 Pathogenicity Analyses...................................................................................................59 Growth Rate Analyses..................................................................................................... 60 Results.....................................................................................................................................61 Phylogenetic Analyses.....................................................................................................61 Pathogenicity Analyses...................................................................................................65 Growth Rate Analyses..................................................................................................... 66 Discussion...............................................................................................................................67 LIST OF REFERENCES...............................................................................................................90 BIOGRAPHICAL SKETCH.......................................................................................................102

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6 LIST OF TABLES Table page 1-1 Taxonomic grouping of Corynespora cassiicola host species. ......................................... 20 1-2 Occurrence and fungalhost interaction of Corynespora cassiicola identified during 2004-2005 Gua m and Florida surveys............................................................................... 21 2-1 Isolate designations, geog raphic location of isolation, hos t of isolation, phylogenetic lineage (PL), type of growth on a sso ciated host, and species of Corynespora used in the phylogenetic analyses.................................................................................................. 72 2-2 Summary of sequence data from four lo ci used to confirm the phylogenetic lineage of Corynespora cassiicola isolates....................................................................................76 2-3 Pathogenicity profiles for 50 Corynespora cassiicola isolates. .........................................77 2-4 Growth rate of Corynespora cassiicola isolates at 23C. .................................................. 79 2-5 Growth rate of Corynespora cassiicola isolates at 33C. .................................................. 81

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7 LIST OF FIGURES Figure page 1-1 Corynespora cassiicola isolate from Cucumis sativus ......................................................45 1-2 Various symptoms caused by Corynespora cassiicola on naturally infected leaves......... 46 2-1 Fifty percent majority rule consensu s tree-phylogram from Bayesian inference analysis of combined data from rDNA ITS, Cc-ga4, Cc-caa5, and Cc-act1 sequences...................................................................................................................... .....83 2-2 Fifty percent majority rule consensu s tree-phylogram from Bayesian inference analysis of rDNA ITS locus. ............................................................................................84 2-3 Fifty percent majority rule consensu s tree-phylogram from Bayesian inference analysis of the Cc-caa5 locus............................................................................................85 2-4 Fifty percent majority rule consensu s tree-phylogram from Bayesian inference analysis of the Cc-ga4 locus..............................................................................................86 2-5 Fifty percent majority rule consensu s tree-phylogram from Bayesian inference analysis of the Cc-act1 locus. .......................................................................................... 87 2-6 UPGMA dendrogram of 50 Corynespora cassiicola isolates based on pathogenicity profiles on eight crop plants:..............................................................................................88 2-7 Demonstration of the C. cassiicola disease rating system ................................................. 89

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8 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy HOST RANGE, PHYLOGENETIC, AND PATHOGENIC DIVERSITY OF Corynespora cassiicola (Berk. & Curt). Wei By Linley Joy Smith August 2008 Chair: Lawrence E. Datnoff Major: Plant Pathology The fungus Corynespora cassiicola (Berk. & Curt.) Wei is a pathogen, endophyte, and saprophyte. It can be found growing on at least 5 30 plant species from 380 genera, primarily in the tropics. Isolates from dive rse hosts were collected or solicited from locations in American Samoa, Brazil, Malaysia, Micronesia, and Florid a, Mississippi, and Tennessee within the United States. Outgroup taxa including C. citricola C melongenea C. olivaceae, C. proliferata C. sesamum and C. smithii were solicited from culture co llections. A multilocus phylogenetic analysis using 143 isolates was performed to investigate how genetic dive rsity correlates with host-specificity, growth rate, a nd geographic distribution. Phyl ogenetic trees were congruent from the rDNA ITS region, two random hypervariable loci ( Cs caa5 and Cs ga4), and the actin encoding locus CC act1, indicating asexual propagation. Fifty isolates had different pathogenicity profiles when tested against eight known C. cassiicola hosts: basil, bean, cowpea, cucumber, papaya, soybean, sweet potato, and toma to. Phylogenetic lineage correlated with pathogenicity profiles, host originality, and growth rate, but not with geographic location. Common fungal genotypes were widely distributed geographically indicating long distance and global dispersal of clonal lineages. This re search reveals an abundance of previously

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9 unrecognized diversity within the species and provides evid ence for redefining species distinctions within Corynespora, which will aid in future disease control strategies.

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10 CHAPTER 1 INDEX OF PLANT HOSTS OF CORYNESP ORA CASSIICOLA Introduction Corynespora cassiicola (Berk. & Curt.) W ei has been commonly reported as a plant pathogenic foliar fungus with a wide host range w ithin tropical and subtropical areas (Holliday 1980; Farr et al 1980; Romruensukharom et al 2005). In addition to being a pathogen, on some hosts C. cassiicola is also reported to grow as an endophyte or saprophyte (Collado 1999; Gond et al 2007; Promputtha et al. 2007; Suryanarayanan et al. 2002; Kingsland 1985; Hyde et al. 2001; Lee et al. 2004; Lumyong et al. 2003). Though the diseases attributed to C. cassiicola are mainly foliar, it may also cause fruit, stem and root diseases (J ones et al. 1991). The generalization that individual C. cassiicola isolates have a wide host range is not supported by the literature because hos t specific isolates, isolates pathogenic to select hosts, and weak pathogens or secondary invaders of senescent ti ssue are known to exist (Onesirosan et al. 1974; Cutrim and Silva et al. 2003; Kingsland 1985; Pere ira et al. 2003). Rarely reported outside the tropics and subtropics, there ar e occasional reports of the f ungus from temperate regions, particularly on soybean (Boosalis and Hamilton 1957; Malvick 2004; Raffel et al. 1999; Seaman et al. 1965). Disease symptoms attributed to C. cassiicola include necrosis, of ten with a surrounding yellow halo (Pernezny and Simone 1993) due to the production of a host specific protein toxin, cassiicolin (Barthe et al. 2007; Kurt 2004). With respect to foliage, young and mature leaves can be affected, although the pathogen is more commonly associated w ith older leaves (Pernezny et al. 2008). Substantial crop losses have been observed in many countries on numerous hosts: southern United States on ornamentals (Alfieri et al. 1984, 1994; Chase 1981,1982, 1984, 1986, 1987, 1993; El-Gholl and Schubert 1990; El-Gho ll et al. 1997; Miller and Alfieri 1973;

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11 McRitchie and Miller 1973; Simone 2000, 2000), cucumber (Abul-Hayja et al. 1978; Blazquez 1967; Strandburg 1971), and tomato (Bliss et al. 1973; Blazquez 1972; Jones and Jones 1984; Pernezny et al. 1996, 2002; Smith et al. 2006, Smith et al. 2008b); Midwestern United States on soybean (Boosalis and Hamilton 1957), cowpea (Olive and Bain 1945) and sesame (Stone and Jones 1960); India on ornamentals (Cheeran 1968; Mallaiah et al. 1981 ; Mehrotra 1987, 1997; Silva et al. 2000; Singh et al. 1982), Hevea rubber trees (Atan and Hamid 2003; Silva et al. 1998), cotton (Lakshmanan et al. 1990), and weed s (Philip et al. 1972); Brazil on ornamentals (Da Silva et al. 2005; Leite and Barreto 2000; Pohltronieri 2003) and weeds (Pereira et al. 2003); Philippines, Nigeria, and U.S. Virgin Is lands on papaya (Quimio and Abilay 1979; Oluma and Amuta 1999; Bird et al. 1966); and Microne sia and Asia on ornamentals (Florence and Sharma 1987; Hasama et al. 1991), cucurbit s (Yudin and Schlub 1998; Tsay and Kuo 1991), tomato (Schlub and Yudin 2002), and pepper (Kwon et al. 2001). Most regions report C. cassiicola diseases on only a few host species, despite the broad host range of the fungus, prompting questions pertaining to isolate host specificity and distribution. Addressing such questions will have implications for disease control and quarantine. The host -specificity and severity of the fungus on Lantana camara in Brazil led to the discovery of a new forma specialis, C. cassiicola f. sp. lantanae, and the use of the isolate as a bioherbicide (Pereira et al. 2003 ). Based on the vast number of weeds that serve as hosts, and past demonstration of host-specificity in some is olates, there is great pot ential for the discovery of additional isolates useful for biological control. Further information on the fungal-host interaction and host range of individual isolates will be useful in the st udy of disease epidemics. The objective of this study was to compile a list of C. cassiicola hosts into a single document, thereby aiding further research on the hos t range of individual isol ates. Prior to this

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12 study, the most complete host list ing is in the fungal database of the ARS/USDA Systematic Mycology and Microbiology Labor atory, which included 257 plan t host species (Farr et al. 2008). This study will provide a more complete index for use by those engaged in phylogenetic analysis of Corynespora spp. and in disease management. Awareness of the potential host range of the fungal species is vital to the determination of the host-speci ficity of individual isolates. The host range of individual isolates has direct implications for disease management, including the identification of potential inoculum sources, recomme ndations for intercropping and crop rotation, weed management, biological control candidacy, and isolate choice for resistance breeding. In order to obtain an estimate of the comp leteness of the list of hosts known to harbor C. cassiicola surveys were conducted to identify hosts in Guam and Florida. Guam is an ideal location to discover new hosts due to its tropi cal climate, wet and dry seasons, and lack heretofore of a Corynespora host survey (Schlub and Yudin 2002). Florida was included because outbreaks of target spot on tomato caused by C. cassiicola are common and it represents a subtropical environment located an ocean and a continent away from Guam. Methods Literature Survey and Host Index An index of plant hosts of C. cassiico la was compiled from a search of world literature for any reference regarding its presen ce on plant tissue. All plant-f ungus associations were included such as pathogenic, endophytic, and saprophytic. Resources included ar ticles in refereed journals, graduate student theses, books, and web-based resources such as annual reports, production guides, and plant clinic lists. The final list of susceptible hosts of C. cassiicola was compiled from the literature and personal observation from surveys in Florida and Guam.

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13 All plant species, genera, and families were named and classified according to the USDA Germplasm Resources Information Network (GRIN) taxonomy, which follows the APGII system. In some cases, the host name given in th e original citation was ch anged to be consistent with GRIN taxonomy. In a few cases, neither the species cited nor a proper synonym was identified using GRIN taxonomy and the species na me was kept as originally cited. Only one reference was provided per host, with emphasis on citing the first known report of that host. For some hosts, the only reference that could be found was a website, and in those cases the website is listed. The number of plan t host species was conservative ly determined by counting only unique species within each genus. Ge nera with unidentified species (e.g. Crossandra spp.) were counted only once when no other named speci es were present within that genus. Guam and Florida Surveys Surveys for the pres ence of C. cassiicola were conducted throughout Guam and Florida. The Guam survey was conducted for one year beginning in January of 2004 and the Florida survey was conducted for one year beginning in January of 2005. Survey areas focused on roadsides, nurseries, and farms. During the c ourse of the survey, leaves from plants with characteristic C. cassiicola foliage disease symptoms were co llected and placed in individual plastic bags. Known hosts of C. cassiicola were sampled more intensely through the additional collection of old and young asymptomatic leaves. An effort was made to sample from an equal number of individual plants and unique plant species in Florida and Guam. To induce sporulation, leaf tissu e was placed abaxial side up in the moisture chamber for 10 days. Moisture chambers were created on the lab bench by placing 10 ml of sterilized distilled water on a paper towel in a 150 mm petri plate. A plant species was identified as a host of C. cassiicola if characteristic structures of the fungus developed within 10 days. An isolate was labeled a pathogen if coni diophores arose from a necrot ic spot and an endophyte if

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14 conidiophores arose from healthy, green tissue. Petri plates were inspected under a dissecting microscope daily for spores and conidiophores of C. cassiicola. Structures were confirmed based on microscopic morphological features su ch as percurrent proliferation of the conidiophores and pseudoseptation. Single spore isolates were obt ained for long-term storage by needle transfer of spores to antibiotic V8 agar agar slants (340 ml V8 juice, 660 ml water, 3 g CaCO3, 17 g agar, 100 g/ml ampicillin or kanamycin). Slants were left at room temperature until colonies reached at least 5 cm in diameter, covered with autoclaved mineral oil, and stored at 5o C until further study. Results Over 900 individual plants were surveyed in both Gua m and Florida from 320 unique plant species in Guam and 289 unique plant species in Florida. Compilation of Corynespora cassiicola hosts from the literature and surveys conducted in Guam and Florida resulted in an index of 530 plant species from 380 genera. The majority of index host species for C cassiicola are herbaceous Eudicotyledonae, but 52 Monocot yledonae, eight Magnoliids, five Filicopsida (ferns), and one cycad are also represented. No hosts were found within the Anthocerotophyta (hornworts), Bryophyta (mosses), Equisetops ida (horsetails, s phenophytes), Lycopsida (lycophytes), or Marchantiomorpha (liverworts) (Table 1-1). Hosts were found in two plant divisions: Filicopsida and Spermatopsida. The five hosts in the Filicopsida include Arachniodes aristata ( Davalliaceae ), Athyrium niponicum ( Dryopteridaceae ), Adiantum cuneatum ( Pteridaceae ), Davallia repens ( Davalliaceae ), and Platycerium spp. ( Pteridaceae ). The plant division Spermatophyta ( Cycadales, Magnoliidae, Monocotolydonae, and Tricolpates) contains 99% of the host spec ies (Table 1-1). There are eight species from the Magnoliidae. Three species are from the Piperaceae ( Piper betle P.

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15 hispidinervum and Perperomia obtusifolia). Three species are from the Magnoliales in the family Annonaceae ( Annona reticulata A. squamosa and Asimina triloba ). Two species are from the Laurales in the Hernandiaceae ( Hernandia ovigera) and the Lauraceae ( Ocotea leucoxylon ) (Table 1-2). The 52 host species from the Monocotolydonae are from 16 families: Araceae (13 species), Poaceae (9 species), Arecaceae (7 species), Dioscoreaceae (5 species, all from the genus Dioscorea), Orchidaceae (4 species), Agavaceae (3 species), Musaceae (2 species), Alismataceae (1 species), Asparagaceae (1 species), Bromeliaceae (1 species), Commelinaceae (1 species), Heliconiaceae (1 species), Hemerocallidaceae (1 species), Marantaceae (1 species), Restonaceae (1 species), and Strelitziaceae (1 species), in decreasing order of host species numbers. The remaining 464 host species are Eudicots. Families that contain the largest number of hosts include Fabaceae (70 species), Lamiaceae (33 species), Malvaceae (32 species), Asteraceae (26 species), Apocynaceae (21 species), Acanthaceae (20 species), Euphorbiaceae (20 species), Verbenaceae (17 species), Convolvulaceae (14 species), Cucurbitaceae (13 species), and Solanaceae (13 species), in decreasing order of host species numbers. Between the two surveys, 91 new hosts specie s were identified, 87 of which were found in the survey conducted on Guam. New hosts were found in 32 families, of which three families had never been reported to harbor the fungus: Hernandiaceae, Moringaceae and Mutingiaceae. Ten new host species were found to harbor the fungus in the survey conducted in F lorida ( Cerinthe major Corchorus aestuans Fatshedera lizei Hibiscus rosa-sinensis Jatropha spp., Salvia farinacea Salvia microphylla Salcia officinalis Sida spinosa, and Stachytarpheta

PAGE 16

16 jamaicensis). Six new hosts were found in both Guam and Florida ( Corchorus aestuans, Salvia farinacea S. microphylla S. officinalis Sida spinosa, and Stachytarpheta jamaicensis ). From the Guam and Florida surveys, C. cassiicola was more often identified as a pathogen than as an endophyte on 191 and 121 plant species, respectively. On 48 hosts, the fungus was identified as both a pathogen and an endophyte. Endophytic isolates of C. cassiicola were most likely recovered from young leaves and pa thogenic isolates from older leaves. Discussion The index produced here contains 530 C. cassiico la host plant species. Four hundred thirty nine species were identified from the literature and 91 new species were identified from the field surveys conducted in Guam and Florida. The number of new hosts found to harbor the fungus in Guam was 87 and in Florida was 10, with six new hosts found in both Guam and Florida. This suggests that there are many add itional host species remaining to be discovered. Although most of the literature on C. cassiicola relates to the diseases it causes, in this study the fungus was often isolated from asymptom atic tissue, indicative of endophytic growth. There are likely many additional endophytic hosts that remain to be discovered considering only healthy leaves from previous ly reported hosts were sampled. The extent to which C. cassiicola was occurring as an endophyte was not appreciated prior to this su rvey. During the course of the Guam survey, C. cassiicola often sporulated from healthy tissue when placed in a moisture chamber instead of necrotic tissue. In these cases, C. cassiicola was likely not the cause of the necrosis because other fungi were ofte n found to sporulate in those areas. There seems to be no clear demarcation as to the presence of C. cassiicola on a particular host and its ability to grow endophytically or pathogenically. Publications on C. cassiicola are usually restricted to a description of symptoms on a particular host or as part of a list of fungi from an endophyte study. Kochs postulates are ra rely completed, and when they are, often the

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17 fungus is not pathogenic on the host it was is olated from without wounding (Kingsland 1985; Pernezny et al. 1996). This study recorded 48 cas es from the Guam and Florida surveys where plants were found to harbor pathogenic isolates of C. cassiicola and in other locations harbor endophytic isolates. It may be that the fungus has the ability to delay symptoms by growing initially as an endophyte. Pathogenic isolates were often found on older leaves indicating that endophytic isolates may become pathogens as the hos t tissue ages or begins senescence. Despite the symptomless nature of an e ndophytic relationship with the host, it is likely that the potential exists for the fungus to switch to an opportunist ic pathogen and/or a saprophyte on the same host because individual hosts were found to har bor both pathogenic and endophytic isolates. The likelihood of finding the fungus as an e ndophyte or as a pathogen may depend on the plant family. In this study, plant families more likely found harboring the fungus growing as an endophyte were Araceae Bignoniaceae, Convolvulaceae Crassulaceae Elaeocarpaceae Hernandaceae Magnoliaceae, Meliaceae, and Moraceae. Magnolia liliifera ( Magnoliaceae ) was recently reported as hosting a Corynespora spp. endophyte with ribosomal DNA (ITS15.8S-ITS2) sequence homology to C. cassiicola (Promputtha et al. 2007) and was therefore included in our list. In the Guam survey, Hernandia sp. ( Magnoliaceae ) was also found to support endophytic growth of C. cassiicola. Families that were likely to support pathogenic growth of the fungus in these surveys were Acanthaceae, Amaranthaceae Apocynaceae, Asteraceae Begoniaceae, Boragniaceae, Gesnariaceae, Lamiaceae and Verbenaceae. Throughout the survey, it was diffi cult to determine whether the Corynespora species observed were in fact C. cassiicola At least one hundred an d thirteen species of Corynespora are currently described, but a m onograph is needed, including mol ecular analyses, in order to assess the validity of these species (Sivanesan 1996). Most species have been named according

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18 to host identity, and only a few species have been described in culture. In addition, single isolates exhibit considerable morphological plasticity that depends on humidity, light, temperature, and substrate; therefore, morphol ogical differences need to be compared with molecular differences. Although the hosts included in this index are restri cted to those reported for C. cassiicola some may actually be hosts of other Corynespora species due to misidentification. Likewise, there may be hosts reported to harbor other species of Corynespora that may, in fact, be harboring C. cassiicola because the morphological distinctions between species are based on overlapping, variable, morphol ogical characters. Phylogenetic analyses of the isolates should help to clarify these issues. Despite these complications, this is the first step taken to consolid ate our knowledge of the potential host range of C. cassiicola, which is vital for further stud ies of the biology of individual isolates and ultimately in future studies of Corynespora species evolution. Although there is no teleomorphic stage currently known for C. cassiicola the Ascomycete species Corynesporasca caryote and Pleomassaria swidae have unknown Corynespora species anamorphs (Sivanesan 1996; Tanaka et al. 2008). There is no evidence to suggest that C. cassiicola is reproducing other than by asexual spores. However, eviden ce for sexual recombination needs to be tested between isolates within and among host species. Insight into the evolut ionary potential of the fungus will lead to a better understanding of how to control its diseases (McDonald 2004). The literature search and surveys elucidated several characteristics of C. cassiicola that warrant further investigation: (1) the inability of some isolates recovered from symptomatic tissue to re-infect the original hosts; (2) the ability to be endophytic, path ogenic, and saprophytic on individual hosts; (3) the wide host range of th e fungal species, yet restricted host ranges of individual isolates; (4) the ability to grow on some members of a plant taxonomic group and not

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19 others; (5) a lack of unde rstanding of the diversity within th e fungal species and how it relates to host range; (6) the taxonomic va lidity of the 113 species of Corynespora considering the high morphological plasticity of indivi dual isolates. Future research should attempt to address these issues and the organization of the plant hosts in a single publication will facilitate this.

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20 Table 1-1. Taxonomic grouping of Corynespora cassiicola host species. Plant Group Number of Host Species in the Index Number of Host Species Sampled in Guam Number of Host Species Sampled in Florida Anthocerotphyta (hornworts) 0 2 3 Bryophyta (mosses) 0 5 2 Filicopsida (ferns) 5 14 21 Spermatopsida (seed plants) 525 299 263 Conifers 0 3 6 Cycads 1 4 5 Gnetales 0 2 1 Angiosperms 524 290 251 Magnoliids 8 6 4 Monocotyledons 52 61 38 Eudicots 464 223 209

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21Table 1-2. Occurrence and f ungal-host interaction of Corynespora cassiicola identified during 2004-2005 Guam and Florida surveys. Host Fungal-Host Interaction LocationReference Acanthaceae Juss. (dicot) Acanthus ilicifolius L. endophytic GU Sadaba et al. 1995 Aphelandra squarrosa Nees pathogenic FL, GU Chase 1982 Asystasia spp. Blume Alfieri et al. 1984 Asystasia gangetica (L.) T. Anders. pathogenic GU Alfieri et al. 1984 Crossandra spp. Salisb. pathogenic FL Alfieri et al. 1994 Eranthemum pulchellum Andrews pathogenic FL Alfieri et al. 1994 Fittonia spp. Coem. pathogenic FL Chase 1982 Fittonia albivenis (Lindl. ex hort. Veitch) Brummitt endophytic, pathogenic FL, GU Chase 1982 Hygrophila spp. R. Br. FL Alfieri et al. 1994 Justicia spp. L. Ellis 1957 Justicia brandegeeana Wasshausen & L.B. Sm. pathogenic FL, GU Alfieri et al. 1994 Justicia carnea Lindl. pathogenic GU Ellis 1957 Justicia ventricosa Wall. ex Hook. Zhuang 2001 Meisosperma oppositifolium endophytic GU Smith et al. 2007 Pachystachys coccinea (Aubl.) Nees Urtiaga 1986 Pachystachys lutea Nees pathogenic FL, GU Alfieri et al. 1994 Peristrophe spp. Nees Alfieri et al. 1994 Pseuderanthemum spp. Radlk. El-Gholl et al. 1997 Pseuderanthemum carruthersii (Seem.) Guillaumin pathogenic GU El-Gholl et al. 1997 Ruellia humboldtiana (Nees) Lindau endophytic, pathogenic FL Urtiaga 2004 Strobilanthes dyerianus M.T. Mast. pathogenic GU Coile and Dixon 1994 Thunbergia fragrans Roxb. Zhuang 2001 Warpuria clandestina Stapf. pathogenic GU Ellis 1957 Actinidiaceae Gilg & Werderm. (dicot) Actinidia chinensis Planch. Peregrine and Ahmad 1982 Adoxaceae E. Mey. (dicot) Viburnum spp. L. Alfieri et al. 1994 Viburnum odoratissimum Ker Gawl. endophytic FL, GU Alfieri et al. 1994

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22Table 1-2. Continued Host Fungal-Host Interaction LocationReference Agavaceae Dumort. (monocot) Agave sisalana Perrine Ellis 1957 Cordyline fruticosa (L.) Chev. endophytic GU Situmorang and Budimen 1984 Dracaena spp. Vand. ex L. Alfieri et al. 1984 Dracaena reflexa Lam. endophytic, pathogenic FL Alfieri et al. 1994 Alismataceae Vent. (monocot) Echinodorus spp. Rich. ex Engelm. Alfieri et al. 1994 Amaranthaceae Juss. (dicot) Achyranthes aspera L. CABI, Herb. IMI 191361 Alternanthera ficoidea (L.) P. Beauv. pathogenic GU first report Amaranthus spp. L. Alfieri et al. 1994 Amaranthus spinosus L. pathogenic FL, GU Alfieri et al. 1994 Amaranthus tricolor L. Peregrine and Ahmad 1982 Celosia argentea L. var. cristata (L.) Kuntze pathogenic GU first report Digera muricata (L.) Mart. Sarma and Nayudu 1970 Anacardiaceae R. Br. (dicot) Lannea coromandelica (Houtt.) Merr. CABI, Herb. IMI 266196 Mangifera indica L. Rajak and Pandey 1985 Schinus spp. L. endophytic, pathogenic FL Alfieri et al. 1984 Spondias purpurea L. pathogenic FL Freire 2005 Vernicia montana Lour. endophytic FL Ellis 1957 Annonaceae Juss. (dicot) Annona reticulata L. Peregrine and Ahmad 1982 Annona squamosa L. endophytic GU first report Asimina triloba (L.) Dunal CABI, Herb. IMI 364250 Apiaceae Lindl. (dicot) Foeniculum vulgare Mill. Peregrine and Ahmad 1982

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23Table 1-2. Continued Host Fungal-Host Interaction LocationReference Apocynaceae Juss. (dicot) Adenium obesum (Forssk.) Roem. & Schult. El-Gholl 1997 Allamanda spp. L. endophytic FL Alfieri et al. 1984 Allamanda cathartica L. pathogenic GU Alfieri et al. 1994 Alstonia scholaris (L.) R. Br. endophytic, pathogenic FL Suryanarayanan et al. 2002 Calotropis procera (Aiton) W. T. Aiton CABI, Herb. IMI 173980 Carissa spp. L. pathogenic FL Alfieri et al. 1994 Catharanthus roseus (L.) G. Don pathogenic FL, GU McGovern 1994 Conopharyngia longiflora (Benth.) Stapf Kranz 1963 Cryptolepis buchananii Schult. CABI, Herb. IMI 221003 Funastrum clausum (Jacq.) Schltr. Urtiaga 2004 Hoya spp. R. Br. pathogenic FL Alfieri et al. 1994 Mandevilla spp. Lindl. Alfieri et al. 1984 Mandevilla splendens (Hook. f.) Woodson pathogenic FL, GU Alfieri et al. 1994 Nerium oleander L. pathogenic FL Alfieri et al. 1994 Plumeria rubra L. forma acutifolia (Poir.) Woodson endophytic, pathogenic GU Ellis 1957 Rauvolfia serpentina (L.) Benth. ex Kurz CABI, Herb. IMI 122395 Tabernaemontana divaricata (L.) R. Br. ex Roem. & Schult. CABI, Herb. IMI 209321 Tabernaemontana sananho Ruiz & Pav. Urtiaga 2004 Tacazzea spp. Decne. Ellis 1957 Telosma cordata (Burm. f.) Merr. endophytic, pathogenic GU first report Thevetia peruviana (Pers.) K. Schum. CABI, Herb. IMI 231448 Trachelospermum jasminoides (Lindl.) Lem. pathogenic FL Alfieri et al. 1984 Vinca spp. L. Alfieri et al. 1994 Aquifoliaceae Bercht. & J. Presl (dicot) Ilex vomitoria Sol. ex Aiton endophytic FL Alfieri et al. 1994 Araceae Juss. (monocot) Aglaonema spp. Schott pathogenic FL Alfieri et al. 1994 Alocasia macrorrhizos (L.) G. Don endophytic, pathogenic GU Mercado et al. 1997 Amorphophallus paeoniifolius (Dennst.) Nicolson Puzari and Saikia 1981

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24Table 1-2. Continued Host Fungal-Host Interaction LocationReference Anthurium spp. Schott pathogenic Alfieri et al. 1994 Anthurium andraeanum Linden ex Andr pathogenic GU Alfieri et al. 1994 Anubias afzelii Schott El-Gholl 1997 Caladium bicolor (Aiton) Vent. endophytic, pathogenic GU first report Colocasia esculenta (L.) Schott endophytic GU Onesirosan et al. 1974 Dieffenbachia spp. Schott endophytic FL Alfieri et al. 1994 Epipremnum pinnatum (L.) Engl. pathogenic FL Alfieri et al. 1984 Philodendron bipinnatifidum Schott ex Endl. endophytic GU first report Syngonium podophyllum Schott pathogenic GU Coile and Dixon 1994 Xanthosoma sagittifolium (L.) Schott endophytic GU Ellis 1957 Zantedeschia spp. Spreng. Raabe et al. 1981 Zantedeschia aethiopica (L.) Spreng. Raabe et al. 1981 Araliaceae Juss. (dicot) Fatshedera spp. Guillaumin Alfieri et al. 1984 Fatshedera lizei (hort. ex Cochet) Guillaumin endophytic FL first report Polyscias balfouriana L.H.Bailey Alfieri et al. 1984 Polyscias fruticosa (L.) Harms pathogenic FL Alfieri et al. 1994 Polyscias scutellaria (Burm. f.) Fosberg pathogenic GU first report Arecaceae Bercht. & J. Presl (monocot) Attalea butyracea (Mutis ex L. f.) Wess. Boer Urtiaga 2004 Calyptronoma plumeriana (Mart.) Lourteig Delgado-Rodriguez and Mena-Portales 2004 Cocos nucifera L. CABI, Herb. IMI 317357 Dypsis lutescens (H. Wendl.) Beentje & J. Dransf. endophytic, pathogenic FL Alfieri et al. 1994 Elaeis guineensis Jacq. Ellis 1957 Licuala ramsayi (Mueler) Domin. Shivas and Alcorn 1996 Rhopalostylis sapida H. Wendl and Drude McKenzie et al. 2004 Asparagaceae Juss. (monocot) Asparagus officinalis L. Urtiaga 2004 Asteraceae Bercht. & J. Presl (dicot) Ageratum conyzoides L. pathogenic GU Smith and Schlub 2004

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25Table 1-2. Continued Host Fungal-Host Interaction LocationReference Aspilia africana (Pers.) C. D. Adams pathogenic Onesirosan et al. 1974 Bidens spp. L. Alfieri et al. 1984 Bidens alba (L.) DC. pathogenic FL, GU Alfieri et al. 1984 Calyptocarpus vialis Less. pathogenic GU Smith and Schlub 2004 Chromolaena odorata (L.) R. M. King & H. Rob. pathogenic GU CABI, Herb. IMI 147913 Chrysanthemum spp. L. endophytic FL Turner 1971 Chrysanthemum indicum L. Peregrine and Ahmad 1982 Elephantopus mollis Kunth endophytic GU first report Elephantopus scaber L. CABI, Herb. IMI 199985 Elephantopus tomentosus L. Zhuang 2001 Emilia sonchifolia (L.) DC pathogenic GU McKenzie 1990 Gaillardia aristata Pursh pathogenic GU Ellis 1957 Lactuca sativa L. pathogenic GU Ellis 1957 Liatris spp. Gaertn. ex Schreb. endophytic, pathogenic FL Alfieri et al. 1994 Melanthera biflora (L.) Wild Ellis 1957 Mikania micrantha Kunth pathogenic GU Smith et al. 2007 Pseudelephantopus spicatus (B. Juss. ex Aubl.) C. F. Baker endophytic GU first report Pseudogynoxys chenopodioides (Kunth) Cabrera endophytic, pathogenic FL Alfieri et al. 1994 Sphagneticola trilobata (L.) Pruski endophytic GU Alfieri et al. 1994 Symphyotrichum novi-belgii (L.) G. L. Nesom Dixon 1997 Synedrella nodiflora (L.) Gaertn. pathogenic GU Onesirosan et al. 1974 Tithonia rotundifolia (Mill.) S. F. Blake Wei 1950 Tridax procumbens L. pathogenic GU first report Verbesina turbacensis Kunth Urtiaga 2004 Vernonia cinerea (L.) Less. pathogenic GU Cutrim and Silva 2003 Zinnia violacea Cav. Urtiaga 2004 Balsaminaceae A. Rich. (dicot) Impatiens balsamina L. pathogenic GU Wei, 1950 Impatiens noli-tangere L. pathogenic FL CABI, Herb. IMI 124564 Impatiens sultanii Hook. f. Urtiaga 2004

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26Table 1-2. Continued Host Fungal-Host Interaction LocationReference Impatiens walleriana Hook. f. Alfieri et al. 1994 Begoniaceae C. Agardh (dicot) Begonia spp. L. Chase 1982 Begonia coccinea Hook. pathogenic GU Chase 1982 Begonia cucullata Willd. pathogenic GU first report Bignoniaceae Juss. (dicot) Bignonia spp. L. Orieux and Felix 1968 Crescentia cujete L. pathogenic FL Alfieri et al. 1994 Handroanthus serratifolius (Vahl) S. Grose Mendes et al. 1998 Newbouldia laevis (P. Beauv.) Seem. ex Bureau endophytic, pathogenic GU Ellis 1957 Radermachera sinica (Hance) Hemsl. endophytic FL Alfieri et al. 1994 Radermachera xylocarpa (Roxb.) K. Schum. endophytic FL Suryanarayanan et al. 2002 Stereospermum colais (Buch.-Ham. ex Dillwyn) Mabb. endophytic FL Murali et al. 2007 Tabebuia spp. Gomes ex DC. Mendes et al. 1998 Tabebuia aurea (Silva Manso) Benth. & Hook. f. ex S. Moore pathogenic FL Alfieri et al. 1984 Tabebuia heterophylla (DC.) Britton endophytic GU Alfieri et al. 1994 Tabebuia pallida (Lindl.) Miers pathogenic FL Alfieri et al. 1994 Tabebuia odontodiscus (Bureau & K. Schum.) Toledo Mendes et al. 1998 Tecoma capensis (Thunb.) Lindl. Urtiaga 2004 Boraginaceae Juss. (dicot) Cerinthe major L. pathogenic FL first report Cordia collococca L. Urtiaga 2004 Cordia curassavica (Jacq.) Roem. & Schult. Urtiaga 2004 Cordia obliqua Willd. Murali et al. 2007 Cordia wallichii G. Don. Murali et al. 2007 Cordia subcordata Lam. pathogenic GU first report Tournefortia argentea L. f. pathogenic GU first report Brassicaceae Burnett (dicot) Brassica rapa L. Peregrine and Ahmad 1982

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27Table 1-2. Continued Host Fungal-Host Interaction LocationReference Bromeliaceae Juss. (monocot) Ananas comosus (L.) Merr. Blazquez 1968 Burseraceae Kunth (dicot) Bursera simaruba (L.) Sarg. endophytic, pathogenic FL Alfieri et al. 1994 Canarium album (Lour.) Raeusch. Zhang and Ji 2005 Cannabaceae Martinov (dicot) Trema micrantha (L.) Blume Arnold 1986 Trema orientalis (L.) Blume CABI, Herb. IMI 256125 Capparaceae Juss. (dicot) Capparis spp. L. CABI, Herb. IMI 259297 Caprifoliaceae Juss. (dicot) Lonicera japonica Thunb. endophytic FL Alfieri et al. 1984 Lonicera sempervirens L. Alfieri et al. 1994 Caricaceae Dumort. (dicot) Carica papaya L. pathogenic FL, GU Beaver 1981 Vasconcellea cauliflora (Jacq.) A. DC. Urtiaga 2004 Vasconcellea pubescens A. DC. Johnston 1960 Celastraceae R. (dicot) Celastrus paniculatus Willd. CABI, Herb. IMI 302698 Elaeodendron glaucum (Rottb.) Pers. Murali et al. 2007 Euonymus spp. L. Alfieri et al. 1994 Salacia senegalensis (Lam.) DC. Ellis 1957 Combretaceae R. Br. (dicot) Anogeissus latifolia (Roxb. ex DC.) Wall. ex Guill. & Perr. Suryanarayanan et al. 2002 Terminalia arjuna (Roxb. ex DC.) Wight & Arn. CABI, Herb. IMI 302839 Terminalia catappa L. endophytic GU first report Terminalia crenulata Roth. Murali et al. 2007 Terminalia elliptica Willd. Suryanarayanan et al. 2002

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28Table 1-2. Continued Host Fungal-Host Interaction LocationReference Commelinaceae Mirb. (monocot) Commelina benghalensis L. pathogenic GU Cutrim and Silva 2003 Convolvulaceae Juss. (dicot) Evolvulus glomeratus Nees & Mart. endophytic GU Alfieri et al.1994 Ipomoea alba L. endophytic, pathogenic GU McKenzie 1990 Ipomoea aquatica Forssk. endophytic GU McKenzie 1990 Ipomoea batatas (L.) Lam. endophytic, pathogenic FL, GU Silva et al. 2003 Ipomoea indica (Burm.) Merr. endophytic, pathogenic GU first report Ipomoea littoralis (L.) Blume endophytic, pathogenic GU first report Ipomoea obscura (L.) Ker Gawl. endophytic, pathogenic GU Smith and Schlub 2004 Ipomoea pes-caprae (L.) R. Br. endophytic GU Hawaiian Ecosys tems at Risk (HEAR) 2008 Ipomoea triloba L. endophytic, pathogenic GU Smith and Schlub 2004 Lepistemon spp. Blume Onesirosan et al. 1974 Merremia aegyptia (L.) Urb. endophytic, pathogenic GU first report Merremia peltata (L.) Merr. endophytic, pathogenic GU first report Operculina turpethum (L.) Silva Manso GU first report Stictocardia tiliifolia (Desr.) Hallier f. endophytic GU first report Cornaceae Bercht. & J. Presl (dicot) Alangium chinense (Lour.) Harms Guo 1992 Cornus florida L. Alfieri et al. 1994 Crassulaceae J. St.-Hil. (dicot) Crassula ovata (Mill.) Druce Alfieri et al. 1994 Kalanchoe spp. Adans. endophytic FL Alfieri et al. 1994 Kalanchoe pinnata (Lam.) Pers. endophytic GU first report Kalanchoe thyrsiflora Harv. endophytic, pathogenic GU first report Sedum spp. L. Chase 1982 Cucurbitaceae Juss. (dicot) Citrullus lanatus (Thunb.) Matsum. & Nakai pathogenic GU Sobers 1966 Coccinia grandis (L.) Voigt endophytic, pathogenic GU Philip et al. 1972 Cucumis anguria L. endophytic GU Cutrim and Silva 2003

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29Table 1-2. Continued Host Fungal-Host Interaction LocationReference Cucumis melo L. endophytic, pathogenic GU Ellis and Holliday 1971 Cucumis sativus L. pathogenic FL, GU Wei 1950 Cucurbita spp. L. Grand 1985 Cucurbita maxima Duchesne Williams and Liu 1976 Cucurbita moschata Duchesne Minter et al. 2001 Cucurbita pepo L. pathogenic GU Cutrim and Silva 2003 Lagenaria siceraria (Molina) Standl. endophytic, pathogenic GU Ellis 1957 Luffa acutangula (L.) Roxb. endophytic, pathogenic GU Onesirosan et al. 1974 Luffa aegyptiaca Mill. Onesirosan et al. 1974 Momordica charantia L. pathogenic GU Alfieri et al. 1994 Sechium edule (Jacq.) Sw. endophytic, pathogenic FL, GU Alfieri et al. 1984 Davalliaceae M. R. Schomb. (dicot) Arachniodes aristata (G. Forst.) Tindale endophytic, pathogenic GU Anderson and Dixon 2004 Davallia spp. Sm. Alfieri et al. 1994 Davallia repens (L. f.) Kuhn pathogenic GU Alfieri et al. 1994 Dioscoreaceae R. Br. (monocot) Dioscorea alata L. CABI, IMI 229871 Dioscorea bulbifera L. endophytic, pathogenic GU Onesirosan et al. 1974 Dioscorea cayenensis Lam. CABI IMI 83832 Dioscorea esculenta (Lour.) Burkill endophytic, pathogenic GU Onesirosan et al. 1974 Dioscorea pentaphylla L. Peregrine and Ahmad 1982 Dryopteridaceae Herter (fern) Athyrium niponicum (Mett.) Hance endophytic GU El-Gholl 1997 Ebenaceae Grke (dicot) Diospyros montana Roxb. Murali et al. 2007 Elaeocarpaceae Juss. ex DC. (dicot) Elaeocarpus joga Merr. endophytic GU first report Elaeocarpus tuberculatus Roxb. Suryanarayanan et al. 2002 Muntingia calabura L. endophytic GU first report

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30Table 1-2. Continued Host Fungal-Host Interaction LocationReference Ericaceae Juss. (dicot) Oxydendrum arboreum (L.) DC. Alfieri et al. 1994 Rhododendron spp. L. Alfieri et al. 1984 Rhododendron canescens (Michx.) Sweet Rhododendron obtusum (Lindl.) Planch. endophytic, pathogenic FL E llis and Holliday 1971 Vaccinium corymbosum L. pathogenic FL Hongn et al. 2007 Erythroxylaceae Kunth (dicot) Erythroxylum monogynum Roxb. Murali et al. 2007 Euphorbiaceae Juss. (dicot) Acalypha macrostachya Jacq. Urtiaga 2004 Bridelia ferruginea Benth. Ellis 1957 Chamaesyce hirta (L.) Millsp. pathogenic GU first report Codiaeum variegatum (L.) A. Juss. endophytic, pathogenic GU CABI, IMI 179212 Cnidoscolus aconitifolius (Mill.) I. M. Johnst. Peregrine and Ahmad 1982 Croton bonplandianus Baill. endophytic, pathogenic FL, GU Sarma and Nayudu 1970 Croton fragrans Kunth. Urtiaga 2004 Drypetes alba Poit. Mercado 1984 Euphorbia spp. L. Ellis 1957 Euphorbia cyathophora Murray endophytic, pathogenic GU Barreto and Evans 1998 Euphorbia pulcherrima Willd. ex Klotzsch pathogenic FL Chase 1986 Euphorbia milii Des Moulins pathogenic GU Smith et al. 2007 Givotia rottleriformis Griff. Murali et al. 2007 Hevea brasiliensis (Willd. ex A. Juss.) Mll. Arg. Silva et al. 1995 Hura crepitans L. Urtiaga 1986 Jatropha spp. L. pathogenic FL first report Jatropha gossypiifolia L. pathogenic GU Smith et al. 2007 Manihot spp. Mill. Malvick 2004 Manihot carthagenensis (Jacq.) Mll. Arg. Onesirosan et al. 1974 Manihot esculenta Crantz endophytic, pathogenic GU Ellis 1957

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31Table 1-2. Continued Host Fungal-Host Interaction LocationReference Phyllanthus amarus Schumach. & Thonn. endophytic, pathogenic GU Mathiyazhagan et al. 2004 Phyllanthus emblica L. Prakash and Garg 2007 Tragia spp. L. Ellis 1957 Fabaceae Lindl. (dicot) Acacia spp. Mill. Situmorang and Budimen 1984 Acacia auriculiformis A. Cunn. ex Benth. endophytic, pathogenic GU first report Afzelia africana Sm. ex Pers. Dade 1940 Albizia lebbeck (L.) Benth. endophytic GU first report Albizia zygia (DC.) J. F. Macbr. Ellis 1957 Alysicarpus vaginalis (L.) DC. endophytic GU first report Arachis hypogaea L. Vyas et al. 1985 Bauhinia spp. L. Alfieri et al. 1994 Bauhinia galpinii N. E. Br. pathogenic GU Smith and Schlub 2004 Bauhinia purpurea L. pathogenic FL, GU Ellis 1957 Bauhinia racemosa Lam. Suryanarayanan et al. 2002 Butea monosperma (Lam.) Taub. Murali et al. 2007 Caesalpinia granadillo Pittier Urtiaga 2004 Cajanus cajan (L.) Millsp. Lenn 1990 Calopogonium mucunoides Desv. pathogenic GU Onesirosan et al. 1974 Cassia fistula L. endophytic GU Suryanarayanan et al. 2002 Clitoria ternatea L. pathogenic GU first report Crotalaria goreensis Guill. & Perr. Hyde and Alcorn 1993 Crotalaria juncea L. GU Wei 1950 Crotalaria micans Link Shaw 1984 Crotalaria pallida Aiton Turner 1971 Crotalaria retusa L. endophytic, pathogenic GU first report Crotalaria spectabilis Roth Malvick 2004 Cyamopsis tetragonoloba (L.) Taub. Spencer 1962 Dalbergia spp. L. f. Ellis 1957

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32Table 1-2. Continued Host Fungal-Host Interaction LocationReference Dalbergia latifolia Roxb. endophytic Suryanarayanan et al. 2002 Dalbergia lanceolaria L. f. Murali et al. 2007 Delonix regia (Bojer ex Hook.) Raf. CABI, Herb. IMI 314022 Desmodium spp. Desv. Lenn, 1990 Desmodium incanum DC. pathogenic GU Smith and Schlub 2004 Desmodium tortuosum (Sw.) DC. pathogenic GU Smith and Schlub 2004 Desmodium triflorum (L.) DC. pathogenic GU Smith and Schlub 2004 Erythrina spp. L. Delgado-Rodriguez et al. 2002 Gliricidia sepium (Jacq.) Kunth ex Walp. Boa and Lenn 1994 Glycine max (L.) Merr. pathogenic FL, GU Olive et al. 1945 Glycine soja Siebold & Zucc. Lenn 1990 Hymenaea courbaril L. Urtiaga 2004 Lens culinaris Medik. Khare 1991 Lupinus albus L. Sobers 1966 Lupinus angustifolius L. Sobers 1966 Lupinus luteus L. Sobers 1966 Lupinus pilosus L. Malvick 2004 Macrolobium spp. Schreb. Kranz 1963 Macroptilium atropurpureum (Moc. & Sess ex DC.) Urban pathogenic GU first report Macroptilium lathyroides (L.) Urban pathogenic GU Smith and Schlub 2004 Mimosa diplotricha C. Wright Silva 1995 Mimosa pudica L. endophytic, pathogenic GU Smith and Schlub 2004 Mucuna pruriens (L.) DC. Sobers 1966 Phaseolus lunatus L. Malvick 2004 Phaseolus vulgaris L. pathogenic GU Wei 1950 Pisum sativum L. pathogenic GU first report Pithecellobium dulce (Roxb.) Benth. pathogenic GU first report Psophocarpus tetragonolobus (L.) DC. Ellis 1957 Pterocarpus indicus Willd. Situmorang and Budimen 1984

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33Table 1-2. Continued Host Fungal-Host Interaction LocationReference Pueraria montana (Lour.) Merr. Peregrine and Ahmad 1982 Ricinus communis L. Spencer and Walters 1968 Saraca indica L. CABI, Herb. IMI 210811 Senna alata (L.) Roxb. Wei 1950 Senna occidentalis (L.) Link pathogenic GU first report Senna surattensis (Burm. f.) H. S. Irwin & Barneby pathogenic GU first report Senna tora (L.) Roxb. Situmorang and Budimen 1984 Sesamum indicum L. endophytic GU Wei 1950 Spathodea campanulata P. Beauv. pathogenic GU Smith et al. 2007 Teramnus labialis (L. f.) Spreng. pathogenic GU Smith et al. 2007 Trifolium repens L. Cho and Shin 2004 Trigonella foenum-graecum L. Komaraiah and Reddy 1986 Tylosema esculentum (Burch.) A. Schreib. Alfieri et al. 1994 Vicia spp. L. Alfieri et al. 1984 Vigna mungo (L.) Hepper Gowda et al. 2001 Vigna radiata (L.) R. Wilczek Malvick 2004 Vigna unguiculata (L.) Walp. subsp. sesquipedalis (L.) Verdc. pathogenic GU Seaman et al. 1965 Vigna umbellata (Thunb.) Ohwi & H. Ohashi Peregrine and Ahmad 1982 Wisteria sinensis (Sims) DC. endophytic FL Alfieri et al. 1984 Fagaceae Dumort. (dicot) Quercus ilex L. Collado et al. 1999 Gesneriaceae Rich. & Juss. (dicot) Aeschynanthus longicaulis Wall. ex R. Br. Chase 1982 Aeschynanthus radicans Jack pathogenic GU Chase 1982 Columnea spp. L. Chase 1982 Episcia cupreata (Hook.) Hanst. pathogenic FL Alfieri et al. 1994 Gloxinia perennis (L.) Fritsch Brooks 2002 Nematanthus spp. Schrad. Chase 1982 Saintpaulia ionantha H. Wendl. pathogenic GU Smith et al. 2007

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34Table 1-2. Continued Host Fungal-Host Interaction LocationReference Sinningia speciosa (Lodd. et al.) Hiern pathogenic FL Alfieri et al. 1994 Streptocarpus spp. Lindl. Alfieri et al. 1994 Streptocarpus rexii (Bowie ex Hook.) Lindl. pathogenic FL, GU Alfieri et al. 1994 Heliconiaceae Nakai (monocot) Heliconia caribaea Lam. Urtiaga 2004 Hemerocallidaceae R. Br. (monocot) Hemerocallis spp. L. Peregrine and Ahmad 1982 Hernandiaceae Blume (dicot) Hernandia spp. L. endophytic GU first report Hernandia ovigera L. endophytic GU first report Hydrangeaceae Dumort. (dicot) Hydrangea spp. L. Alfieri et al. 1984 Hydrangea macrophylla (Thunb.) Ser. pathogenic FL Sobers 1966 Lamiaceae Martinov (dicot) Ajuga spp. L. Alfieri et al. 1984 Ajuga reptans L. pathogenic FL Alfieri et al. 1984 Anisochilus carnosus (L. f.) Wall. ex Benth. CABI, Herb. IMI 151008 Coleus barbatus (Andrews) Benth. pathogenic FL, GU Fernandes and Barreto 2003 Congea tomentosa Roxb. Peregrine and Ahmad 1982 Clerodendrum inerme (L.) Gaertn. Ahmad 1969 Clerodendrum infortunatum L. CABI, Herb. IMI 112265 Clerodendrum speciosissimum Van Geert ex C. Morren Urtiaga 1986 Hyptis suaveolens (L.) Poit. endophytic GU Smith et al. 2007 Leucas aspera (Willd.) Link Sarma and Nayudu 1970 Mentha arvensis L. endophytic, pathogenic GU Cheeran 1968 Mentha piperita L. Williams and Liu 1976 Moluccella spp. L. Alfieri et al. 1984 Moluccella laevis L. Alfieri et al. 1984 Monarda punctata L. Alfieri et al. 1994

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35Table 1-2. Continued Host Fungal-Host Interaction LocationReference Ocimum basilicum L. endophytic, pathogenic GU Taba et al. 2002 Ocimum tenuiflorum L. Sarma and Nayudu 1970 Origanum vulgare L. pathogenic FL, GU Perilla frutescens (L.) Britton pathogenic GU Hasama et al. 1991 Plectranthus amboinicus (Lour.) Spreng. pathogenic GU Miller 1991 Plectranthus barbatus Andrews Smith et al. 2007 Plectranthus parviflorus Willd. pathogenic FL Alfieri et al. 1994 Premna serratifolia L. pathogenic GU first report Premna tomentosa Willd. Murali et al. 2007 Rosmarinus officinalis L. Alfieri et al. 1994 Salvia spp. L. Peregrine and Ahmad 1982 Salvia farinacea Benth. pathogenic FL, GU first report Salvia leucantha Cav. pathogenic FL Riley 1960 Salvia microphylla Kunth pathogenic FL, GU first report Salvia officinalis L. pathogenic FL, GU first report Salvia splendens Sellow ex Schult. pathogenic FL Chase 1982 Solenostemon scutellarioides (L.) Codd pathogenic FL, GU Alfieri et al. 1994 Stachys floridana Shuttlew. ex Benth. Alfieri et al. 1994 Thymus vulgaris L. pathogenic FL Silva 1995 Tectona grandis L. f. Murali et al. 2007 Teucrium canadense L. El-Gholl 1997 Lauraceae Juss. (dicot) Ocotea leucoxylon (Sw.) Laness. Delgado-Rodriguez et al. 2002 Lecythidaceae A. Rich. (dicot) Careya arborea Roxb. Murali et al. 2007 Lecythis ollaria Loefl. Urtiaga 2004 Loganiaceae R. Br. ex Mart. (dicot) Buddleja asiatica Lour. pathogenic GU Smith and Schlub 2004 Strychnos potatorum L. f. Murali et al. 2007

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36Table 1-2. Continued Host Fungal-Host Interaction LocationReference Lythraceae J. St.-Hil. (dicot) Lagerstroemia indica L. pathogenic FL Alfieri et al. 1994 Lagerstroemia microcarpa Wight Murali et al. 2007 Lagerstroemia parviflora Roxb. Murali et al. 2007 Pemphis acidula Forst. & Forst. endophytic GU first report Magnoliaceae Juss. (dicot) Magnolia champaca (L.) Baill. ex Pierre CABI, Herb. IMI 254407 Magnolia liliifera (L.) Baill. endophytic FL Promputtha et al. 2007 Malpighiaceae Juss. (dicot) Malpighia glabra L. Poltronieri et al. 2003 Malvaceae Juss. (dicot) Abelmoschus esculentus (L.) Moench pathogenic GU Wei 1950 Abutilon theophrasti Medik. endophytic, pathogenic GU Spencer and Walters 1969 Ceiba pentandra (L.) Gaertn. endophytic GU Mehrotra 1989 Ceiba speciosa (A. St.-Hil.) Ravenna Ferreira 1989 Corchorus aestuans L. pathogenic FL, GU Smith and Schlub 2004 Corchorus capsularis L. pathogenic GU Wei 1950 Corchorus olitorius L. endophytic, pathogenic GU Ellis 1957 Desplatsia spp. Bocq. Ellis 1957 Durio zibethinus L. Williams and Liu 1976 Gossypium barbadense L. endophytic, pathogenic GU Jones 1961 Gossypium hirsutum L. Jones 1961 Grewia tiliifolia Vahl Suryanarayanan et al. 2002 Helicteres isora L. Murali et al. 2007 Hibiscus spp. L. Urtiaga 2004 Hibiscus cannabinus L. Shaw 1984 Hibiscus mutabilis L Kwon and Park 2003 Hibiscus rosa-sinensis L. endophytic FL first report Hibiscus sabdariffa L. endophytic GU Wei 1950

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37Table 1-2. Continued Host Fungal-Host Interaction LocationReference Kydia calycina Roxb. CABI, Herb. IMI 264454 Pavonia spp. Cav. Urtiaga 2004 Pseudobombax septenatum (Jacq.) Dugand Urtiaga 2004 Sida acuta Burm. f. pathogenic GU Smith and Schlub 2004 Sida glomerata Cav. Urtiaga 2004 Sida rhombifolia L. pathogenic GU CABI, Herb. IMI 180198 Sida spinosa L. pathogenic FL, GU first report Sida urens L. Ellis 1957 Sterculia apetala (Jacq.) H. Karst. Urtiaga 2004 Talipariti tiliaceum (L.) Fryxell endophytic GU first report Theobroma cacao L. Duarte et al. 1978 Thespesia populnea (L.) Soland. ex Correa endophytic pathogenic GU first report Triumfetta rhomboidea Jacq. endophytic GU Onesirosan et al. 1974 Waltheria indica L. endophytic GU CABI, Herb. IMI 123575 Urena lobata L. pathogenic GU first report Marantaceae R. Br. (monocot) Maranta leuconeura E. Morren pathogenic FL Alfieri et al. 1994 Marcgraviaceae Bercht. & J. Presl (dicot) Norantea guianensis Aubl. endophytic GU Wei 1950 Meliaceae Juss. (dicot) Chukrasia velutina M. Roem. endophytic GU first report Guarea guidonia (L.) Sleumer Urtiaga 2004 Melia azedarach L. endophytic GU first report Moraceae Gaudich. (dicot) Artocarpus altilis (Parkinson) Fosberg CABI, Herb IMI 351978 Broussonetia spp. L'Hr. ex Vent. Pollack and Stevenson 1973 Broussonetia papyrifera (L.) L'Hr. ex Vent. endophytic GU Alfieri et al. 1994 Ficus spp. L. Ellis 1957 Ficus benjamina L. endophytic GU Chase 1984

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38Table 1-2. Continued Host Fungal-Host Interaction LocationReference Ficus elastica Roxb. ex Hornem. endophytic GU Chase 1987 Ficus exasperata Vahl Onesirosan et al. 1974 Ficus hispida L. f. CABI, Herb IMI 311137 Ficus lyrata Warb. endophytic FL Alfieri et al. 1994 Ficus racemosa L. Gilson 2002 Ficus religiosa L. CABI, Herb. IMI 217075 Moringaceae Martinov (dicot) Moringa oleifera Lam. endophytic GU Smith et al. 2007 Muntingiaceae C. Bayer et al. (dicot) Muntingia calabura L. pathogenic GU first report Musaceae Juss. (monocot) Musa sapientum L. Blazquez 1968 Musa acuminata Colla Lumyong et al. 2003 Myrsinaceae R. Br. (dicot) Ardisia foetida Willd. Urtiaga 2004 Myrtaceae Juss. (dicot) Eucalyptus spp. L'Hr. Eucalyptus grandis W. Hill ex Maiden C.M.I. No. 303 Eucalyptus tereticornis Sm. Vittal and Dorai 1994 Eugenia uniflora L. pathogenic GU CABI, Herb IMI 99533 Psidium guajava L. Alfieri et al. 1984 Syzygium aromaticum (L.) Merr. & L. M. Perry Saikia and Sarbhoy 1981 Syzygium cumini (L.) Skeels pathogenic GU Sarbhoy et al. 1971 Syzygium jambos (L.) Alston pathogenic GU Smith et al. 2007 Nyctaginaceae Juss. (dicot) Bougainvillea spectabilis Willd. endophytic GU first report Mirabilis jalapa L. CABI, Herb IMI 259283 Nymphaeaceae Salisb. (dicot) Nymphaea ampla (Salisb.) DC. Urtiaga 2004

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39Table 1-2. Continued Host Fungal-Host Interaction LocationReference Nyssaceae Juss. ex Dumort. (dicot) Nyssa spp. L. Alfieri et al. 1994 Oleaceae Hoffmanns. & Link (dicot) Chionanthus retusus Lindl. & Paxton Alfieri et al. 1994 Jasminum spp. L. Alfieri et al. 1984 Jasminum laurifolium Roxb. forma nitidum (Skan) P. S. Green Alfieri et al. 1994 Jasminum multiflorum (Burm. f.) Andrews Alfieri et al. 1994 Jasminum sambac (L.) Aiton CABI Herb. IMI 111858 Jasminum simplicifolium G. Forst. pathogenic FL Alfieri et al. 1994 Ligustrum lucidum W. T. Aiton Alfieri et al. 1994 Ligustrum japonicum Thunb. Alfieri et al. 1994 Ligustrum sinense Lour. endophytic GU Alfieri et al. 1994 Orchidaceae Juss. (monocot) Cattleya spp. Lindl. Simone 2000 Dendrobium spp. Sw. Alfieri et al. 1994 Phalaenopsis spp. Blume Alfieri et al. 1994 Vanilla planifolia Andrews Urtiaga 2004 Passifloraceae Juss. ex Roussel (dicot) Passiflora spp. L. Pernezny and Simone 1993 Passiflora edulis Sims endophytic FL Alfieri et al. 1994 Passiflora foetida L. pathogenic GU Smith et al. 2007 Passiflora suberosa L. endophytic GU first report Pedaliaceae R. Br. (dicot) Josephinia imperatricis Vent. Hyde and Alcorn 1993 Martynia annua L. CABI, Herb IMI 264260 Sesamum indicum L. Riley 1960 Piperaceae Giseke (dicot) Piper betle L. endophytic, pathogenic GU Acharya et al. 2003

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40Table 1-2. Continued Host Fungal-Host Interaction LocationReference Piper hispidinervum C. DC. Poltronieri et al. 2003 Peperomia obtusifolia (L.) A. Dietr. pathogenic FL Chase 1982 Poaceae Barnhart (monocot) Arundinaria pygmaea (Miq.) Asch. & Graebn. LSU Ag Center 2008 Bambusa vulgaris Schrad. ex J. C. Wendl. endophytic GU first report Dendrocalamus spp. Nees Lu et al. 2000 Oryza sativa L. CABI, Herb IMI 280017 Ottochloa nodosa (Kunth) Dandy Situmorang and Budimen 1984 Panicum repens L. Situmorang and Budimen 1984 Pennisetum glaucum (L.) R. Br. Lenn 1990 Megathyrsus maximus (Jacq.) B. K. Simon & S. W. L. Jacobs endophytic GU Smith and Schlub 2004 Sorghum bicolor (L.) Moench Mendes et al. 1998 Polypodiaceae Bercht. & J. Presl (fern) Platycerium spp. Desv. pathogenic FL Alfieri et al. 1994 Polygonaceae Juss. (dicot) Coccoloba fallax Lindau Urtiaga 2004 Pteridaceae E. D. M. Kirchn. (fern) Adiantum spp. L. Situmorang and Budimen 1984 Adiantum tenerum Sw. pathogenic FL Alfieri et al. 1984 Restionaceae R. Br. (monocot) Ischyrolepis subverticillata Steud. Lee et al. 2004 Rhamnaceae Juss. (dicot) Colubrina retusa (Pittier) Cowan Urtiaga 2004 Ziziphus cyclocardia S.F. Blake pathogenic FL Urtiaga 2004 Ziziphus mauritiana Lam. pathogenic GU first report Ziziphus xylopyrus (Retz.) Willd. Murali et al. 2007 Rosaceae Juss. (dicot) Malus pumila Mill. CABI, Herb IMI 284207 Pyrus communis L. Alfieri et al. 1984

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41Table 1-2. Continued Host Fungal-Host Interaction LocationReference Rubiaceae Juss. (dicot) Guettarda speciosa L. endophytic GU first report Ixora coccinea L. CABI, Herb. IMI 129296 Ixora nigricans R. Br. ex Wt. & Am. Murali et al. 2007 Morinda citrifolia L. endophytic GU first report Nauclea diderrichii (De Wild.) Merr. CABI, Herb. IMI 126192 Pentas lanceolata (Forssk.) Deflers pathogenic GU first report Spermacoce spp. L. Situmorang and Budimen 1984 Rutaceae Juss. (dicot) Aegle marmelos Gond et al. 2007 Naringi crenulata (Roxb.) Nicolson Murali et al. 2007 Salicaceae Mirb. (dicot) Casearia decandra Jacq. Urtiaga 2004 Sapindaceae Juss. (dicot) Acer negundo L. El-Gholl 1997 Acer rubrum L. Alfieri et al. 1994 Cupaniopsis anacardioides (A. Rich.) Radlk. Alfieri et al. 1994 Dodonaea viscosa Jacq. Singh et al. 1982 Litchi chinensis Sonn. Matayba scrobiculata (Kunth) Radlk. Urtiaga 2004 Saxifragaceae Juss. (dicot) Saxifraga stolonifera Curtis El-Gholl 1997 Tolmiea spp. Torr. & A. Gray Alfieri et al. 1984 Tolmiea menziesii (Pursh) Torr. & Gray pathogenic FL Alfieri et al. 1984 Scrophulariaceae Juss. (dicot) Alectra sessiliflora (Vahl) Kuntze Urtiaga 2004 Antirrhinum majus L. Alfieri et al. 1994 Buchnera americana L. pathogenic GU Smith and Schlub 2004 Digitalis spp. L. Alfieri et al. 1994

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42Table 1-2. Continued Host Fungal-Host Interaction LocationReference Paulownia spp. Siebold & Zucc. Mehrotra 1997 Paulownia tomentosa (Thunb.) Steud. endophytic GU Mehrotra 1997 Russelia equisetiformis Schltdl. & Cham. endophytic, pathogenic FL, GU Alfieri et al. 1984 Simaroubaceae DC. (dicot) Ailanthus excelsa Roxb. CABI, Herb IMI 337615 Solanaceae Juss. (dicot) Capsicum annuum L. endophytic GU Kwon et al. 2001 Capsicum frutescens L. Pernezny and Simone 1993 Nicotiana glutinosa L. Tsay and Kuo 1991 Nicotiana tabacum L. pathogenic GU Fajola and Alasoadura 1973 Petunia hybrida hort. ex E. Vilm. pathogenic GU Alfieri et al. 1994 Petunia integrifolia (Hook.) Schinz & Thell. Peregrine and Ahmad 1982 Solanum erianthum D. Don Shaw 1984 Solanum lycopersicum L. pathogenic FL, GU Wei 1950 Solanum melongena L. endophytic GU Onesirosan et al. 1974 Solanum nigrum L. endophytic FL, GU Sarma and Nayudu 1971 Solanum torvum Sw. endophytic GU Onesirosan et al. 1974 Solanum tuberosum L. Peregrine and Ahmad 1982 Solanum viarum Dunal Casady 1994 Strelitziaceae Hutch. (monocot) Strelitzia spp. Aiton Alfieri et al. 1994 Strelitzia reginae Aiton pathogenic FL, GU Alfieri et al. 1994 Theaceae Mirb. (dicot) Camellia sinensis (L.) Kuntze endophytic GU El-Gholl et al. 1997 Turneraceae Kunth ex DC. (dicot) Turnera ulmifolia L. Urtiaga 2004 Urticaceae Juss. (dicot) Boehmeria nivea (L.) Gaudich. Cecropia peltata L. Minter et al. 2001

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43Table 1-2. Continued Host Fungal-Host Interaction LocationReference Cecropia schreberiana Miq. Minter et al. 2001 Laportea aestuans (L.) Chew Alfieri et al. 1994 Pilea spp. Lindl. Chase 1982 Pilea cadierei Gagnep. & Guillaumin pathogenic GU Alfieri et al. 1994 Pilea microphylla (L.) Liebm. pathogenic GU Smith and Schlub 2004 Pilea nummulariifolia (Sw.) Weddell pathogenic FL, GU Alfieri et al. 1994 Verbenaceae J. St.-Hil. (dicot) Callicarpa americana L. Alfieri et al. 1994 Citharexylum spinosum L. El-Gholl 1997 Clerodendrum buchananii (Roxb.) Walp. pathogenic GU first report Clerodendrum paniculatum L. pathogenic FL Ellis 1957 Clerodendrum quadriloculare (Blanco) Merr. pathogenic GU first report Clerodendrum thomsoniae Balf. pathogenic FL Daughtrey 2000 Gmelina arborea Roxb. endophytic GU Florence and Sharma 1987 Lantana camara L. pathogenic FL, GU Pereira et al. 2003 Petrea spp. L. Ellis 1957 Stachytarpheta angustifolia (Mill.) Vahl. pathogenic GU Ellis 1957 Stachytarpheta cayennensis (Rich.) Vahl pathogenic GU McKenzi 1990 Stachytarpheta jamaicensis (L.) Vahl pathogenic FL, GU Smith and Schlub 2004 Vitex agnus-castus L. Alfieri et al. 1994 Vitex negundo L. CABI, Herb. IMI 244917 Vitex parviflora Juss. pathogenic GU Smith and Schlub 2004 Vitex pinnata L. Ellis 1957 Vitex trifolia L. pathogenic GU McKenzie 1996 Vitaceae Juss. (dicot) Cissus spp L. Alfieri et al. 1994 Cissus alata Jacq. Alfieri et al. 1994 Tetrastigma voinierianum (Baltet) Pierre ex Gagnep. Alfieri et al. 1994 Vitis spp. L. Alfieri et al. 1994

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44 Table 1-2. Continued Host Fungal-Host Interaction LocationReference Zamiaceae Horan. (gymnosperm) Encephalartos spp. Lehm. Alfieri et al. 1994 Host plants are listed alphabetically by family (in bold). Each species is followe d by the first known reported reference. Fu ngal-host interaction refers to the endophytic or pa thogenic nature of the fungus and was only reported for hosts that were collected dur ing the Guam (GU) and Florida (FL) surveys. Location refers to whether the plant species was found as a host of C. cassiicola in FL or GU. Forty of the hosts were found on the CABI online database website (http://194.203.77.76/herbIMI/Disp layResults.asp?strName=Corynespora+cassiicola CABI Databases: Herb. IMI records for Fungus: Corynespora cassiicola).

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45 Figure 1-1. Corynespora cassiicola isolate from Cucumis sativus A) sporulating on naturally infected leaf tissue after 24 hours in the moisture chamber, B) germinating spore on water agar, and C) growing on V8 agar afte r single spore isolation (images are not shown to scale).

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46 Figure 1-2. Various symptoms caused by Corynespora cassiicola on naturally infected leaves of A) Vaccinium corymbosum B) Carica papaya C) Ageratum conyzoides D) Allamanda spp., E) Macroptilium lathyroides, F) Abutilon theophrasti G) Bidens alba, H) Euphorbia cyathophora I) Chromolaena odorata Continued. J) Corchorus aestuans K) Passiflora foetida L) Ipomoea pes-caprae M) Ipomoea obscura N) Lantana camara, O) Merremia peltata P) Bauhinia galpinii Q) Catharanthus roseus R) Phyllanthus amarus S) Hydrangea macrophylla and T) Salvia farinacea Images are not to scale.

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47 Figure 1-2. Continued.

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48 CHAPTER 2 GENETIC AND PATHOGENIC DIVERSITY OF CORYNESPORA CASSIICOLA Introduction Target spot, caused by the fungal pathogen Corynespora cassiicola (Berk. & Curt.) Wei, is common in the tropics, subtropics and greenhouses (Chase 1987). C. cassiico la is reported to infect 530 plant species from 380 genera, includi ng monocots, dicots, ferns, and one cycad (Chapter 1, this dissertation). Isolate characterization is needed to determine which hosts might serve as sources of inoculum for target spot of tomato species and other hosts since there is much variability concerning the host range of i ndividual isolates. Some isol ates show pathogenicity to a wide range of hosts, whereas others exhibit host specificity, and some are only pathogenic when associated with wounding (Chase 1982; Cutrim and Silva 2003; Kingsland 1985; Onesirosan et al. 1973, 1974; Pereira et al. 2003; Poltronieri et al. 2003; Seaman et al. 1965; Smith and Schlub 2004; Smith and Schlub 2005; Spencer and Walters 1969; Volin and Pohronezny 1989). At least two ra ces of the fungus have been distinguished based on their differential pathogenicity response on soybean and cowpea (Olive and Bain 1945; Spencer and Walters 1969). However, isolates from soybea n, sesame, cowpea and cotton in Mississippi were alike in pathogenicity (Jones 1961) A more extensive study found eight different pathogenicity profiles among 28 isolates from soybean in Mexic o, cucumber in Florida, and diverse hosts in Nigeria (Onesirosan et al 1974). Furukawa et al. (2008) found that an isolate from Salvia splendens was not pathogenic to cucumber, green pe pper or hydrangea; howev er isolates from these hosts were pathogenic to Salvia splendens Furukawa et al (2008), therefore, demonstrated that isolates with different pathogenicity profiles can be found on the same host. Since the 1960s, a leaf and fruit spot disease of tomato caused by C. cassiicola has become increasingly serious in tropical countries worldwide (Jones and Jones 1984). It was first

PAGE 49

49 reported in Florida in 1972 and ha s since become one of states most damaging foliage and fruit diseases (Blazquez 1972; Pernezny et al. 1993, 1996, 2000, 2002). Under warm, humid, conditions the disease leads to heavy defoliation and significant losses in yield (Volin and Pohronezny 1989). Currently, there are no resi stant tomato cultivars available, although resistance found in PI 120265 ( Lycopersicon esculentum ) and PI 11215 ( L. pimpinellifolium ) and was controlled by a single recessive gene (Bli ss et al. 1973). Understanding the genetic and pathogenic diversity of the pathogen and its distribution is vital to isolate selection for resistance screening. Kingsland (1985) compared three isolates from tomato, cucumber and papaya debris and found that tomato and cucumber were susceptible to all isolates, but the isolate from papaya debris was not pathogenic on papaya indicating that it was possibl y growing as a saprophyte. In many studies, isolates were found to be non-pa thogenic on the hosts from which they were isolated, further indicating that C. cassiicola can grow as a saprophyte (Chase 1982; Kingsland 1985; Onesirosan et al. 1974; Hyde et al. 2001; Lee et al. 2004). Other studies show that isolates are only secondary invaders, or inva ders of senescent tissue. Isol ates from the ornamental hosts Aeschynanthus pulcher (lipstick vine), Aphelandra squarrosa (zebra plant), azalea and hydrangea were pathogenic on all hosts in cros s-pathogenicity trials when wounded; however, only A. pulcher was susceptible without wounding (Chase 1982). Silva et al. (1998) compared pathogenicity of 16 isolates from rubber trees in Sri Lanka and five isolates from diverse hosts in Australia. Papaya isolates from Australia were pathogenic to tomato and rubber, but not cowpea and eggplan t. Mimosa and thyme isolates from Australia were pathogenic to eggplant, rubber, and tomato, but not cowpea. Isolates from Sri Lanka

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50 collected from different rubber clones were eith er pathogenic to all hosts (cowpea, eggplant, rubber, and tomato), or pathogeni c to all hosts but eggplant. The host specificity and severity of the fungus on Lantana camara in Brazil has led to the discovery that C. cassiicola may be useful as a bioherbicide (Pereira et al. 2003). Based on the vast number of weeds that serve as hosts of th e fungus, there is great potential for the discovery of several more isolates useful for biological cont rol of weeds. Consideri ng the wide variation in isolate pathogenicity that has been previously reported, additional studies are needed to further understand the host range of individual isolates from different hosts and locations. Prior research on the genetic characterization of C. cassiicola is limited to restriction fragment length polymorphism (RFLP) of ITS rDNA and random amplified polymorphic DNA (RAPD) studies. No variati on between five isolates of C. cassiicola collected from mimosa, papaya, and thyme in Australia was found based on RFLP of ITS (S ilva et al. 1995). Silva et al. (1995) concluded that RFLP of the ITS regions of rDNA can be used to distinguish between Corynespora and the morphologically similar genus Helminthosporium but not different isolates of C. cassiicola However, the three isolates from papa ya had identical RAPD patterns, growth rate, isolate color, and pathoge nicity profiles, which were di fferent from the isolates from mimosa and thyme, indicating an ongoing process of host specialization on papaya (Silva et al. 1995). RAPD analyses from 27 isolates collected from Hevea brasiliensis in Sri Lanka revealed correlations between host locati on, host genotype, isolate mor phology, and isolate pathogenicity (Silva et al. 1998). Silva et al (1998) concluded that a progenitor strain may have been spread in India by distribution of live plant material. Prior outbreaks of the disease on the susceptible

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51 rubber clone RRIC 103 in other countries, and the sudden appearance and severity of target spot on the same clone in Sri Lanka in 1985, is evidence for such dissemination. Silva et al. (2003) characterized 42 is olates from bitter gourd, cocoa, manihot, papaya, rubber, sweet potato, tomato, and wing-bean from various regions in India based on RAPD analyses. RAPD groups did not correlate with geographic origin, but isolates obtained from rubber clone RRIC 103 grouped together. This stra in might be responsible for several recent outbreaks on this clone. In addition, all but one of the isolates from rubber clone RRIC 110 clustered in 2 RAPD groups, which may identify th e strain that caused the outbreak on this clone in 1995. Silva et al (2003) concluded that correlation of RAPD groups with pathogenicity was needed to help develop resistant cl ones against all pathogenic isolates. Atan and Hamid (2003) characterized nine C. cassiicola isolates from Hevea brasiliensis in Malaysia using RAPD of genomic DNA and RFLP of amplified ITS regions. RFLP analyses with three restriction enzymes yielded monomorphic patterns. However, isolate OPEN 1 from clone RRIM 2020 had a distinct RFLP pattern from the other eight isolates after digestion with Hae III. RAPD results indicated the presence of at least two genetical ly distinct races that infect rubber. Seven isolates pathogenic to clones RRIM 600, RRIM 2009, and two unidentified rubber clones were molecularly similar and identi fied as Race 1. The remaining two isolates, both pathogenic on clone RRIM 2020, had identical banding patterns and we re considered Race 2. Unfortunately, the majority of the diversity assessments are limited to rubber isolates from Malaysia and Sri Lanka and are ba sed on RAPD techniques, which is problematic with respect to repeatability and homology assessm ent (Isabel et al. 1999). In addition, all the RFLP studies used the ITS rDNA region which has minimal vari ation among isolates (Silva et al. 1995, 1998).

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52 Investigations into th e genetic variation among C. cassiicola isolates using more reliable molecular methods and more dive rse isolates are needed. In this study, we collected and solicited 143 is olates from diverse hosts and locations. To test whether C. cassiicola is panmictic throughout its range, a llelic genealogies were constructed from four loci including the rDNA IT S region, two random hypervariable loci, Cc caa5 and Cc ga4, and the single copy actin -encoding nuclear gene, Cc act1 Fifty of these isolates were spray inoculated on seedlings of eight crop plants to te st pathogenicity profiles. Correlations among an isolates pathogenicity profile, it s host of origin, and genotype we re investigated. The purpose of this research is to gain knowledge of the diversity within the species C. cassiicola because of its implications for resistance breeding and disease management of target spot of basil, bean, cowpea, cucumber, papaya, soybean, sweet potat o, tomato, and potentially other crops. Methods Collection and Solicitation of Fungal Isolates C. cassiico la isolates were collected from diverse plant hosts during 5-day collecting trips to locations in the Pacific: American Samo a (AS), Hawaii (HI), Palau (PW), Pohnepei (PH), Saipan (SN), and Yap (YP) in the summer of 2005. More extensive surveys were conducted to collect the fungus in Florid a (FL) and Guam (GU) betw een 2004-2006 (see Chapter 1). Farms, nurseries, and roadsides were surveyed fo r plants with target spot symptoms. First, second, and third priority was given to crops, weeds, and naturalized or indigenous hosts of C. cassiicola respectively. Symptomatic le aves were put into individual plastic bags in the field and later placed abaxial side up in petri dishes with moistened paper towels in a laboratory. After 24 hours in the moisture chamber, petri plat es were placed under th e dissecting microscope and suspected spores and conidiophores of C. cassiicola were confirmed microscopically.

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53 Single spores were captured at the end of a teasing needle an d transferred to antibiotic V8 agar (340 ml V8 juice, 660 ml water, 3g CaCO3, 17g agar, 100 g/ml Ampicillin or Kanamycin) slants, left at room temperature until the colony re ached at least 5 cm in diameter, whereby it was covered with autoclaved mine ral oil, and stored at 5o C until further study. Sporulation from non-symptomatic leaf material was noted, po ssibly indicating non-pathogenic growth. To obtain globally diverse isolates, individual researchers in Brazil (BZ), Malaysia (MY), Mississippi (MS), and Tennessee (TN) were solicited for additional C. cassiicola cultures. Isolates from BZ on lantana (JMP216), papa ya (DOA16b), soybean (RWB321) and tomato (JMP217) came from Alvaro Almeida, EMBRAPA Isolates CBPP, CLN 16 and CSB1 2 were received from MY off of rubber from Dr. Safiah Atan, Malaysian Rubber Board. Isolate TN13-3 was received from Nashville, TN on greenhouse Afri can violet from Justin S. Clark, University of Tennessee. Isolate MS01 was received from MS on greenhouse tomato leaves from David Ingram, Central MS Research and Extension Center. Isolates of different specie s were also solicited from culture collections to serve as outgroups. Cultures from Commonwealth Agricu ltural Bureaux Interna tional (CABI) in the United Kingdom included C smithii IMI 5649b and C citricola IMI 211585. Cultures from National Institute of Agrobiological Sciences (NIAS) in Japan included C citricola MAFF No. 425231, C melongenea MAFF No. 712045, and C sesamum MAFF No. 305095. Cultures from Centraalbureau voor Schimmelcultures (CBS) in the Netherlands included C proliferata CBS 112393, C citricola CBS 169.77, and C olivaceae CBS 291.74. Cultures were single-spored after they were received. A complete list of isolates used in these st udies, along with the plant host, geographic location and the type of a ssociation with the hos t plant (endophytic or pathogenic growth) can be found in Table 2-1.

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54 Primer Development for Random Hypervariable Loci Three C. cas siicola isolates from long-term stor age (FL31, GU112, and PW56) were chosen based on unique host and location. A sm all piece of mycelium from the monosporic cultures was extracted with tweezers and placed onto a V8 agar plate. The isolates were grown under constant fluorescent light for 7 days. Aerial mycelium was scraped from the agar surface, placed in 1.5 ml microcentrifuge tubes, lyophilized overnight, and then frozen in liquid nitrogen. Genomic DNA was purified using the DNeasy plant Mini Kit (Quiagen, Inc.) according to the manufacturers specifications. Genomic DNA combined from all three isolates was digested with the Sau 3AI restriction enzyme (7.2 l of DNA from each of the three isolates; 2.5 l 10X buffer; 1.0 l 10 U/ l Sau 3A I enzyme; incubated at 37oC for 2 hours). The digested genomic DNA was fractionated to remove fragments less than 400 bp using a Chroma Spin column (Chroma Spin + TE 400, Clonetech Laboratories, Inc.) a ccording to the manufacturers specifications. The digested fractionated DNA was quantified and ligated to Sau 3AI linkers and incubated at 16oC overnight. Excess linkers were removed using the same Chroma Spin column as above. The linkerligated fragments were PCR amplified using SauL-A primers and a program consisting of initial denaturation for 3 min at 94oC, followed by 25 cycles of 94oC for 1 min, 68oC for 1 min, and 72oC for 2 min, and a final amplification at 72oC for 10 min. The amplified genomic PCR library (compos ed of 400-1500 bp fragments) was enriched for fragments containing two differe nt microsatellite repeats, (CAA)n and (GA)n. The denatured genomic PCR library was hybridized to the following biotinylated oligoprobes: [5(CAA)15TATAAGATA-Biotin] and [5(GA)15TATAAGATA-Biotin] (Tepnel Lifecodes Corporation) and incubated at 48oC overnight. The PCR fragments that hybridized to the repeat

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55 probes were captured and eluted using two VECT REX Avidin D matrix columns (cat. No. A2020, Vector Laboratories, Burlingame, CA) accordi ng to the manufacturers specifications. The two mixtures containing genomic fragments enriched for the (CAA)n tri-repeat and the (GA)n direpeat were PCR amplified using SauL-A primer s following the same PCR conditions as above. PCR products from the amplification of the en riched microsatellite library were ligated into a plasmid vector (pCR 2.1-TOPO vector; Invitrogen, Inc.) a nd transformed into E. coli (One ShotTM TOP 10 Cells, Invitrogen, In c.) using the TOPO TA Cl oning Kit (Invitrogen, Inc.) according to the manufacturers instructions. Transformed colonies were lifted and crosslinked onto nylon membranes in an UV chamber (GS Gene LinkerTM, Bio-Rad Laboratories, Inc., Hercules, CA) using the optimal crosslink program. Nylon membranes were hybridized with al kaline phosphatase-label ed repeat probes ((CAA)n and (GA)n) and the Quick-LightTM hybridization Kit (Te pnel Lifecodes Corp.) according to the manufacturers recommendation. Colonies containing plasmids that tested positive for inserts with repeats were sequenced in one direction. Primers were designed to amplify 300-500 base pair fragments flanking lo w repeat number (<10) sequences using Primer3 (v. 0.4.0). Sequences with repeats of less than 10 were likely to be non-va riable microsatellite loci, but may contain polymorphic flanking sequences. Sequences were screened for polymorphisms using five isolates fr om different hosts and locations. Primers that amplified the Cc ga4 and Cc caa5 loci were chosen for further study because they amplified sequences with relatively high levels of polymorphism (>5%). Fungal Cultures and Extraction of Genomic DNA Genom ic DNA from 143 isolates (Table 2-2) in long-term storage was purified and amplified using Extract-N-Amp (Sigma-Aldrich) according to the manufacturers specifications.

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56 The following primers were used for PCR amplification: ITS1 and ITS4 (White et al. 1990) for the internal transc ribed spacer region, including the 5.8 rRNA coding region; ACT512F and ACT-783R (Carbone and Kohn 1999) fo r the single copy nuc lear actin locus Cc act1 ; GA4-F (5-CCT GCT CCG ACT TTG TTG AG-3 ) and GA4-R (5-GTC TGG GAG CAG CAA AGA CT-3) for the random hypervariable Cc ga4 locus; CAA5-F (5-GTC CAC AAG TGG AAC CTC GT-3) and CAA5-R (5-CCT CGT CTG CCA GTT CTT CT-3) for the random hypervariable Cc caa5 locus. Hot-start PCR was performed with a MyCyclerTM thermocycler (BioRad) with a program consisting of initial denaturation for 3 min at 94oC, followed by 30 cycles of 30 sec at 94oC, 30 sec at 58oC, and 30 sec at 72oC, and a final cycle of 5 min at 72oC for the ITS, Cc ga4 and Cc caa5 loci. For the Cc act1 locus, the program was identical except for an annealing temperature of 61oC. PCR products were puri fied using the QIA quick PCR purification Kit (QIAGEN Inc.) according to the manufacturers instructions. The purified products were then quantified on 1% ethidium bromide-stained agarose gels. Sequencing of the DNA samples was done at the University of Florida DNA Sequencing Core Laboratory using ABI Prism BigDye Terminator cycle sequenc ing protocols (part number 4303153) developed by Applied Biosystems (Perkin-Elmer Corp., Foster City, CA). The excess dye-labeled terminators were removed using MultiScreen 96-well filtration system (Millipore, Bedford, MA, USA). The purified extension products were dried in SpeedVac (ThermoSavant, Holbrook, NY, USA) and then suspended in Hi-di formamide. Se quencing reactions were performed using POP-7 sieving matrix on 50-cm capillaries in an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, US A) and were analyzed by ABI Sequencing Analysis software v. 5.2 and KB Basecaller.

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57 Phylogenetic Analyses Four loci (rDNA ITS, Cc caa5, Cc ga4, and Cc act1 ) from 143 isolates were sequenced. Forward and reverse sequences from each P CR product were concatenated in SequencherTM 4.8 and trimmed to include only bases sequenced in bo th directions. Samples with ambiguities were sent for re-sequencing. Multiple alignments fr om each locus were executed separately with Clustal X (1.83.1) and the alignments were insp ected and adjusted manually using MacClade 4.08 OS X (Maddison and Maddison 2005). Data from ITS rDNA, Cc ga4, Cc caa5 and Cc act1 loci were partitioned to facilitate different permutations of combined analysis. A partitionhomogeneity test (incongruence le ngth-difference test or ILD) was implemented to evaluate the homogeneity of different data partition subs ets using PAUP* v4.0b10 (Swafford 2002). The test implemented 1,000 replicates (heuristic search ; random simple sequence additions; TBR; maxtrees = 1,000). Comparisons were evaluated using a threshold of p < 0.001 and were made between all data partitions. With the ILD test indicating the combinability of all molecular data, neighbor joining (NJ) and maximum parsimony (MP) analyses were conducted for each data partition and the combined data set using PAUP* (Swafford 2002). C smithii IMI 5649b, C citricola IMI 211585, C proliferata CBS 112393, C citricola CBS 169.77, and C olivaceae CBS 291.74 were defined as outgroups. Cultures from Nationa l Institute of Agrobiol ogical Sciences (NIAS) in Japan ( C citricola MAFF No. 425231, C melongenea MAFF No. 712045, and C sesamum MAFF No. 305095) were not included as outgroups because they grouped with C. cassiicola isolates in phylogenetic analyses (see Results below). For the NJ analyses, default settings were used except ties were broken randomly by initial seed. Due to long computational time, MP analyses were conducted in th e following manner. An initial heuristic search was conducted w ith one random addition replicate, TBR (tree-

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58 bisection-reconnection) branch swapping, and the MulTre es option (saving a ll optimal trees) in effect. A second heuristic search was conducte d using 1000 random addition replicates with the above settings and saving no more than 10 trees with a sc ore greater than or e qual to the best tree score from the first replicate in the previous an alysis. In all analyses, gaps were treated as missing data. Strict consensus trees were generated from analyses with multiple equally parsimonious trees. For all MP analyses, st atistical support for node s was estimated using maximum parsimony bootstrap (BS) replicates (Felse nstein 1985). For the combined data set, BS estimates were obtained using 1,000 replicates, each with 100 random taxon addition replicates and saving no more than 1,500 trees pe r bootstrap replicate, TBR branch swapping and the MulTrees option in effect. All data were also analy zed by Bayesian inference (BI) methods with MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and Hu elsenbeck 2003). An a ppropriate model of evolution (under the AIC criterion) was selected for each data partition using the program Modeltest v3.4 (Posada and Crandall 1998). All Bayesian analyses (individual loci and combined data) were conducted while retaining the appropriate model for each data partition. Markov Chain Monte Carlo was implemented with four heated chains and trees were sampled every 1,000th generation for one million generations. The first 25 percent of the total number of generations was discarded as burn-in. A 50 percent majority rule consensus tree was generated from the remaining trees, in which the percenta ge of nodes recovered represented their posterior probability (PP). Congruent nodes resultin g from the NJ, MP, and BI analyses of the combined molecular data was used to assign isolates to a phylogenetic lineage (PL). On ly isolates that fell within clades of high support (BS value >70 a nd PP value > 95) were assigned to a PL.

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59 Pathogenicity Analyses Fifty out of the 143 Corynespora isolates were used for pathogenicity profiling (Table 2-3) on eight crop plants. Isolates originally isolated from crop pl ants and from all phylogenetic lineages were chosen. Each isolate was spray-in oculated onto four replicate plants of eightweek-old: basil Italian La rge Leaf (Ba), bean Bush Kentucky Wonder (Be), cowpea California black-eye (Co), cucumber Straigh t 8 (Cu), soybean AG00901 (So), and tomato Rutgers (To) seedlings; 8-week-old sweet potat o Beauregard (Sw) cuttings; and 12-week-old papaya HI Sunrise (Pa) seedlings. Cultivars were chosen based on their known susceptibility in the survey regions. To increase colony sporulation for inoculum preparation, aerial mycelium from 10-day-old V8 agar plates was gently scraped with a gla ss cover slip to flatten mycelium and then placed under constant cool-white fluorescent light (One sirosan et al. 1975). Three days later, the surface of the agar was scraped with a glass cove r slip and the resulting mycelia was blended in 200 ml sterile distilled water fo r two seconds and filtered through three layers of cheesecloth. Spores were counted under a hemacytometer and the concentration was adjusted to 20,000 spores/ml. One drop of Tween 20 per 100 ml was added to the inoculum. Plants were sprayed with the spore suspension until leaf run off (a bout 500 ml), making sure that both leaf surfaces were fully covered. Plants were kept on a mist bench to maintain constant leaf moisture 3 days prior to inoculation and for the remainder of th e experiment. Plants we re rated 7 days after inoculation using the rating system developed by Onesirosan et al. (1973): (0) symptomless, no lesions on leaves or stems; (1) non pathogeni c hypersensitive response, a few to many nonexpanding pinpoint lesions; (2) moderately vi rulent, many expanding lesions, some coalescing, but not resulting in blight; (3) hi ghly virulent, lesions spreading to form large areas of dead tissue resulting in a blighting e ffect. Incidence (I), defined as the number of plants showing symptoms

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60 (with ratings of 1, 2, or 3), and severity (S), defined as the av erage rating for all symptomatic plants, were recorded. The experiment was repeated. Each isolate was assigned a pathogenicity profile (PP), which is a list of susceptible hosts. Hosts were considered susceptible if at least one of th e replicates from the two experiments (total of eight plants) received a rating of 2 or 3. PPs were converted to a binary character matrix so that each isolate received a zer o (non-pathogenic, all reps with ratings of 0 or 1) or a one (pathogenic, at least one rep with a rating of 2 or 3) for each host. Unweighted pair group method with arithmetic mean (UPGMA) trees we re constructed from the binary matrix and internal support for nodes was estimated using bootstrap analyses with 1,000 reps and a UPGMA algorithm. The tree topology was visually compared to the PL designation of each isolate tested (Figure 2-6). PPs were also visually mapped on the four-locus combined BI phylogenetic tree (Figure 2-1). Growth Rate Analyses Seventy-seven isolates were test ed for growth rate at two te mperatures (23 C and 33 C). A sm all piece of aerial mycelium was extracted from the monosporic cultures in long-term storage with tweezers and placed onto a V8 agar plate. After 5 days, the 77 colonies had grown beyond the mineral oil and six 4 mm agar plugs were cut from actively growing mycelium at the colony edge. A single plug was placed in the center of six V8 agar plates. Three replicate plates of each isolate were immediately placed in growth chambers at 23 C and 33 C under 12 hours of alternating fluorescent light (ca. 25 lux) and dark. The average of two colony diameters at 90 degrees from each other was recorded at 48, 72, 96, 120, 144, and 166 hours. Average colony diameter was plotted against time and a line of best fit was generated for each replicate. The slope of the line of best fit (R2>0.98) was used to compare variation within reps to variation between isolates in SAS Statistical Software

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61 (Version 8, 1999). The experiment was repeated with five isolates with no statistically significant variation (data not shown). A co rrelation between isolate growth rate and phylogenetic lineage was tested us ing SAS statistical software. Results Phylogenetic Analyses The General Tim e Reversible model (GTR + I + ) was selected by Modeltest for each of the four gene partitions. The corresponding model for each locus wa s applied to all BI analyses and the combined dataset was partitioned. The final combined dataset contains 2,136 aligned characters used for analyses. Tree topologies re sulting from NJ, MP, and BI analyses recovered essentially the same well-supported nodes. The analyses reveal four ma jor phylogenetic lineages (PL) with high statistical support (BS value >70 and PP value > 95) (Figure 2-1). All major PLs contain isolates from diverse lo cations, indicating their global dispersal. PL1 contains a distinct clade with high stat istical support (designate d PL1.1) containing only isolates collected from papaya from around the world indicating specialization on this host. PL1.2 contains two isolates from Stachytarpheta jamaicensis collected from Guam and Palau, indicating potential specializati on on this host. This supports pathogenicity studies showing isolate specificity to this host (Smith and Schl ub 2005). Isolates from diverse hosts are present in PL1 including crops (basil, bitter melon, eggp lant, cowpea, cucumber, oregano, pumpkin, rubber, soybean, sweet potato, watermelon), ornamentals ( Buddleja Catharanthus Codiaeum Coleus Episcia and Tabebouia ), and weeds ( Bidens, Buchnera Clerodendrum Commelina Lantana, Macroptilium Meisosperma, Vitex ). Tomato isolates are missing from PL1, indicating that isolates in this lineage may not be pathogenic to tomato. Isolates in PL2 are also globa lly distributed and include crops (cucumber, rubber, sweet potato), ornamentals (African violet, Allamanda Catharanthus Pilea ), and weeds ( Piper Pilea ).

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62 There is also a lack of tomato isolates in PL2 indicating that isolates from this lineage may be nonpathogenic on this host. Though PL2 was highl y supported (BS and PP values of 100), its sister relationship to PL1, PL 3, and PL4 remains unresolved. Globally distributed isolates from PL3 incl ude crops (basil, bitter melon, cucumber, pumpkin, soybean, tomato), ornamentals ( Bauhinia, Moringa, Pachystachys Plectranthus, Saintpaulia ), and weeds ( Acanthus Asystasia Calopogonium Coccinia Euphorbia Luffa Passiflora Teramnus ). These are hosts that may harbor is olates pathogenic to tomato. PL5 and PL6 group with PL3 with low support (MPBS value of 60). PL5 contain C. cassiicola isolates from African violet in Guam and Tennessee that ar e very similar in sequence, especially at the Cc-caa5 locus, indicating specialization on this host. African violet isolates from Saipan and Yap are found in PL3. PL6 is highly supporte d and contains isol ates from Brazil on Coleus Palau on cowpea, and Saipan on Asystasia The majority of tomato isolates group in PL 4 from diverse locations including American Samoa, Brazil, Florida, Guam, Mississippi, Palau, a nd Saipan. These twelve tomato isolates also group with isolates from crops (bean, cassa va, cucumber, sweet potato), ornamentals ( Bauhinia Cassia, Coleus Eugenia Ficus Jatropha, Salvia Syzygium ), and common weeds ( Calopogonium Calyptocarpus Chromolaena, Euphorbia Hyptus Lantana Mikania Spathodea ), which are likely inoculum sources for the initiation of disease on tomato. The rDNA ITS region (Figure 2-2) is composed of 1,013 characters, 400 of which are an insertion in the outgroup taxa C. smithii Of the 612 remaining characters, 141 are variable and 107 are informative. The rDNA ITS sequences re veal the m isidentification of three outgroup taxa from the NIAS culture collection. C sesamum 305095, C citricola 425231, and C melongenea 712045 should be reclassified as C cassiicola based on rDNA ITS sequences.

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63 When the outgroup taxa C. citricola C. olivaceae, C. proliferata and C. smithii are removed from the analyses, the rDNA ITS sequences of C. cassiicola contain only three informative characters out of 584 bases. Two of these characters separate the isolat es into three distinct phylogenetic lineages that correlate with the PLs in the combined analysis. These two characters are base pair 158 (C or T), and base pair 497 (A or G) of the C. cassiicola rDNA ITS alignment. Three haplotypes are represented by these two ch aracters: CA, CG, and TG (no haplotype TA); all isolates with haplotype CA group in PL4, isolates with hapl otype TG group in PL1, isolates with haplotype CG group in PL2, PL3, PL5 and PL6. CG is also the ance stral haplotype, present in all outgroups except for C. proliferata (haplotype CA). The third informative character in the rDNA ITS sequences of C. cassiicola is base pair 123, which is a T in the majority of isolates, but a C in isolates PW101 (PL5), RWB321 (PL5), SN64 (PL5.1), and TN13-3 (PL6). It is this character (bp 123) that caused the polymorphic band pattern obs erved by Atan and Hamid (2003) in their RFLP analysis of the rDNA ITS region of rubber isolates using HaeIII (recognition sequence GGCC). The sister relationships between the phyl ogenetic lineages remain unresolved in the analyses of the individual loci and in the combined analyses. In addition, the ITS rDNA region was the only locus that showed good support for C citricola C olivaceae C proliferata and C smithii as sister taxa to the ingroup of C. cassiicola isolates. The phylogenetic placement of the outgroup taxa was not well supported in the combined analyses, or the GA4 locus. The CAA5 locus showed support for C olivaceae C proliferata and C smithii as basal to PL1, PL2, PL3, PL5, and PL6, but PL4 and C. citricola fell basal to that group. The apparent paraphyly of C. cassiicola at the Cc-caa5 locus may be a result of character variation that occurred at this locus

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64 before the species evolved. Additional loci c ontaining characters that reveal the sister relationships of the different PLs ar e needed in future analyses. The Cc-caa5 locus (Figure 2-3) reveals similar tree topologies to the combined analyses with high support for the four major PLs. Differen ces at this locus in PL1 include the lack of PL1.1 that distinguishes papaya isol ates from other isolates in PL1 in the combined analyses. In addition, PL1.2, which includes two rubber isolates from Malaysia, has low support. GU70 and SN59, isolates basal to PL1 in th e combined analyses, groups with other isolates in PL1 at this locus. The Cc-caa5 locus shows strong support for PL2 with the same nine isolates as in the combined analysis. The Cc-caa5 locus does not resolve PL3.1 or PL3.3 as distinct from PL3, although the five isolates in PL 3.2 group together with strong support. This locus does not distinguish isolates FL2920, GU120 GU136 as distinct from other isolates in PL4. PL5 and PL5.1 isolates are group basal to PL3, but with low support. PL6, which includes African violet isolates from Guam and Tennesse e, group with isolate NIAS 712045 w ith high support. Isolates FL50 ( Hydrangea macrophylla ) and FL51 ( Vaccinium corymbosum ) are unresolved at this locus as well as in combined analyses. The Cc-ga4 locus (Figure 2-4) highly suppor ts PL1, PL2, PL4, and PL6, although the sister relationships between th e PLs are unresolved. Isolates in PL3 form a clade with low support. The Cc-ga4 locus did reveal a shared haplotype between papaya isolates with a point mutation from an A to a G at base 74. C cassiicola isolates are not monophyletic at this locus because the outgroups C. proliferata C. olivaceae, and C. smithii fall basal to PL1, PL2, and PL6 with low support. This may be a result of character variation before speciation, a high incidence of homoplasious characters, or the convergent evolution of specific adaptations.

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65 The Cc-act1 locus could not be amplified in the outgroup taxa C citricola C. proliferata and C. smithii perhaps due to mutations in the primer annealing site. The locus did amplify in C olivaceae and shows high variation from C cassiicola isolates (Figure 2-5). There is good support for PL3, PL2, and PL4 at this locus, al though only marginal support for PL1 (BS value of 68). Again, the sister relationships between th e PLs are unresolved. The Cc-act1 locus also reveals a shared haplotype between papaya isolat es with a point mutation from an A to a G at base 229. Pathogenicity Analyses As a result of screening f ifty isolates fo r pathogenicity on eight index hosts, 16 unique pathogenicity profiles (PP) were developed (Table 2-3). The most common PP was CuTo, followed by Pa and CuSwTo. Cucumber was the most susceptible host, with all isolates producing symptoms and an average severity rati ng of 2.3. Tomato was also highly susceptible with 49 out of 50 isolates show ing symptoms with an average severity rating of 1.8. Even though only eight isolates were pathogenic on papaya, the average severity rating was 2.1 indicating that pathogenic isolates were highly virulent. Isolates pathogenic to basil, bean, cowpea, soybean and sweet potato were less virulent on these hosts with average severity ratings less than 1.5. There was a strong correlation be tween PP and PL (Figure 2-6). Seven out of ten isolates with PP CuTo were from PL4 and all isolates with PP CuSwTo and BeCuSwTo were from PL4. In PL4, all isolates but SN37 were highly virule nt on tomato (average severity ratings ranging from 2.5 to 3) and all isolates but GU28 were pa thogenic to cucumber (average severity ratings ranging from 1.3 to 3). In addition, the only is olates pathogenic to bean were from PL4, although these five isolates were weakly virulent (average severity ra tings ranging from 1.3 to 1.9).

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66 In PL3, all isolates were strongly pathogenic to cucumber (average se verity ratings ranging from 2.3 to 2.8) and six out of seven isolates were strongly pathogenic to tomato (average severity ratings ranging from 2.5 to 3). Four out of the six isolates also were pathogenic to basil and all isolates with PP BaCuTo were from PL3. Pathogenicity profile CuSw was unique to isolat es from PL2 and all isolates tested from PL2 had this profile. In additi on, isolates collected in the field from papaya in PL1.1 were specific to papaya in pathogenicity studies, although all isolat es were weakly virulent on cucumber with average severity ratings of 1.3 or less. All isolates from PL1 were pathogenic to cucumber with average severity ratings ranging from 1.3 to 3. Nine out of the 13 isolates from PL1 were pathogenic to cowpea and seven were pathogenic to basil. The only other host susceptible to isolates from PL1 was soybea n, which was only weakly susceptible when inoculated with isolate PW87. Growth Rate Analyses The null hypotheses of no growth rate diffe rences am ong isolates, phylogenetic lineage, and temperatures were rejected (P <0.0001), while the null hypothe ses of no growth rate differences among repetitions was accepted with a probability of 0.7546. The 77 isolates tested all grew faster at 23 C than 33 C. At 23 C, average isolate growth rate (average of three repetitions) was between 0.1479 and 0.474 with an ove rall mean of 0.3855 (Table 2-4). At 33 C, average isolate growth rate was between 0.1382 and 0.4153, with an overall mean of 0.2958 (Table 2-5). At 23 C, there we re 29 significantly different growth rates and at 33 C there were 39 significantly different growth rates. Among the fastest growing isolates at both temperatures were FL37, GU90, GU99, AS67, and HI01. The slowest growing isolates were very different at the two temperatures. Slow growing isolat es at 23 C were PH01, JMP216a, GU120, FL15, and

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67 DOA16b. Slow growing isolates at 33 C were AS119, AS117, JMP217, AS49, GU120, and GU112. Though there were not enough replicates to test for interactions among effects, growth rate alone correlated with lo cation, phylogenetic lineage, and with host. Isolates from Oahu and Palau grew the fastest at both te mperatures. Isolates from Braz il, Florida and Malaysa tended to have slower growth rates at bot h temperatures. Surprisingly, American Samoan isolates grew proportionately much faster at 23 C than 33 C, despite its tropical climate. Isolates from PL6 and PL1 grew the fastest at both temperatures. Isolates from PL 2 and PL4 grew the slowest at 23 C and isolates from PL5 and PL3 grew the slowest at 33 C. All isolates from Clerodendrum Commelina, Ficus Macroptilium pumpkin, and Stachytarpheta were fast growing at both temperatures. In addition, isolates from Allamanda Coleus eggplant, Lantana, and tomato isolates had slower growth ra tes at both temperatures. Discussion The current study presents the first robust, global phylogeny of the species Corynespora cassiicola Based on sequence data from four unique loci, there is eviden ce for high genetic diversity within the species. The highly clonal nature of C. cassiicola is demonstrated in the congruence of the phylogenetic trees from distinct loci. All loci disti nguish four major clonal lineages within C. cassiicola The low level of sequence va riation at the rDNA ITS region within the species relative to other Corynespora species suggests that these lineages are in fact clonal populations, rather than taxon omically distinct species. As reported previously, the pa ttern of distribution of the diversity within the species correlates with the host (Smith et al. 2008a). Identical hapl otypes are widely distributed geographically. The lack of correlation be tween phylogenetic data and location provides evidence for the recent global dispersal of isolat es from all four phylogenetic lineages. In

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68 addition, geographically diverse is olates from the same host plan t shared identical haplotypes, potentially indicating host specialization. For example, isolates co llected from tomato in Brazil, Florida, Guam, Mississippi, Palau, and Saipan had identical haplotype s at all four loci. Isolates collected from Lantana in Florida and Brazil were also iden tical at all four loci. Isolates collected from African violet in Guam and Tenne ssee were unique from all other isolates and nearly identical to each other. Perhaps the mo st compelling evidence for host specialization is the shared identical sequences of all isolates collected from papaya from very diverse locations. Tomato isolates from diverse locations incl uding North and South America and the Pacific Islands are found in only two of the five major phylogenetic lineages (PL3 and PL4). Isolates from other hosts that fall into these same PLs are likely pathogenic to tomato and may serve as source hosts or altern ative hosts for target spot of tomato. Tomato isolates are genetically similar to isolates from common crops (basil, bean, bitter melon, cassava, cu cumber, papaya, pumpkin, soybean, sweet potato), weeds (Acanthus, Calopogonium Calyptocarpus Chromolaena, Coccinia Euphorbia, Lantana, Macroptilium Mikania Momordica, Passiflora, and Teramnus ), and ornamentals ( Asystasia Bauhinia, Cassia, Coleus Eugenia, Euphorbia Ficus Hyptus Jatropha, Luffa Moringa Pachystachys Plectranthus, Saintpaulia Salvia Spathodea, and Syzygium ). Based strictly on these data, control of ta rget spot should involve isolation of tomato fields from these plant species, when possible. Pathogenicity testing, in addi tion to phylogenetics, should be used to determine which hosts might serve as sources of inoculum for the in itiation of target spot of tomato. There are at least sixteen unique pathoge nicity profiles within C. cassiicola on the eight crop plants that were tested. Isolates from the same lineages show similar but not identical prof iles (Figure 2-1 and Figure 2-6). For example, all but two isolates in PL3 and PL4 are pat hogenic to tomato, and

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69 isolates from all other lineages are nonpathogenic to tomato. All is olates pathogenic to basil are from PL1 and PL3, but not all isolates in these cl ades are pathogenic to ba sil. Interestingly, the majority of isolates, excluding the isolates collected from papaya, were pathogenic on cucumber. Though there were no isolates collected from toma to that grouped in PL1 and PL2, isolates from these lineages produced a hypersen sitive response on tomato, showi ng pinpoint lesions that were given a disease rating of one. These data are similar to pathogenicity tests using 18 C. cassiicola isolates from Nigeria, the Southern U.S., and Mexico (Onesirosan et al. 1973) in that both studies found isolates specific to papaya and cucumber. Likewise, bo th studies found that isolates pathogenic to tomato also were likely to be pathogenic on several other hosts. The number of isolates screened compared to the number of unique pathogenicity pr ofiles in both studies i ndicates that gains and losses of pathogenicity are common. Growth rate at different temper atures has provided evidence for isolates adapted to tropical and temperate environments. Using an isolate collected from toma to in Florida, Pernezny et al. (2000) found the best colony growth occurred at 32C, whereas Sobers (1966) reported an optimum growth rate at 24C for Florida isolates collected from hydrangea and azalea. Jones and Jones (1984) report higher disease severity on tomato inoculated a nd maintained at temperatures between 20-23 C. In this study, two temperatur e extremes (23 C and 33 C) were chosen in attempt to discern between isolates adapted to temperate and tropical climates. Though the majority of isolates were collected from tropical c limates, all isolates grew faster at 23 C than 33 C. Growth rate also strongly correlated with phylogenetic lineage. Isol ates from PL2 and PL4 may be more adapted to warmer temperatures, and isolates from PL5 and PL3 might be more adapted to cooler temperatures. Such phys iological traits, incl uding growth rate and

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70 pathogenicity profile, correlate with phylogene tic data and may be useful for isolate classification. These studies have shown that the rDNA ITS sequence will be useful for the initial screening of isolates and for isolate selection for resistance breeding. The rDNA ITS region was useful for the grouping of isolates into three grou ps (PL1 (haplotype TG), PL4 (haplotype CA), and PLs 2, 3, 5, and 6 (haplotype CG)) that correlate with phylogenetic data from the combined four locus data set. For example, isolates fr om PL1 (rDNA ITS haplotype TG) should be used to screen for resistance to target spot in papaya. In contrast, isolates from PL2 and PL4 (rDNA ITS haplotypes CA and CG) should be used to screen for resistance to target spot in tomato. In addition, genotyping by restriction digest of the amplified ITS region is possible now that specific polymorphisms have been identified an d mapped. For example, use of the enzyme HpyCH4V (recognition sequence TGCA) will cut in two positions in haplotypes CA and CG, but only one position in haplotype TG. Additionally, this research found isolates with the same unique genotype found in Atan and Hamids (2003) RFLP analysis of the rDNA ITS region using HaeIII. However, only four of the 143 isolates we sequenced shared this polymorphism at base pair 123, rendering RFLP analysis of the rDNA ITS region using HaeIII ineffective for distinguishing among the majority of isolates. Despite evidence for host specificity (on African violet, Lantana papaya, and Stachytarpheta for example), the combined pathogenici ty and phylogenetic data indicate that there are many hosts with the potential to harbor C. cassiicola isolates pathogenic to susceptible crops such as basil, cucumber, and tomato. Studi es that incorporate many isolates from the same host across diverse locations, the sequencing of additional loci, and subsequent pathogenicity screening, will no doubt reveal additional ge netic diversity and host specificities.

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71 It is hoped that this research will aid ot hers in unraveling the many complexities that remain to be discovered with respect to C. cassiicola and its disease development in the field. For example, more studies are needed to explain why C. cassiicola is rare in Hawaii on all cultivated crops except basil, if there are isolates adapted to tr opical and temperate climates, and how isolate genotype and pathogeni city profiles are corre lated using more diverse isolates and hosts.

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72 Table 2-1. Isolate designations geographic location of isolati on, host of isolation, phylogenetic lineage (PL), type of growth on a ssociated host, and species of Corynespora used in the phylogenetic analyses. Isolate ID Location Host PL Growth Species CABI 211585 New Zealand Poncirus trifoliatus O endophytic C. citricola CBS 169.77 New Zealand Poncirus trifoliatus O endophytic C. citricola NIAS 425231 Japan Ocimum basilicum 1 pathogen C. citricola NIAS 712045 Japan Solanum melongenea ? pathogenic C. melongenae CBS 291.74 Netherlands Tilia spp. O saprophyte C. olivacea CBS 112393 Italy Fagus sylvatica O endophytic C. proliferata NIAS 305095 Japan Sesamum indicum 1 pathogenic C. sesamum CABI 5649b England Fagus sylvatica O saprophyte C. smithii AS49 Amer. Samoa Solanum lycopersicum 3 pathogenic C. cassiicola AS50 Amer. Samoa Solanum lycopersicum 3 pathogenic C. cassiicola AS54 Amer. Samoa Vigna unguiculata 1 saprophyte C. cassiicola AS58 Amer. Samoa Vigna unguiculata 1 saprophyte C. cassiicola AS65 Amer. Samoa Solanum melongenea 6 saprophyte C. cassiicola AS67 Amer. Samoa Commelina benghalensis 1 pathogenic C. cassiicola AS71 Amer. Samoa Cucurbita pepo 1 saprophyte C. cassiicola AS78 Amer. Samoa Ocimum basilicum 1 pathogenic C. cassiicola AS80 Amer. Samoa Ocimum basilicum 3.1 pathogenic C. cassiicola AS81 Amer. Samoa Clerodendrum quadriloculare 1 pathogenic C. cassiicola AS92 Amer. Samoa Cucumis sativus 6 pathogenic C. cassiicola AS98 Amer. Samoa Cucumis sativus 1 pathogenic C. cassiicola AS117 Amer. Samoa Carica papaya fruit 3.1 saprophyte C. cassiicola AS119 Amer. Samoa Cucurbita pepo 3.1 saprophyte C. cassiicola DOA16b Brazil Carica papaya 1.1 pathogenic C. cassiicola JMP216a Brazil Lantana camara 6 pathogenic C. cassiicola JMP217 Brazil Solanum lycopersicum 6 pathogenic C. cassiicola JMP218 Brazil Glycine max 1 pathogenic C. cassiicola RWB321 Brazil Coleus barbatus 4 pathogenic C. cassiicola FL09 FL, USA Lantana camara 6 pathogenic C. cassiicola FL11 FL, USA Carica papaya 1.1 pathogenic C. cassiicola FL12 FL, USA Solanum lycopersicum 6 pathogenic C. cassiicola FL15 FL, USA Salvia farinacea 6 pathogenic C. cassiicola FL21 FL, USA Bauhinia galpinii 6 pathogenic C. cassiicola FL34 FL, USA Tabebouia pallida 1 pathogenic C. cassiicola FL36 FL, USA Catharanthus roseus 2 pathogenic C. cassiicola FL37 FL, USA Clerodendrum paniculatum 1 pathogenic C. cassiicola FL50 FL, USA Hydrangea macrophylla ? pathogenic C. cassiicola FL51 FL, USA Vaccinium corymbosum ? pathogenic C. cassiicola FL62 FL, USA Coleus barbatus 1 pathogenic C. cassiicola FL757 FL, USA Origanum vulgare 1 pathogenic C. cassiicola FL2920 FL, USA Solanum lycopersicum 6 pathogenic C. cassiicola MS31 MS, USA Solanum lycopersicum 6 pathogenic C. cassiicola TN3-3 TN, USA Saintpaulia ionantha 5 pathogenic C. cassiicola GU01 Guam Cassia fistula 6 saprophyte C. cassiicola

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73 Table 2-1. Continued. Isolate ID Location Host PL Growth Species GU06 Guam Hyptus suarelens 6 endophytic C. cassiicola GU08 Guam Lantana camara 1 pathogenic C. cassiicola GU10 Guam Codiaeum variegatum 1 endophytic C. cassiicola GU11 Guam Citrullus vulgaris 1 saprophyte C. cassiicola GU12 Guam Calopogonium mucunoides 6 pathogenic C. cassiicola GU14 Guam Calyptocarpus vialis 6 pathogenic C. cassiicola GU16 Guam Asystasia gangetica 3 pathogenic C. cassiicola GU21 Guam Buddleja asiatica 1 pathogenic C. cassiicola GU23 Guam Ipomoea batatas 6 endophytic C. cassiicola GU25 Guam Buchnera floridana 1 pathogenic C. cassiicola GU28 Guam Solanum lycopersicum 6 pathogenic C. cassiicola GU32 Guam Euphorbia heterophylla 3 endophytic C. cassiicola GU38 Guam Allamanda cathartica 2 pathogenic C. cassiicola GU41 Guam Eugenia uniflora 6 endophytic C. cassiicola GU42 Guam Bidens alba 1 pathogenic C. cassiicola GU44 Guam Jatropha curcas 6 endophytic C. cassiicola GU49 Guam Syzygium jambos 6 endophytic C. cassiicola GU51 Guam Meisosperma oppositifolium 1 endophytic C. cassiicola GU55 Guam Calopogonium mucunoides 3.1 pathogenic C. cassiicola GU65 Guam Passiflora foetida 3 endophytic C. cassiicola GU68 Guam Moringa oleifera 3 endophytic C. cassiicola GU70 Guam Solanum melongenea 1.3 endophytic C. cassiicola GU79 Guam Acanthus ilicifolius 3 endophytic C. cassiicola GU83 Guam Euphorbia heterophylla 6 endophytic C. cassiicola GU90 Guam Stachytarpheta jamaicensis 1 pathogenic C. cassiicola GU92 Guam Carica papaya 1.1 pathogenic C. cassiicola GU93 Guam Capsicum annum 1 endoph ytic C. cassiicola GU98 Guam Spathodea campanulata 6 pathogenic C. cassiicola GU99 Guam Saintpaulia ionantha 5 pathogenic C. cassiicola GU101 Guam Euphorbia milii 6 saprophyte C. cassiicola GU102 Guam Phaseolus vulgaris 6 saprophyte C. cassiicola GU103 Guam Pilea nummulariifolia 2 endophytic C. cassiicola GU104 Guam Macroptilium atropurpureum 1 pathogenic C. cassiicola GU107 Guam Mikania micrantha 6 pathogenic C. cassiicola GU109 Guam Bauhinia galpinii 3 pathogenic C. cassiicola GU110 Guam Plectranthus ambionicus 3 pathogenic C. cassiicola GU111 Guam Manihot esculenta 6 endophytic C. cassiicola GU112 Guam Glycine max 3 endophytic C. cassiicola GU114 Guam Teramnus labialis 3 endophytic C. cassiicola GU115 Guam Vitex parviflora 1 pathogenic C. cassiicola GU120 Guam Coleus barbatus 6 pathogenic C. cassiicola GU128 Guam Solanum lycopersicum 6 pathogenic C. cassiicola GU136 Guam Ficus benjamani 6.1 endophytic C. cassiicola HI01 Oahu, Hawaii Ocimum basilicum 1 pathogenic C. cassiicola CBPP Malaysia Hevea brasiliensis clone unk. 1.2 pathogenic C. cassiicola CLN16 Malaysia Hevea brasiliensis RRIM 2020 1.2 pathogenic C. cassiicola

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74 Table 2-1. Continued. Isolate ID Location Host PL Growth Species CSB12 Malaysia Hevea brasiliensis RRIM 725 2 pathogenic C. cassiicola GU136 Guam Ficus benjamani 6.1 endophytic C. cassiicola HI01 Oahu, Hawaii Ocimum basilicum 1 pathogenic C. cassiicola CBPP Malaysia Hevea brasiliensis clone unk. 1.2 pathogenic C. cassiicola CLN16 Malaysia Hevea brasiliensis RRIM 2020 1.2 pathogenic C. cassiicola CSB12 Malaysia Hevea brasiliensis RRIM 725 2 pathogenic C. cassiicola PH01 Pohnpei Carica papaya 1.1 endophytic C. cassiicola PW01 Palau Carica papaya 1.1 pathogenic C. cassiicola PW12 Palau Carica papaya 1.1 pathogenic C. cassiicola PW17 Palau Carica papaya 1.1 pathogenic C. cassiicola PW20 Palau Carica papaya 1.1 pathogenic C. cassiicola PW25 Palau Carica papaya 1.1 pathogenic C. cassiicola PW27 Palau Carica papaya 1.1 pathogenic C. cassiicola PW34 Palau Carica papaya 1.1 pathogenic C. cassiicola PW37 Palau Carica papaya 1.1 pathogenic C. cassiicola PW38 Palau Carica papaya 1.1 pathogenic C. cassiicola PW43 Palau Carica papaya 1.1 pathogenic C. cassiicola PW48 Palau Carica papaya 1.1 pathogenic C. cassiicola PW53 Palau Carica papaya 1.1 pathogenic C. cassiicola PW56 Palau Carica papaya 1.1 pathogenic C. cassiicola PW57 Palau Solanum lycopersicum 6 pathogenic C. cassiicola PW63 Palau Solanum lycopersicum 6 pathogenic C. cassiicola PW69 Palau Piper betle 2 endophytic C. cassiicola PW79 Palau Pilea microphylla 2 pathogenic C. cassiicola PW80 Palau Saintpaulia ionantha 1 pathogenic C. cassiicola PW83 Palau Saintpaulia ionantha 1 pathogenic C. cassiicola PW87 Palau Cucumis sativus 1 pathogenic C. cassiicola PW89 Palau Chromolaena odorata 6 endophytic C. cassiicola PW91 Palau Luffa acutangula 1 endophytic C. cassiicola PW92 Palau Catharanthus roseus 1 pathogenic C. cassiicola PW94 Palau Stachytarpheta jamaicensis 1 pathogenic C. cassiicola PW99 Palau Momordica charantia 3 pathogenic C. cassiicola PW101 Palau Vigna unguiculata 4 saprophyte C. cassiicola SN03 Saipan Momordica charantia 1 pathogenic C. cassiicola SN05 Saipan Ipomoea batatas 1 pathogenic C. cassiicola SN06 Saipan Luffa acutangula 3.1 endophytic C. cassiicola SN07 Saipan Carica papaya 1.1 endophytic C. cassiicola SN18 Saipan Carica papaya 1.1 pathogenic C. cassiicola SN24 Saipan Solanum lycopersicum 6 pathogenic C. cassiicola SN27 Saipan Solanum lycopersicum 6 pathogenic C. cassiicola SN30 Saipan Solanum lycopersicum 6 pathogenic C. cassiicola SN37 Saipan Vigna unguiculata 6 saprophyte C. cassiicola SN40 Saipan Cucumis sativus 6 pathogenic C. cassiicola SN43 Saipan Saintpaulia ionantha 3 pathogenic C. cassiicola SN48 Saipan Coccinia grandis 3 endophytic C. cassiicola SN53 Saipan Carica papaya 1.1 pathogenic C. cassiicola

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75 Table 2-1. Continued. Isolate ID Location Host PL Growth Species SN59 Saipan Lantana camara 1.4 pathogenic C. cassiicola SN64 Saipan Asystasia gangetica 4.1 pathogenic C. cassiicola SN69 Saipan Pachystachys lutea 3 pathogenic C. cassiicola YP01 Yap Carica papaya 1.1 pathogenic C. cassiicola YP08 Yap Carica papaya 1.1 pathogenic C. cassiicola YP17 Yap Carica papaya 1.1 pathogenic C. cassiicola YP26 Yap Cucumis sativus 1 pathogenic C. cassiicola YP27 Yap Cucumis sativus 2 pathogenic C. cassiicola YP29 Yap Cucumis sativus 1 pathogenic C. cassiicola YP41 Yap Saintpaulia ionantha 2 pathogenic C. cassiicola YP42 Yap Solanum lycopersicum 3 pathogenic C. cassiicola YP51 Yap Vigna unguiculata 1 saprophyte C. cassiicola YP59 Yap Ipomoea batatas 2 endophytic C. cassiicola Information on Corynespora cassiicola isolates used in this study including location, original host, phylogenetic lineage (PL), type of growth in associa tion with the host (endophytic or pathogenic), and the Corynespora species. The first eight isolates were solic ited from culture collections as outgroups (O). Three isolates from the NIAS culture collection (305095, 425231, and 712045) are likely misidentified because they grouped with C. cassiicola isolates according to sequence data. They are labeled here according to the original culture collection desi gnations, though they shoul d be re-classified as C. cassiicola The remaining isolates were collected as part of this study or solicited from other researchers and are listed according to geographic location.

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76 Table 2-2. Summary of sequence da ta from four loci used to c onfirm the phylogenetic lineage of Corynespora cassiicola isolates. Locus Total Variable Informative Tree ScoreNo. MP Trees Combineda 2136 248 1743307430 rDNA ITS 1013 135 1001589990 rDNA ITSb 584 4 34 1 Cc-ga4 414 31 25409530 Cc-ga4b 414 28 25369560 Cc-caa5 366 38 3452 40 Cc-caa5b 366 37 3246 12 Cc-act1 343 44 1549 11 Cc-act1b 343 16 1517 4a Combined loci: rDNA ITS, Cc-ga4, Cc-caa5, and Cc-act1 b Locus analyzed with only C. cassiicola taxa represented (no outgroups).

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77Table 2-3. Pathogenicity profiles for 50 Corynespora cassiicola isolates. Path Pro PL Isolate Host Ba Be Co Cu Pa So Sw To I S I S I S I S I S I S I S I S BaCoCu 1 AS78 Ba 52.6 041.8 62.3 0011 21 BaCoCu 1 AS58 Co 62.3 032.3 63 00071 BaCoCu 1 YP29 Cu 72.1 031.7 83 0000BaCoCu 1 AS71 Pu 82.1 022 71.3 011 021 BaCoCuSo 1 PW87 Cu 52.2 051.6 82.5 021.5 071 BaCu 1 HI01 Ba 42.5 000 73 0011 81 BaCu 1 SN05 Sw 71.9 11 00 62.7 00021 BaCuTo 3 AS50 To 52.2 021 62.7 011 21 83 BaCuTo 3 YP42 To 62.3 0072.6 00082.6 BaCuTo 3.1 AS80 Ba 51.8 0082.5 011 082.8 BaCuTo 3.1 AS117 Sap 71.9 021 82.8 021 81 72.9 BeCoCuSw 4 SN37 Co 71 51.6 51.4 82.8 021 31.3 81 BeCuSwTo 4 JMP217 To 51 71.9 082.9 0012 82.5 BeCuSwTo 4 GU102 Be 071.3 082.6 0021.5 82.5 BeCuSwTo 4 SN40 Cu 061.3 082.5 0012 82.6 BeCuTo 4 PW57 To 061.7 082.1 021 083 CoCu 1 AS98 Cu 051 61.8 82.6 00071 CoCu 1 YP26 Cu 071 41.5 72.4 0081 81 CoCu 1 JMP218 So 0072.1 83 0071 71 CoCu 1 GU08 La 0031.7 73 00081 Cu 1 AS54 Co 011 083 011 81 11 Cu 1 YP51 Co 00083 0071 11 CuPa 1.1 DOA16b Pa 71 0081.1 82.3 081 11 CuPa 1.1 FL11 Pa 051 081.3 72.6 0081 CuPa 1.1 PH01 Pa 11 11 071.1 61.8 0081 CuSo 3 GU112 So 0051 82.8 012 11 21 CuSw 2 YP27 Cu 11 021 62.8 0051.4 21 CuSw 2 YP59 Sw 21 011 72.6 0061.4 71 CuSw 2 SN59 La 11 51 082.8 0071.3 71 CuSwTo 4 PW63 To 41 071 82.6 0012 83 CuSwTo 4 SN24 To 00082.5 0021.5 82.6 CuSwTo 4 SN27 To 51 0083 0051.2 82.9

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78 Path Pro PL Isolate Host Ba Be Co Cu Pa So Sw To I S I S I S I S I S I S I S I S CuSwTo 4 GU23 Sw 0 0 7 1 82.4 00 51.8 82.9 CuSwTo 4 FL09 La 0 1 1 0 81.4 02 1 31.3 82.8 CuTo 3 AS49 To 0 0 0 42.8 00 11 72.6 CuTo 3.1 AS119 Pu 0 0 0 82.3 01 1 71 82.5 CuTo 4 FL12 To 0 5 1 5 1 83 02 1 11 83 CuTo 4 FL2920 To 4 1 0 0 82 00 082.6 CuTo 4 MS31 To 0 7 1 8 1 82.8 00 11 82.3 CuTo 4 GU128 To 0 1 1 0 82.9 02 1 11 82.5 CuTo 4 SN30 To 0 0 0 83 02 1 21 83 CuTo 4 AS92 Cu 2 1 1 1 0 82.8 00 11 82.9 CuTo 4 JMP216a La 7 1 0 0 71.3 00 21 83 CuTo 5 PW101 Co 0 0 4 1 72.9 00 071.3 Pa 1.1 GU92 Pa 2 1 0 0 11 72.1 0 11 71 Pa 1.1 PW01 Pa 0 0 0 81 82.4 0 081 Pa 1.1 PW12 Pa 0 0 1 1 11 41.4 2 1 011 Pa 1.1 SN03 Pa 1 1 0 0 81 62.3 0 11 11 Pa 1.1 YP01 Pa 0 7 1 0 11 81.9 0 081 To 4 GU28 To 0 0 0 81 00 11 83 Path Pro (Pathogenicity Profile) : A list of susceptible hosts, or plants with an average disease rating greater than 1. PL : Phylogenetic lineage designation based on combined se quence analysis of ITS rDNA, CAA5, GA4, and ACT. Isolate : Corynespora cassiicola isolate code. Host : Original host the isolate was collected from. Ba ( Ocimum basilicum ), Be ( Phaseolus vulgarus ), Co ( Vigna unquiculata ), Cu ( Cucumis sativus ), La ( Lantana camara ), Pa ( Carica papaya), Pu ( Cucurbita pepo), Sw ( Ipomoea batatas ), To ( Solanum lycopersicum ). I (Incidence): Number of plants (out of 8 reps) that s howed symptoms seven days after inoculation with 20,000 C. cassiicola spores per ml. S (Severity): Average rating of symptomatic plants (these rated 1, 2, or 3). Plants were rated with the following scale: (0) symptomless; (1 ) non pathogenic hypersensitive response, a few to many non-expanding pinpoint lesions; (2) moderately virulent, many expanding lesio ns, some coalescing, but not resulting in blight; (3 ) highly virulent, lesions spreading to fo rm large areas of dead tissue resulting in a blighting effect.

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79 Table 2-4. Growth rate of Corynespora cassiicola isolates at 23C. Iso. ID PL Location Host Avg GR LSD GU99 6 Guam Saintpaulia 0.4743a AS81 1 Samoa Clerodendron 0.4639ab GU90 1 Guam Stachytarpheta 0.4528abc HI01 1 Oahu Basil 0.4521abcd PW94 1 Palau Stachytarpheta 0.4521abcd FL37 1 Florida Clerodendron 0.4514abcd GU104 1 Guam Macroptilium 0.4507abcde AS67 1 Samoa Commelina 0.4479bcde AS54 1 Samoa Bean 0.4444bcdef GU08 1 Guam Lantana 0.4438bcdef AS71 1 Samoa Pumpkin 0.4410bcdef SN03 1 Saipan Bitter melon 0.4389cdef YP26 1 Yap Cucumber 0.4375cdef GU136 4 Guam Ficus 0.4375cdef PW80 1 Palau Saintpaulia 0.4375cdef SN05 1 Saipan SwPotato 0.4375cdef AS58 1 Samoa Bean 0.4368cdef YP29 1 Yap Cucumber 0.4361cdef YP51 1 Yap Bean 0.4326cdef SN37 4 Saipan Bean 0.4313cdef GU115 1 Guam Vitex 0.4278defg PW92 1 Palau Catharanthus 0.4264efg AS78 1 Samoa Basil 0.4229fgh GU21 1 Guam Buddleja 0.4215fgh AS80 3 Samoa Basil 0.4202fgh PW91 1 Palau Luffa 0.4202fgh AS50 3 Samoa Tomato 0.4063ghi FL34 1 Florida Tabebouia 0.4055ghi YP08 1 Yap Papaya 0.4000hij PW79 2 Palau Pilea 0.3951ijk SN59 1 Saipan Lantana 0.3951ijk RWB321 5 Brazil Coleus 0.3945ijk JMP218 1 Brazil Soybean 0.3924ijkl CSB12 2 Malaysia Rubber 0.3917ijklm SN06 3 Saipan Luffa 0.3903ijklmn PW37 1 Palau Papaya 0.3896ijklmn FL2920 4 Florida Tomato 0.3882ijklmn SN07 1 Saipan Papaya 0.3868ijklmno PW101 5 Palau Bean 0.3833ijklmno PW99 3 Palau Bitter melon 0.3833ijklmno SN64 5 Saipan Asystasia 0.3822ijklmno GU109 3 Guam Bauhinia 0.3820ijklmno AS98 1 Samoa Cucumber 0.3819ijklmno CLN16 1 Malaysia Rubber 0.3778jklmnop PW89 4 Palau Chromolaena 0.3778jklmnop YP59 2 Yap SwPotato 0.3778jklmnop

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80 Table 2-4. Continued. Iso. ID PL Location Host Avg GR LSD AS49 3 Samoa Tomato 0.3771jklmnopq GU107 4 Guam Mikania 0.3729klmnopqr CBPP 1 Malaysia Rubber 0.3695lmnopqrs AS119 3 Samoa Papaya 0.3687lmnopqrst YP42 3 Yap Tomato 0.3674mnopqrstu YP01 1 Yap Papaya 0.3667nopqrstu AS92 4 Samoa Cucumber 0.3632opqrstu GU112 3 Guam Bean 0.3632opqrstu FL12 4 Florida Tomato 0.3556pqrstuv GU98 4 Guam Spathodea 0.3549pqrstuv GU92 1 Guam Papaya 0.3529qrstuvw FL09 4 Florida Lantana 0.3521rstuvw YP17 1 Yap Papaya 0.3521rstuvw GU102 4 Guam Bean 0.3507rstuvwx GU38 2 Guam Allamanda 0.3507rstuvwx PW57 4 Palau Tomato 0.3500rstuvwx YP41 2 Yap Saintpaulia 0.3480stuvwxy AS117 3 Samoa Papaya 0.3458stuvwxy SN40 4 Saipan Cucumber 0.3444tuvwxy AS65 4 Saipan Eggplant 0.3437uvwxy PW01 1 Palau Papaya 0.3368vwxyz GU41 4 Guam Eugenia 0.3361vwxyz YP27 2 Yap Cucumber 0.3340vwxyz FL36 2 Florida Catharanthus 0.3312vwxyz JMP217 4 Brazil Tomato 0.3299wxyz SN24 4 Saipan Tomato 0.3264xyz DOA16b 1 Brazil Papaya 0.3250yz FL15 4 Florida Salvia 0.3146z GU120 4 Guam Coleus 0.2792A JMP216a 4 Brazil Lantana 0.2535B PH01 1 Pohnpei Papaya 0.1479C PL: Phylogenetic Lineage based on sequence data from 4 loci. Avg GR: Average slope (growth ra te) of three replicate plates. LSD: Average slope (growth rate) values followed by different letters ar e significantly different from one another according to l east significant difference test ( P <0.05).

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81 Table 2-5. Growth rate of Corynespora cassiicola isolates at 33C. Iso. ID PL Location Host Avg GR LSD AS71 1 Samoa Pumpkin 0.4153a FL37 1 Florida Clerodendron 0.3972b AS78 1 Samoa Basil 0.3965b SN37 4 Saipan Bean 0.3917bc PW92 1 Palau Catharanthus 0.3813bcd SN03 1 Saipan Bitter melon 0.3799bcd GU90 1 Guam Stachytarpheta 0.3778cd GU99 6 Guam Saintpaulia 0.3771cde AS67 1 Samoa Commelina 0.3764cde PW79 2 Palau Pilea 0.3722def PW80 1 Palau Saintpaulia 0.3715defg HI01 1 Oahu Basil 0.3680defgh GU136 4 Guam Ficus 0.3674defghi YP51 1 Yap Bean 0.3653defghi SN05 1 Saipan SwPotato 0.3597efghij GU115 1 Guam Vitex 0.3577fghij GU21 1 Guam Buddleja 0.3576fghij YP26 1 Yap Cucumber 0.3569fghij YP29 1 Yap Cucumber 0.3548fghij AS54 1 Samoa Bean 0.3542ghijk AS58 1 Samoa Bean 0.3542ghijk GU104 1 Guam Macroptilium 0.3542ghijk PW91 1 Palau Luffa 0.3514hijkl GU08 1 Guam Lantana 0.3500ijklm AS98 1 Samoa Cucumber 0.3451jklmno SN07 1 Saipan Papaya 0.3368klmno YP08 1 Yap Papaya 0.3354lmno AS92 4 Samoa Cucumber 0.3327mnop PW94 1 Palau Stachytarpheta 0.3292nopq JMP218 1 Brazil Soybean 0.3285nopq GU98 4 Guam Spathodea 0.3278nopq PW89 4 Palau Chromolaena 0.3278nopq YP17 1 Yap Papaya 0.3236opqr PW37 1 Palau Papaya 0.3224opqr GU107 4 Guam Mikania 0.3215opqr PH01 1 Pohnpei Papaya 0.3174pqrs DOA16b 1 Brazil Papaya 0.3132qrst GU102 4 Guam Bean 0.3125qrstu PW01 1 Palau Papaya 0.3063rstuv GU92 1 Guam Papaya 0.3028stuv YP01 1 Yap Papaya 0.3014stuv PW57 4 Palau Tomato 0.2993tuvw GU41 4 Guam Eugenia 0.2959tuvwx AS81 1 Samoa Clerodendron 0.2952uvwx SN40 4 Saipan Cucumber 0.2951uvwx PW101 5 Palau Bean 0.2951uvwx

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82 Table 2-5. Continued. Iso. ID PL Location Host Avg GR LSD YP41 2 Yap Saintpaulia 0.2903vwxy FL2920 4 Florida Tomato 0.2889vwxy SN64 5 Saipan Asystasia 0.2829wxyz CLN16 1 Malaysia Rubber 0.2792xyz CSB12 2 Malaysia Rubber 0.2771yz CBPP 1 Malaysia Rubber 0.2736yz YP59 2 Yap SwPotato 0.2688zA FL15 4 Florida Salvia 0.2686zA YP27 2 Yap Cucumber 0.2667zA FL36 2 Florida Catharanthus 0.2521AB PW99 3 Palau Bitter melon 0.2486BC GU109 3 Guam Bauhinia 0.2438BC SN24 4 Saipan Tomato 0.2431BC FL12 4 Florida Tomato 0.2326CD AS80 3 Samoa Basil 0.2313CD GU38 2 Guam Allamanda 0.2312CD SN06 3 Saipan Luffa 0.2250DE RWB321 5 Brazil Coleus 0.2188DE FL09 4 Florida Lantana 0.2097EF AS65 4 Saipan Eggplant 0.2076EF AS50 3 Samoa Tomato 0.1993FG YP42 3 Yap Tomato 0.1938GFH SN59 1 Saipan Lantana 0.1875GHI FL34 1 Florida Tabebouia 0.1763HIJ JMP216a 4 Brazil Lantana 0.1750IJ GU112 3 Guam Bean 0.1709IJK GU120 4 Guam Coleus 0.1688JKL AS49 3 Samoa Tomato 0.1660JKL JMP217 4 Brazil Tomato 0.1549KLM AS117 3 Samoa Papaya 0.1521LM AS119 3 Samoa Papaya 0.1382M PL: Phylogenetic Lineage based on sequence data from 4 loci. Avg GR: Average slope (growth ra te) of three replicate plates. LSD: Average slope (growth rate) values followed by different letters ar e significantly different from one another according to l east significant difference test ( P <0.05).

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83 Figure 2-1. Fifty percent majority rule consen sus tree-phylogram from Bayesian inference analysis of combined data from rDNA ITS, Cc-ga4, Cc-caa5, and Cc-act1 sequences. Numbers above branches indicate maximum parsimony bootstrap > 70% and numbers below branches indicate posterior probability values > 0.90. Pathogenicity profiles on eight crop plants: basil (Ba), bean (Be), cowpea (Co), cucumber (Cu), papaya (Pa), soybean (So), sweet potato (Sw), and tomato (To), and phylogenetic lineage (PL) are indicated.

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84 Figure 2-2. Fifty percent majority rule consen sus tree-phylogram from Bayesian inference analysis of rDNA ITS locus. Numbers above branches indicate maximum parsimony bootstrap > 70% and numbers below branches indicate posterior pr obability values > 0.90. 100,000 maximum parsimony trees were a result of only 3 informative characters within C. cassiicola Phylogenetic lineages (PL) are indicated.

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85 Figure 2-3. Fifty percent majority rule consen sus tree-phylogram from Bayesian inference analysis of the Cc-caa5 locus. Numbers above branches indicate maximum parsimony bootstrap > 70% and numbers below branches indicate posterior probability values > 0.90. Phylogene tic lineages (PL) are indicated.

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86 Figure 2-4. Fifty percent majority rule consen sus tree-phylogram from Bayesian inference analysis of the Cc-ga4 locus. Numbers above branches indicate maximum parsimony bootstrap > 70% and numbers below branches indicate posterior pr obability values > 0.90. Phylogenetic lineages (PL) are indicated.

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87 Figure 2-5. Fifty percent majority rule consen sus tree-phylogram from Bayesian inference analysis of the Cc-act1 locus. Numbers above br anches indicate maximum parsimony bootstrap > 70% and numbers below branches indicate posterior probability values > 0.90. Phylogene tic lineages (PL) are indicated.

PAGE 88

88 Figure 2-6. UPGMA dendrogram of 50 Corynespora cassiicola isolates based on pathogenicity profiles on eight crop plants: basil (Ba), bean (Be), cowpea (Co), cucumber (Cu), papaya (Pa), soybean (So), sweet potato (Sw) tomato (To). Isolates are labeled with their phylogenetic lineag e (PL) designation to demonstrat e that isolates from the same PL cluster together. Statistical s upport for nodes by 1,000 UPGMA Bootstrap repetitions is indicated.

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89 Figure 2-7. Demonstration of the C. cassiicola disease rating system. Symptoms on A) basil, B) bean, C) cowpea, and D) tomato plants se ven days after inoculation with different isolates of Corynespora cassiicola. Plants were rated with the following scale: (0) symptomless; (1) non pathogenic hypers ensitive response, a few to many nonexpanding pinpoint lesions; (2) moderately virulent, many expanding lesions, some coalescing, but not resulting in blight; (3) hi ghly virulent, lesions spreading to form large areas of dead tissue re sulting in a blighting effect.

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90 LIST OF REFERENCES Abul-Hayja ZP, W illiams H, Peterson CE (1978) Inheritance of resistan ce to anthracnose and target leaf spot in cucumbers. Plant Dis Rep 62:43-45 Acharya B, Mishra SK, Acharya A, Mohapatr a KB, and Das, AK (2003) Bioassay of culture metabolites of Corynespora cassiicola ( Berk. Curt. ) Wei on the detached plant parts of betelvine ( Piper betle L.). Orissa J Hortic 31:8-9 Ahmad S (1969) Fungi of West Pa kistan. Biological Society of Pakistan Monograph 5(Sup.1):1110 Alfieri SA Jr, Langdon KR, Wehlburg C, Kimbrough JW (1984) Index of Plant Diseases in Florida. Florida Dept. of Agriculture and C onsumer Sciences, Div. Of Plant Industry. Bull. No. 11 (Revised), pp 389 Alfieri SA Jr, Langdon KR, Kimbrough JW, El-G holl NE, Wehlburg C (1994) Diseases and Disorders of Plants in Florida. Florida Department of Agriculture and Consumer Services, pp 1114 Anderson PJ, Dixon WN (2004) Florida Department of Agriculture and Consumer Services Plant Pathology Section, Ornamentals, Folia ge Plants, Tri-ology, Vol. 43, No. 1 Arnold GRW (1986) Lista de Hongos Fitopatogenos de Cuba. Ministerio de Cultura Editorial Cientifico-Tecnica, 207 pp Atan S, Hamid NH (2003) Differentiating races of Corynespora cassiicola using RAPD and internal transcribed spacer markers. J Rub Res 6:58-64 Barreto RW, Evans HC (1998) Fungal pathogens of Euphorbia heterophylla and E. hirta in Brazil and their potential as weed biocontrol agents Mycopathologia 141:21-36 Barthe P, Pujade-Renauld V, Breton F, Gargani D, Thai R, Roumestand C, de Lamotte F (2007) Structural analysis of cassiicolin, a host-selective protein toxin from Corynespora cassiicola. J Mol Biol 367:89-101 Beaver RG (1981) Guam Agricultural Experiment Station Annual Report, 36 pp Bird J, Krochmal A, Zentmyer G, Adsuar J (1966 ) Fungus diseases of papaya in the U.S. Virgin Islands. J Agric Univ Puer Rico 50:186-200 Blazquez CH (1967) Corynespora leaf spot of cucumber. Fla Agric Exp Stn J Ser No 2858, pp 177-182 Blazquez CH (1968) Corynespora cassiicola on bananas. Phytopath ology 52:1347 (Abstr.) Blazquez CH (1972) Target spot of tomato. Plant Dis Rep 56:243-245

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91 Bliss FA, Onesirosan PT, Arny DC ( 1973) Inheritance of resistance in tomato to target leaf spot. Phytopathology 63:837-840 Boa E, Lenn J (1994) Diseases of Nitrogen Fixing Trees in Developing Countries. An annotated list. Natural Resources Inst., Kent, United Kingdom, pp 82 Boosalis MG, Hamilton RI (1957) Root and stem rot of soybean caused by Corynespora cassiicola Plant Dis Rep 41:8:696-698 Brooks F (2002) List of plant di seases in American Samoa. Land Grant Technical Report No. 38. 50 CABI Databases (2008, July 21). Herb. IMI records for Fungus: Corynespora cassiicola. Retrieved July 21, 2008 from: http://194.203.77.76/herbIMI/DisplayResults .asp? strName=Corynespora+cassiicola Carbone I, Kohn LM (1999) A me thod for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91:553-556 Casady W (1994) Florida Department of Agricu lture and Consumer Services, P94-5328, Triology, Vol. 33, No.6, Nancy C. Coile Managi ng Editor. Retrieved May 12, 2008 from: http://www.doacs.state.fl .us/pi/enpp/94-11&12all.htm Chase AR (1981) Com parison of Myrothecium sp. and Corynespora cassiicola leaf spots of two cultivars of Aphelandra squarrosa. Proc Fla State Hortic Soc 94:115-116 Chase AR (1982) Corynespora leaf spot of Aeschynanthus pulcher and related plants. Plant Dis 66:739-740 Chase AR (1984) Leaf spot disease of Ficus benjamina caused by Corynespora cassiicola Plant Dis 68:251 Chase AR (1986) Corynespora bract spot of Euphorbia pulcherrima in Florida. Plant Dis 70:1074 Chase AR (1987) Compendium of Ornament al Foliage Plant Diseases. American Phytopathological Society Press, St. Paul, 92 pp Chase AR (1993) Corynespora l eaf spot and stem rot of Salvias. CFREC-Apopka Research Report, RH-93-12 Cheeran A (1968) Leaf and stem bli ght of Japanese Mentha caused by Corynespora cassiicola Agric Res J Karala 6:141

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92 Cho WD, Shin HD (2004) List of Plant Diseases in Korea. Fourth ed. Korean Society of Plant Pathology, 779 pp Coile NC, Dixon WN (1994) Florida Department of Agriculture and Consumer Services Plant Pathology Section, Ornamentals, Folia ge Plants, Tri-ology, Vol. 33, No. 5 Collado J, Platas G, Gonzalez I, and Pelaez F (1999) Geographical and seasonal influences on the distribution of fungal endophytes in Quercus ilex New Phytol 144:525-532 Cutrim FA, Silva GS (2 003) Pathogenicity of Corynespora cassiicola to different plant species. Fitopatol Brasil 28:193-194 Da Silva JL, Soares DJ, Barreto RW (2005) Eye-spot of Rudbeckia laciniata caused by Corynespora cassiicola in Brazil. Br it Soc Plant Pathol, New Dis Rep No. 12 Dade HA (1940) A revised list of Gold Coast fungi and plant diseases. XXIX. Bull. Misc. Inform. Kew 6:205-247 Daughtrey M (2000) Diseases of bleeding heart ( Clerodendrum thomsoniae Balf.). APSnet: Common names of plant diseases. Plant Pathology Online. http://www.apsnet.org/online/ common/names/bleedhrt.asp Delgado-Rodriguez G, Mena-Porta les J (2004) Hifomicetos (hongos anamorficos) de la reserva ecologica "alturas de banao" (Cuba). Bol Soc Micol Madrid 28:115-124 Delgado-Rodriguez G, Mena-Portales J, Cal duch M, Decock C (2002) Hyphomycetes (hongos mitosporicos) del area protegida mil cumbre s, Cuba Occidental. Cryptog Mycol 23:277-293 Dixon WN (1997) Florida Department of Agricu lture and Consumer Services Plant Pathology Section, Ornamentals, Foliage Pl ants, Tri-ology, Vol. 36, No 2 Duarte MLR, Albuquerque FC, Prabhu AS (1978) A new leaf disease of cacao plants ( Theobroma cacao ) caused by the fungus Corynespora cassiicola. Fitopatol Brasil 3:259-265 El-Gholl NE, Schubert TS (1990) Corynespora leaf spot of Tabebuia Fla Dept Agric & Consumer Serv. Division of Plant Industry. Plant Pathol Circ No 328 El-Gholl NE, Schubert TS, Coile NC (1997) Diseases and disorders of plants in Florida. Bulletin No. 14 Supplement No 1. Fla Dept of Agric and Cons Serv, pp 90-91 Ellis MB (1957) Some species of Corynespora. Mycological Papers 65:1-15 Ellis MB, Holliday P (1971) Corynespora cassiicola (Berk. & Curt.) Wei. CMI Descriptions of Fungi and Bacteria No 31, Sheet 303

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93 Fajola AO, Alasoadura SO (1973) Corynespora leaf spot, a new disease of tobacco ( Nicotiana tabacum ). Plant Dis Rep 57:375-378 Farr DF, Rossman AY, Palm ME, McCray EB. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Re trieved May 24, 2008, from http://nt.arsgrin.gov/fungaldatabases/ Felsenstein J (1985) Confidence limit on phylog enies: an approach using the bootstrap. Evolution; International Journa l of Organic Evolution 39:783-791 Fernandes RC, Barreto RW (2003) Corynespora cassiicola causing leaf spots on Coleus barbatus Plant Pathol 52:786 Ferreira FA (1989) Principais Doencas Florestais no Brasil. Patologia Florestal. Vicosa. MG Minas Gerais, 570 pp Florence EJM, Sharma JK (1987) Corynespora cassiicola : a new leaf pathogen for Gmelina arborea in India. J Trop For 3:181-182 Freire FCO (2005) An updated list of plant fungi from Cear state (Brazil) I Hyphomycetes. Revista Cincia Agronmica 36:364-370 Furukawa T, Ushiyama K, and Kishi K (2008) Cor ynespora leaf spot of scarlet sage caused by Corynespora cassiicola J Gen Plant Pathol 74:117-119 Gond SK, Verma VC, Kumar A, Kumar V, Kh arwar RN (2007) Study of endophytic fungal community from different parts of Aegle marmelos Correae ( Rutaceae) from Varanasi (India). World J Microbiol Biotechnol 23:1371-1375 Gowda CLL, Ramakrishna A, Rupela OP, Wa ni SP (2001) Legumes in Rice-Based Cropping Systems in Tropical Asia. Andhra Pradesh, India, pp 11-25 Grand LF (1985) North Carolina Plant Disease I ndex. North Carolina Agric Res Serv Techn Bull 240:1-157 Guo YL (1992) Foliicolous hyphomycetes of G uniujiang in Anhui Province II. Mycosystema 5:109-112 Hasama W, Morita S, Kato T (1991) Cor ynespora leaf spot of Perilla caused by Corynespora cassiicola Annals Phytopathol Soc Jap 57:732-736 Hawaiian Ecosystems at Risk (HEAR). (2008, Ju ly 11). Pathogens of Plants of Hawaii, Corynespora cassiicola Retrieved July 11, 2008, from: http://www.hear.org/pph/pathogens/1065.htm

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94 Holliday P (1980) Fungus Diseases of Tropical Cr ops. Cambridge University Press. Cambridge, UK Hongn S, Ramallo A, Baino O, Ramallo JC (2007) First Report of Target Spot of Vaccinium corymbosum caused by Corynespora cassiicola Plant Dis 91:771 Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogeny. Bioinformatics (Oxford, England) 17:754-755 Hyde KD, Alcorn JL (1993) Some disease-asso ciated microorganisms on plants of Cape York Peninsula and Torres Strait Islands. Australas Plant Pathol 22: 73-83 Hyde KD, McKenzie EHC, Dalisay TU (2001) Saprobic fungi on bamboo culms. Fungal Divers 7:35-48 Isabel N, Beaulieu J, Theriault P, Bousquet J (1999) Direct evid ence for biased gene diversity estimates from dominant random amplified polymorphic DNA (RAPD) fingerprints. Molecular Ecology 8:477-483 Johnston A (1960) A supplement to a host list of plant diseases in Ma laya. Mycol Pap 77:1-30 Jones JP (1961) A leaf spot of cotton caused by Corynespora cassiicola Phytopathology 51:305308 Jones JP, and Jones JB (1984) Target spot of to mato: epidemiology, and control. Proc Fla State Hortic Soc 97:216-218 Jones JB, Jones JP, Stall RE, Zitter TA (1991) Co mpendium of Tomato Diseases. APS Press, St. Paul, MN, 100 pp Khare MN (1991) Lentil diseases with special re ference to seed quality. Indian J Mycol Plant Pathol 21:1-13 Kingsland GC (1985) Pathogenicity and epidemiology of Corynespora cassiicola in the Republic of the Seychelles. Ac ta Hortic (ISHS) 153:229-230 Komaraiah M, Reddy SM (1986) Production of cellulases by Corynespora cassiicola Wei, a seed borne fungus of methi. Acta Botan Ind 14:133-138 Kranz J (1963) Fungi collected in the Republic of Guinea, Colle ctions from the Kindia area in 1962. Sydowia 17:174-185 Kurt S (2004) Host-specific toxin production by the tomato target leaf spot pathogen Corynespora cassiicola Turk J Agric and Fores 28:389-395

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97 Onesirosan PT, Arny DC, Durbin RD (1975) Increasing sporulation of Corynespora cassiicola Mycopathologia 55:121-123 Onesirosan PT, Arny DC, Durbin RD (1974) Host specificity of Nigerian and North American isolates of Corynespora cassiicola. Phytopathology 64:1364-1367 Onesirosan PT, Arny DC, Durbin RD (1973) Target Spot of Tomato Incited by Corynespora cassiicola (Berk. & Curt.) Wei. Ph. D. Thesis, University of Wisconsin, Madison, 93 pp Orieux L, Felix S (1968) List of plant dise ases in Mauritius. Phytopathol Pap 7:1-48 Peregrine WTH, Ahmad KB (1982) Brunei: A first annotated list of plant diseases and associated organisms. Phytopathol Pap 27:1-87 Pereira JM, Barreto RW, Ellison CA, Maffia LA (2003) Corynespora cassiicola f. sp. lantanae : a potential biocontrol agent from Brazil for Lantana camara BiolControl 26:21-31 Pernezny K, Simone GW (1993) Target spot of several vegetable crops. PP-39, A series of the Plant Pathology Department, Fla Coop Ext Ser, IFAS, Univ of Fla Pernezny K, Datnoff LE, Mueller T, Collins J (1 996) Losses in fresh-market tomato production in Florida due to target spot and bacterial spot and the benefits of protectant fungicides. Plant Dis 80:559-563 Pernezny K, Datnoff LE, Rutherford B, Carroll A (2000) Relationship of temperature to growth, sporulation, and infection of tomato by the target spot fungus. Florida Tomato Committee Tomato Research Report for 2000, pp 16-19 Pernezny K, Stoffella P, Collins J, Carroll A, Bean ey A (2002) Control of target spot of tomato with fungicides, systemic acquired resistance ac tivators, and a biocontro l agent. Plant Prot Sci 38:81-88 Pernezny KL, Datnoff LE, Smith LJ, Schlub RL (2008) An overview of target spot of tomato caused by Corynespora cassiicola. Acta Hort xxx: Second International Symposium on Tomato Diseases (accepted) Philip S, Ramakrishnan CK, Menon MR (1972) Leaf blight of Coccinia indica (Wight & Arn.) caused by Corynespora cassiicola. Agric Res J Kerala 10:196 Pollack FG, Stevenson JA (1973) A fungal pathogen of Broussonetia papyrifera collected by George Washington Carver. Plant Dis Rep 57:296-298 Poltronieri LS, Duarte MLR, Alfenas AC, Trindade DR, Albuquerque FC (2003) Three new pathogens infecting Antilles Cherry in the state of Para. Fitopatol Brasil 28:424-426

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98 Posada D, Crandall KA (1998) Modeltest: test ing the model of DNA substitution. Bioinformatics 14:817-81 Prakash O, Garg N (2007) A new report of Corynespora casiicola causing black rot of aonla seedlings. J Mycol Plant Pathol 37:120-121 Promputtha I, Lumyong A, Dhanasekaran V, McKenzie EHC, Hyde KD, Jeewon R (2007) A phylogenetic evaluation of whether endophytes become saprotrophs at host senescence. Micro Ecol 53:579-590 Puzari KC, Saikia UN (1981) Amorphophallus campanulatus a new host of Corynespora cassiicola Indian Phytopathol 34:537-538 Quimio RH, Abilay LE (1979) Note: Corynespora di sease of papaya in the Philippines. Philipp Phytopathology 15:158-161 Raabe RD, Conners IL, Martinez AP (1981) Checklist of plant dis eases in Hawaii. College of Tropical Agriculture and Human Resources, Univer sity of Hawaii. Information Text Series No. 22. Hawaii Inst Trop Agric Human Resources, 313 pp Raffel SJ, Kazmar ER, Winberg R, Oplinger ES Handelsman J, Goodman RM, Grau CR (1999) First report of root ro t of soybeans caused by Corynespora cassiicola in Wisconsin. Plant Disease 83:696 Rajak RC, Pandey AK (1985) Fungi from Jabalpu r-II. Indian J Mycol Plant Pathol 15:186-194 Riley EA (1960) A revised list of plant diseases in Tanganyika Territory. Mycol Pap 75:1-42 Romruensukharom P, Tragoonrung S, Vanavichit A, Toojinda T (2005) Genetic variability of Corynespora cassiicola populations in Thai land. J Rub Res 8:38-49 Ronquist F, Huelsenbeck JP (2003) MrBayes3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572-1574 Sadaba RB, Vrijmoed LLP, Jones EBG, Hodgkiss IJ (1995) Observations on vertical distribution of fungi associated w ith standing senescent Acanthus ilicifolius stems at Mai Po Mangrove, Hong Kong. Hydrobiologia 295:119-126 Saikia UN, Sarbhoy AK (1981) Corynespora leaf spot of Eugenia caryophyllata Indian Phytopathol 34:401-402 Sarbhoy AK, Lal G, Varshney JL (1971) Fungi of India. Navyug Traders, New Delhi, 148 pp Sarma YR, Nayudu MV (1970) Corynespora leaf s pot of Brinjal. Proc Indian Acad Sci B(LXXIV):92-97

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99 Schlub RL, Yudin L (2002) Eggplant, pepper, and tomato production guide for Guam. Guam Cooperative Extension Publication, 188 pp Seaman WL, Shoemaker RA, Peters on EA (1965) Pathogenicity of Corynespora cassiicola on soybean. Can J Bot 43:1461-1469 Shaw DE (1984) Microorganisms in Papua New Guinea. Dept. Primary Ind., Res Bull 33:1344 Shivas RG, Alcorn JL (1996) A checklist of plant pathogenic and other microfungi in the rainforests of the wet tropics of northern Queensland. Australas Mycol 25:158-173 Silva WPK, Deverall BJ, Lyon BR (1995) RFLP a nd RAPD analyses in the identification and differentiation of isolates of the leaf spot fungus Corynespora cassiicola Austral J Bot 43:609-618 Silva WPK, Deverall BJ, Lyon BR (1998) Mo lecular, physiological and pathological characterization of Corynespora l eaf spot from rubber plantations in Sri Lanka. Plant Pathol 47:267-277 Silva WPK, Karunanayake EH, Wijesundera RLC, Priyanka UMS (2003) Genetic variation in Corynespora cassiicola : a possible relationship between hos t origin and virulence. Mycol Res 107:567-571 Silva WPK, Wijesundera RLC, Karunanayake EH, Jayasinghe CK, Priyanka UMS (2000) New hosts of Corynespora cassiicola in Sri Lanka. Plant Dis 84:202 Simone GW (2000) Diseases of Cattleya Lindl. spp. APSnet: Common Names of Plant Diseases. http://www.apsnet.org/online/common/names/cattleya.asp Simone GW (2000) Diseases of Pointsettia ( Euphorbia pulcherrina ). APSnet: Common Names of Plant Diseases. http://www.apsnet .org/online/common/names/poinsett.asp Singh KP, Shukla RS, Kumar S, Hussain A (1982) A leaf-spot disease of Dodonaea viscosa caused by Corynespora cassiicola in India. Ind Phytopathol 35:325 Situmorang A, Budiman A (1984) Corynespora cassiicola (Berk. And Curt.) Wei, penyebab penyakit gugur duan pada karet. Kumpulan Ma kalah Lokakarya Karet. PNP/PTP Wilayah 1 dan P4TM, Medan Sivanesan A (1996) Corynesporasca caryote gen. et sp. nov. with a Corynespora anamorph, and the family Corynesporascaceae. Mycol Res 100:783-788 Smith LJ, Datnoff LE, Rollins JA, Pernezny KL Scott JW, Schlub RL (2008a) High genetic diversity within Corynespora cassiicola based on multilocus sequence data, pathogenicity, and growth rate. Acta Hort xxx: Second In ternational Symposium on Tomato Diseases (accepted)

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100 Smith LJ, Datnoff LE, Pernezny KL, Rollins J, Schlub RL (2008b) Phylogenetic analyses of diverse Corynespora cassiicola isolates indicate an evolutionary corr elation with host not geography. 9th European Conference on Fungal Genetics Meeting Abstracts Smith LJ, Datnoff LE, Rollins JA, Pernezny KL, Schlub RL (2007) Phylogenetic analysis of Corynespora isolates from diverse hosts a nd locations. Phytopathology 97:S109 Smith LJ, Datnoff LE, Pernezny KL, Roberts PD, Rollins JA, Schlub RL, Scott JW (2006) Characterization and host-range of the tomato target spot fungus, Corynespora cassiicola and resistance of tomato cultivars. Florida To mato Committee, Tomato Research Report for 2004-2005, pp 14-20 Smith LJ, Schlub RL (2005) Foliar fungi on weeds of Guam and the potential for Corynespora cassiicola as a bioherbicide for Stachytarpheta jamaicensis. Phytopathology 95:S93 Smith LJ, Schlub RL (2004) Host range of Corynespora cassiicola and its occurrence on weeds, ornamentals and crops of Guam. Phytopathology 92:S77 Sobers EK (1966) A leaf spot di sease of azalea and hydrangea caused by Corynespora cassiicola Phytopathology 59:455-457 Spencer JA (1962) Stud y of variations in Corynespora cassiicola (Berk. & Curt.) Wei. M. S. Thesis, University of Arkansas, Fayetteville, 31 pp Spencer JA, Walters HJ (1969) Variations in certain isolates of Corynespora cassiicola Phytopathology 59:58-60 Stone WJ, Jones JP (1960) Corynespora bl ight of sesame. Phytopathology 50:263-266 Strandberg JO (1971) Evaluation of cu cumber varieties for resistance to Corynespora cassiicola. Plant Dis Rep 55:142-144 Suryanarayanan TS, Murali TS, Venkatesan G (2002) Occurrence and distribution of fungal endophytes in tropical forests across a rainfall gradient. Can J Bot 80:818-826 Swofford DL (2002) PAUP*. Phylogenetic anal ysis using parsimony (*and other methods). Version4. Sunderland: Sinauer Associates Taba S, Ooshiro A, Takaesu K (2002) Black stem and root rot of basil Ocimum basilicum L. caused by Corynespora citricola Ann Phytopathol Soc Japan 68:43-45 Tanaka K, Yasuyoshi O, Hatakeyama S, Harada Y, Barr ME (2008) Pleos porales in Japan (5): Pleomassaria, Asteromassaria, and Splanchnonema. Mycoscience 46:248-260

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101 Tsay JG, Kuo CH (1991) The occurrence of Coryne spora blight of cucumber in Taiwan. Plant Prot Bull 33:227-229 Turner GJ (1971) Fungi and Plant Diseas e in Sarawak. Phytopathol Pap 13:1-55 Urtiaga R (1986) Indice de enfe rmedades en plantas de Venezuela y Cuba. Impresos en Impresos Nuevo Siglo. SRL, Ba rquisimeto, Venezuela, 202 pp Urtiaga R (2004) Indice de en fermedades en plantas de Venezuela y Cuba, 2nd Ed, 301 pp Vittal BPR, Dorai M (1995) Studies on litter fung i VIII. Quantitative studies of the mycoflora colonizing Eucalyptus tereticornis Sm. Litter. Kavaka 22/23: 35-41 Volin RB, Pohronezny K (1989) Severe spotting of fresh market tomato fruit incited by Corynespora cassiicola after storm-related inju ry. Plant Dis 73:1018-1019 Vyas SC, Shastry PP, Shukla BN, Varma RK (1985) Two new leaf blight diseases of groundnut. Plant Prot Bull 33:121-122 Wei CT (1950) Notes on Corynespora. Mycol Papers 34:1-10 White TJ, Bruns T, Lee S, Taylor J (1990) Am plification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. P CR Protocols: A Guide to Methods and Applications. Innis MA, Gelfand DH, Sninsky JJ, White TJ (ed.) Academic Press, Inc, New York, NY, ch 38, pp 315-322 Williams TH, Liu PSW (1976) A host list of plant diseases in Sabah, Malaysia. Phytopathol Pap 19:1-67 Yudin L, Schlub RL (1998) Guam Cucurbit Guid e. Guam Cooperative Extension Publication, 64 pp Zhang XG, Ji M (2005) Taxonomic studies of Corynespora from Yunnan, China. Mycotaxon 92:425-429 Zhuang WY (2001) Higher Fungi of Tropica l China. Mycotaxon, Ltd., Ithaca, NY, pp 485

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102 BIOGRAPHICAL SKETCH I was born in Pom pano Beach, FL to Janis Th errell and Kenneth Wayne Smith on October 15, 1977. I have an older sister, Allison, and tw o younger brothers, Scott and Reid. Our family moved to Baltimore, MD when I was nine and I attended Baltimore Friends School, where my father was head of the Middle School. Though I have always loved biology and gardening, my interest in agriculture took off in high school when I attended Maine Coast Semester, a small school for students in their junior year locate d on a coastal farm in Wiscasset, Maine. I received my B. A. at Colorado College in 2000 with a major in Biology, while fostering my interest in farming through summer jobs at nurseries, CSAs, and internships. My sophomore year in college, I traveled abroad to East Africa through The School for Field Studies where I learned the importance of economic value in conservation by focusing on wildlife ranching, national parks, and medicinal plant use as case studies. In 2002, I received my Masters Degree from West Virginia University in Plant Pathology as part of the Organic Farm Project by studying the effect of intercropping on diseases caused by Alternaria solani and Meloidogyne incognita I then spent two years in Micronesia on the island of Guam as a Research Assistant documenting pathogens of agronomically important weeds and working in the diagnostic clinic. It was in Guam where I first became aware of Corynespora cassiicola as an agent of disease. An opportunity pr esented itself to continue the work begun on this pathogen at the University of Florida in the Fall of 2004. At the University of Florida, I became well trained in Phylogenetics and this ha s become my specialty. I plan to continue studying fungal systematics beginning in Septem ber, 2008 as a post-doc with the USDA in Beltsville, MD.