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Development of a Real-Time Polymerase Chain Reaction Diagnostic Technique for Fusarium oxysporum f. sp. canariensis on P...

Permanent Link: http://ufdc.ufl.edu/UFE0023881/00001

Material Information

Title: Development of a Real-Time Polymerase Chain Reaction Diagnostic Technique for Fusarium oxysporum f. sp. canariensis on Palm Species
Physical Description: 1 online resource (97 p.)
Language: english
Creator: Vitoreli, Anne
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: canariensis, canary, cepheid, chain, date, fusarium, island, oxysporum, palms, pcr, phoenix, polymerase, proliferatum, reaction, real, time
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Fusarium oxysporum f. sp. canariensis is the causal agent of Fusarium wilt of Canary Island date palm. Fusarium wilt is an important disease that results in the loss of mature palm specimens. Management options involve removing infected trees as quickly as possible to try to prevent spread to other palms. Rapid and accurate detection is vital to the management of this disease. The pathogen is identified with a PCR protocol. We evaluated and modified the detection protocol to correct for false positive results obtained from Fusarium proliferatum which is a common saprophyte on palms. New DNA extraction kits were compared with the previously published extraction technique. Additional PCR primer sets were developed and multiplexed with the previous set to distinguish between the two species. Real-time PCR primers and probes also were evaluated as part of a new detection protocol for the Cepheid SmartCycler system. The new extraction methods and PCR protocols were evaluated and found to reliably identify F. oxysporum f. sp. canariensis. The real-time PCR protocol also reliably detected the pathogen. The updated protocols distinguished between F. oxysporum f. sp. canariensis, forma specialis other than canariensis, and F. proliferatum. The last chapter of the thesis is a standard operating procedure for diagnosticians who wish to use the updated protocols.
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 Anne Vitoreli.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Harmon, Phillip.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0023881:00001

Permanent Link: http://ufdc.ufl.edu/UFE0023881/00001

Material Information

Title: Development of a Real-Time Polymerase Chain Reaction Diagnostic Technique for Fusarium oxysporum f. sp. canariensis on Palm Species
Physical Description: 1 online resource (97 p.)
Language: english
Creator: Vitoreli, Anne
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: canariensis, canary, cepheid, chain, date, fusarium, island, oxysporum, palms, pcr, phoenix, polymerase, proliferatum, reaction, real, time
Plant Pathology -- Dissertations, Academic -- UF
Genre: Plant Pathology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Fusarium oxysporum f. sp. canariensis is the causal agent of Fusarium wilt of Canary Island date palm. Fusarium wilt is an important disease that results in the loss of mature palm specimens. Management options involve removing infected trees as quickly as possible to try to prevent spread to other palms. Rapid and accurate detection is vital to the management of this disease. The pathogen is identified with a PCR protocol. We evaluated and modified the detection protocol to correct for false positive results obtained from Fusarium proliferatum which is a common saprophyte on palms. New DNA extraction kits were compared with the previously published extraction technique. Additional PCR primer sets were developed and multiplexed with the previous set to distinguish between the two species. Real-time PCR primers and probes also were evaluated as part of a new detection protocol for the Cepheid SmartCycler system. The new extraction methods and PCR protocols were evaluated and found to reliably identify F. oxysporum f. sp. canariensis. The real-time PCR protocol also reliably detected the pathogen. The updated protocols distinguished between F. oxysporum f. sp. canariensis, forma specialis other than canariensis, and F. proliferatum. The last chapter of the thesis is a standard operating procedure for diagnosticians who wish to use the updated protocols.
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 Anne Vitoreli.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Harmon, Phillip.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0023881:00001


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1 DEVELOPMENT OF A REAL-TIME POLYME RASE CHAIN REACTION DIAGNOSTIC TECHNIQUE FOR Fusarium oxysporum f. sp canariensis ON PALM SPECIES By ANNE MARIE VITORELI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Anne Marie Vitoreli

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3 To all plant pathology diagnosticians

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4 ACKNOWLEDGMENTS I thank my committee (Dr. Jeff Rollins and Dr. Monica Elliott) and my chair (Dr. Philip Harmon) for their guidance, advice and encourag ement. I thank Dr. James Downer from the University of California, Davis for conducting pat hogenicity testing on some of my isolates; Dr. Robert Stamps from UF/IFAS Mid-Florida REC for providing Dracaena marginata plants; Barry Logan from Whisper Palms Nursery for providing Phoenix canariensis seedlings for pathogenicity tests; Vessela Mavrodieva from th e USDA for her help in optimizing real-time PCR, and Dr. David Geiser for his help with PCR and Fusarium sequencing. I thank my family for all of the love and suppor t they have given me. I especially thank my husband, Odenis Vitoreli, Jr., and my parents, Walter and Teresa Benner for all of their encouragement. I thank all of my friends, co-w orkers, and teachers for their help and support especially Richard Cullen, Dr. Robert Mc Govern, Oscar Ruiz, Eduardo Canova, Amanda Watson, and Carrie Harmon.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 LIST OF ABBREVIATIONS..........................................................................................................9 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 LITERATURE REVIEW.......................................................................................................12 History........................................................................................................................ ............12 Fusarium oxysporum ..............................................................................................................13 Fusarium oxysporum f. sp albedinis ..............................................................................14 Fusarium oxysporum f. sp elaeidis .................................................................................14 Fusarium oxysporum f. sp canariensis ...........................................................................15 Fusarium proliferatum ............................................................................................................16 Polymerase Chain Reaction....................................................................................................17 Nested Polymerase Chain Reaction.................................................................................18 Multiplex Polymerase Chain Reaction............................................................................18 Real-Time Polymerase Chain Reaction...........................................................................19 Plant Disease Clinic Protocol.................................................................................................20 2 REVISION OF THE PLY LER PCR PROTOCOL................................................................23 Materials and Methods.......................................................................................................... .24 Fungal Isolates................................................................................................................ .24 DNA Extraction...............................................................................................................24 The CTAB extraction prot ocol (Plyler Method)......................................................24 Qiagen extraction protocol.......................................................................................25 Sigma extraction protocol........................................................................................26 Reaction Mix, Thermocycler Prog ram, and Gel Visualization.......................................26 New Taq Polymerases..............................................................................................27 Invitrogen Taq..........................................................................................................27 Sigma Taq................................................................................................................28 Development of Fusarium proliferatum Primers............................................................28 Multiplex of Fusarium oxysporum f. sp. canariensis and Fusarium proliferatum Primers........................................................................................................................ .30

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6 Results........................................................................................................................ .............30 DNA Extraction...............................................................................................................30 PCR Mix........................................................................................................................ ..31 Primer Development........................................................................................................31 Conclusions.................................................................................................................... .........31 3 DEVELOPMENT OF A REAL-TIME PCR PROTOCOL....................................................38 Materials and Methods.......................................................................................................... .38 Fusarium oxysporum f. sp. canariensis ...........................................................................38 Fusarium proliferatum ....................................................................................................40 Multiplex Real -Time PCR...............................................................................................41 Results........................................................................................................................ .............42 Fusarium oxysporum f. sp. canariensis ...........................................................................42 Fusarium proliferatum ....................................................................................................42 Multiplex Reaction..........................................................................................................42 Conclusions.................................................................................................................... .........43 4 STANDARD OPERATI NG PROCEDURES........................................................................52 Isolation...................................................................................................................... ............52 Conventional Polymerase Chain Reaction.............................................................................52 Real-Time Polymerase Chain Reaction..................................................................................54 APPENDIX A PRIMER SEQUENCES.........................................................................................................56 B SEQUENCING..................................................................................................................... ..60 C HK SEQUENCE COMPARISONS.......................................................................................62 D TRANSCRIBED ELONGATION FA CTOR1 ALPHA SEQUENCES..............................68 E KOCHS POSTULATES.......................................................................................................85 Material Preparation........................................................................................................... ....85 Inoculation Procedure.......................................................................................................... ...86 Results........................................................................................................................ .............87 Dracaena marginata .......................................................................................................87 Phoenix canariensis .........................................................................................................87 LIST OF ISOLATES............................................................................................................... ......90 LIST OF REFERENCES............................................................................................................. ..93 BIOGRAPHICAL SKETCH.........................................................................................................97

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7 LIST OF TABLES Table page 2-1 Isolates used in optimization of protocol...........................................................................33 3-1 Isolates used in optimization of protocol...........................................................................45 F-1 Isolates used in Koch s Postulate and Sequencing............................................................91

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8 LIST OF FIGURES Figure page 2-1 Comparison of genomic DNA extraction methods............................................................34 2-2 Comparison of Taq using HK66 and HK 67 primers........................................................35 2-3 Fusarium proliferatum primers FP1 and 2......................................................................36 2-4 Multiplex of HK66 & 67 with Fp-1 & 2............................................................................37 3-1. Gel electrophoresis of PCR amp licons using primers FOC3 and FOC4...............................46 3-2. Real-time PCR results us ing primers FOC3 and FOC4........................................................47 3-3. Gel electrophoresis of amplicons obtained with primers FP3 and FP4.................................48 3-4. Real-time PCR results using primers FP3 and FP4 with the FP Texas Red probe...............49 3-5. Real-time multiplex PCR results-FAM channel....................................................................50 3-6. Real-time multiplex PCR resultsTexas Red channel..........................................................51 A-1. Comparison of amplicons sequ enced using HK66 and HK67 primers................................56 A-2. Sequence and primer selection for Fusarium proliferatum primers FP1 and FP2...............57 A-3. Sequence and primer/probe selection for Fusarium oxysporum f. sp canariensis primers FOC3 and FOC4 and FOC Probe1.......................................................................58 A-4. Sequence and primer/probe selection for Fusarium proliferatum primers FP3 and FP4 and FP Probe 1................................................................................................................. ..59 E-1. Inoculated plants pre-inoculation........................................................................................ ..88 E-2. Inoculated plants 5-6 months post-inoculation.....................................................................89

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9 LIST OF ABBREVIATIONS A Adenine APDA Acidified potato dextrose agar BBL Baltimore Biological Laboratories BP Base pairs C Cytosine CLA Carnation leaf agar Ct Cycle Threshold EDTA Ethylenediaminetetraacetic acid ERIC Enterobacterial repetitive intergenic consensus Foa Fusarium oxysporum f. sp. albedinis Foc Fusarium oxysporum f. sp. canariensis Foe Fusarium oxysporum f. sp. elaeidis Fp Fusarium proliferatum G Guanine ICBR Interdiciplinary Center for Biotechnology Research MGA Malachite green agar NTC No template control PCR Polymerase Chain Reaction PDA Potato dextrose agar PDC Florida Extension Plant Disease Clinic in Gainesville, FL QPDA Quarter strength pot ato dextrose agar SCAR Smart Cycler Additive Reagent SNA Spezieller Nhrstoffarmer agar T Thymine

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10 TAE Tris(hydroxymethyl)aminomethane-Ac etateEthylenediaminetetraacetic acid TEF Translation elongation factor TRIS Tris(hydroxymethyl)aminomethane VCG Vegetative compatibility groupings WA Water agar

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11 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DEVELOPMENT OF A REAL-TIME POLYME RASE CHAIN REACTION DIAGNOSTIC TECHNIQUE FOR Fusarium oxysporum f. sp canariensis ON PALM SPECIES By Anne Marie Vitoreli December 2008 Chair: Philip Harmon Major: Plant Pathology Fusarium oxysporum f. sp. canariensis is the causal agent of Fusarium wilt of Canary Island date palm. Fusarium wilt is an important disease that results in the loss of mature palm specimens. Management options involve removing inf ected trees as quickly as possible to try to prevent spread to other palms. Rapid and accurate detection is vital to the management of this disease. The pathogen is identi fied with a PCR protocol. We evaluated and modified the detection protocol to correct for false positive results obtained from Fusarium proliferatum which is a common saprophyte on palms. New DNA extraction kits were compared with the previously published extracti on technique. Additional PCR prim er sets were developed and multiplexed with the previous set to distinguis h between the two species Real-time PCR primers and probes also were evaluated as part of a ne w detection protocol for the Cepheid SmartCycler system. The new extraction methods and PCR prot ocols were evaluated and found to reliably identify F. oxysporum f. sp. canariensis The real-time PCR protocol also reliably detected the pathogen. The updated protocols distinguished between F. oxysporum f. sp. canariensis forma specialis other than canariensis and F. proliferatum The last chapter of the thesis is a standard operating procedure for diagnosticians w ho wish to use the updated protocols.

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12 CHAPTER 1 LITERATURE REVIEW History Fusarium is in the Domain Eukaryota, Kingdom Fungi: Dikarya, Phylum Ascomycota, Subphylum Pezizomycotinia, Class Sordariomycet es, Order Hypocreales, Family Nectriaceae. The genus Fusarium is divided into approximately 16 s ections according to morphological and cultural characteristics (27 ). With the advent of new mo lecular techniques, taxonomical groupings have changed somewhat and remain in a state of flux (24 ). The genus Fusarium was first identified by H.F. Link in 1809 and includes species that can cause disease in plants, animals and humans (24 ). Link based his initial description of the genus on the sporodochium (5 ). Prior to 1935 there were over 1000 species listed for Fusarium (24 ). Since then, several attempts to organize the genu s into an orderly arrangement have been made by splitters, moderates, and lumpers (28 ). The modern system of Fusarium taxonomy was developed by splitters Wollenweber and Reinking in 1935 (28 ). Wollenweber and Reinking used a set of characteristics to identify Fusarium that were arranged into 16 sections, 65 species, 55 varie ties and 22 forms. They used characteristics from microconidia, macroconid ia, chlamydospores, growth on several types of media as well as other characteristics like pigm ent. The system produced from the work of Wollenweber and Reinking was extremely complex and a key for identification was impractical to develop (28 ). Snyder and Hansen reduced the number of Fusarium species to nine in the 1940s by using single-sporing technique (24 ). They found that there was great variability from conidia produced from a single parent and with the Wollenweber a nd Reinking system they may have been placed in separate subsections (28 ). Snyder and Hansen lumped seve ral of the species together and

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13 proposed an informal nomenclature system using forma specialis to distinguish the host which was previously identified by the species name (28 ). Currently a combination of the Wollenweber and Reinking and Snyder and Hansen concepts of Fusarium taxonomy are in use today (24 ). A combination of morphological, bi ological and phylogenetic met hods is used to separate Fusarium into species and formae speciales Fusarium oxysporum Fusarium oxysporum is in the Elegans section and is distributed world-wide causing important wilt diseases. There are more than 120 forma specialis of F. oxysporum with corresponding host ranges (9 ). The fungus is commonly isolated from soil and plant tissue. No sexual stage is known for F. oxysporum Morphological and culture characteristics of Fusarium oxysporum, F. subglutans and F. solani are difficult to differentiate (24 ). Isolates of F. oxysporum on potato dextrose agar (PDA) are very diverse. Mycelia can be abundant and floccose to pionnatal (wet looking with no aerial mycelia) and range in color from hyaline to violet. Pigment may be produced in certain growth media. Pigment color varies from mauve to a dark violet or almost black in color. On carnation leaf agar (C LA), pale or orange sporodochia may be formed that contain macroconidia that are approximately 25m in length, thin walled, usually thin and straight with a slight hooked apical end, and f oot-shaped to pointed basal end. Macroconidia are usually 3-sept ate. Microconidia are produced abundantly in false heads on short monophialides, whereas microconidia of F. solani are produced on long monophialides and microconidia of F. subglutans are formed on polyphialides. Micr oconidia are oval to reniform (kidney) in shape and approxima tely 10m in size. Chlamydospor es are produced abundantly in 2 to 4 weeks in CLA with most isolates. Vegetative compatibility groupings (VCG) a nd enterobacterial re petitive intergenic consensus polymerase chain reaction (ERIC PCR) have been utilized as a DNA fingerprinting

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14 method to categorize isolates of F. oxysporum from Australia (19 ). These tests are complicated, time consuming and cannot accurately differentiate formae speciales. Sequencing of the transcribed elongation f actor 1-alpha (TEF-1 ) is a method for differentiating between species and forma specialis (18 ). This procedure is not feasible for everyday diagnostic purposes. Fusarium oxysporum f. sp albedinis Bayoud disease was first reported on Phoenix dactylifera, in 1870 in Morocco (42 ). Fusarium oxysporum f. sp. albedinis (Foa) was determined to be th e cause of the death of more than 20 million trees since it was initially identified in Morocco and Algeria (17 ). As of 2000, Bayoud disease was limited to the region of Morocco and Algeria but continue s to spread to the east via products made from infected plant tissue (17 ). As of 2004, Bayoud disease has been reported on P. canariensis but it is believed these may have been the forma specialis canariensis (10 ). Symptomless hosts of F. oxysporum f. sp. albedinis include Lawsonia inermis L. (henna) and Medicago sativa L. (alfalfa) (17 12 ). Symptoms of Bayoud disease such as the one-sided progressive death of fronds are similar to Fusarium wilt, (29 12 ). Molecular markers indicate the F. oxysporum f. sp. albedinis and F. oxysporum f. sp canariensis are genetically distinct (15 ). Fusarium oxysporum f. sp elaeidis Fusarium oxysporum f. sp. elaedis the causal agent of oil palm wilt (or fusariose) was first reported in 1946 on Elaeis guineensis in the Belgian Congo (41 ) with later reports in Cameroon, Congo, Ghana, Ivory Coast and Nigeria and is olated reports in Brazil and Ecuador (16 ). The host range for Fusarium oxysporum f. sp. elaeidis includes the Elaeis guineensis and E. oleifera with symptomless hosts Cyperus alterifolius L., Eupatorium odoratum L., and Amaranthus spinosus L. (12 ). Two types of disease symptoms are reported in adult palms in the field. The first disease syndrome is acute wilt where the leaves dry out rapidly, but remain erect until broken off. The host rapidly declines and palm death us ually occurs within 2 to 3 months (16 ). The second

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15 syndrome reported is chronic wilt where the host becomes stunted, and the older, outer fronds dry and wilt with the younger fronds successively becoming affected. The host remains alive for several months (16 ) Fusarium oxysporum f. sp canariensis Fusarium oxysporum was first reported in 1970 on Phoenix canariensis in France (25 ). The causal agent was classified as the forma specialis canariensis by Mercier and Louvet (25 ). Fusarium oxysporum f. sp. canariensis (Foc) was reported on Phoenix canariensis in Italy in 1974 (15 ), Japan in 1977 (3 ), Greece and Sardinia in 2004 (26 10 ). In1980, Fusarium wilt was first reported in Sydney in Eastern Australia wh ere it has slowly spread across the southern region (37 ) reaching Akuna Station, South Australia in 2000 and Melbourne, Victoria in 2003 (38 ). The first report of Fusarium wilt caused by Fusarium oxysporum f. sp canariensis (Foc) in the United States was in California in 1976 along the southern California coast. (14 ). Foc was first isolated from palms in Manatee County, Fl orida in 1994 (PDC records). As of 2008, Foc has been isolated from palms as far north as Okaloosa County, FL. Symptoms of Fusarium wilt include internal discoloration of the rachis and petiole, a reddish-brown stripe on the peti ole, one-sided death of leaflets and progressive death of the leaves (11 ). Management options for this disease ar e limited once a palm is diagnosed with Fusarium wilt. Foc is thought to be transmitte d primarily via transplanting infected trees, contaminated soil, and contaminated pr uning tools. Susceptible hosts include Phoenix canariensis and P. dactylifera, (12 ) with P. reclinata, and P. sylvestris diagnosed by PCR (PDC records). There are currently no known fungicides to cure or prevent Fusarium wilt. Preventative measures include removal of dead or dying trees to a landfill. All equipment used for pruning symptomatic palms or for removal need to be thoroughly cleaned and di sinfected between each

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16 palm with a 10% household bleach solution, a solution of Pinesol, or equivalent sanitizing agent. Palms that are susceptible to Fusarium wilt should not be replanted in an area from which an infected tree was removed (M cGovern, personal communication). A conventional polymerase chain reaction (PCR)-based diagnostic technique was developed at the University of Florida to distinguish Fusarium wilt of palm from other Fusarium spp that infect or otherwise colonize palms (31 ). In the original research, seventy-one isolates of F. oxysporum f. sp canariensis were used to develop the primers for this assay. Isolates were obtained from samples submitted to the Florida Extension Plant Disease Clinic (PDC) in Gainesville, Florida from locati ons in Florida and California. Fusarium proliferatum Fusarium proliferatum is in the Liseola section of Fusarium Morphologically, F. proliferatum is easily confused with F. oxysporum on PDA but can be distinguished by chlamydospores in F. oxysporum cultures and chains of microconidia in F. proliferatum (24 ) Fusarium proliferatum cannot be differentiated from F. fujikuroi (section Gibberella ) (24 ) In order to distinguish between F. proliferatum and F. fujikuroi mating tests or DNA sequencing is required and even then, differentia tion may not be possible. Since F fujikuroi is a rice pathogen and has not been reported on palm s, no distinction needs to be made between these two species for diagnostic purposes Common synonyms for F. proliferatum are Gibberella fujikuroi mating population D, and Gibberella fujikuroi var. intermedia Fusarium proliferatum cultures on CLA have microconidia that are formed in chains and in some cases, in false heads from monophialides and polyphialides. The chains of microconidia are moderate in length. The microconidia are usually pyriform (club-shaped) in form. The macroconidia are thin and almost straight, usually with three to five septa. Macroconidia are

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17 formed in pale orange sporodoc hia that are difficult to find. F. proliferatum does not produce chlamydospores. Another common media used to evalua te morphological ch aracteristics of Fusaria is PDA. Fusarium proliferatum isolates form abundant aerial mycelia which are usually white in color on PDA, but may change to purple with age. Pigment in the agar can be nearly colorless to blackusually a shade of purple. Blue-b lack sclerotia may be indicative of a high level of sexual female fertility but is not diagnostic of F. proliferatum Microconidia are pyrifo rm with no septa and are abundant in the aerial mycelia forming moderate chains or aggregates. M acroconidia are usually thin-walled, three to five septat e, straight and slender. The ap ical cell of the microconidia is curved and the basal cell is poorly developed. Most of the research on F. proliferatum has been on strains pathogenic to asparagus (24 ). F. proliferatum was first reported on Date pa lms in Saudi Arabia in 2000 (1 ) and has since been reported on Majesty Palms in Sardinia (32 ), Central Australian Fan Palms in Australia (29 ), and various palms including Phoenix spp. and Washingtonia spp. in Spain (2 ). Polymerase Chain Reaction Polymerase Chain Reaction (PCR) was invent ed by Kary Banks Mullis in 1983. Dr. Mullis won the Nobel Prize in chemistry for his invention of PCR in 1993 (40 ). PCR involves the denaturation, annealing, and exte nsion of a target DNA sequence (22 ). An initial step of 95C for approximately 1-4 minutes allows a heat st able DNA polymerase (Taq) to become active. Denaturation occurs when the DNA is heated to approximately 95C to break the hydrogen bonds between the bases of the DNA strands. The primers are then annealed to the single stranded DNA at approximately 50-65C. The Taq enzymes then extend the primers to complement the DNA strand. The cycle is repeat ed approximately 30-40 times which results in

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18 the exponential amplification of the target DNA sequence. The DNA is then visualized using gel electrophoresis (22 ). Successful PCR results are dependent upon th e design of the primers and PCR reaction mixture. The primers are required to be unique and conserved to the organism of interest (21 ). Multiple copy target sequences increa se the likelihood of amplification (21 ). Cross-amplification of a closely related species or non-unique sequence may result in false positives (4 ). False negatives may occur for various reasons, such as when primer design is not optimal, primer concentrations are not adequate, or the PCR product is too long (33 ). To distinguish between false positives/negatives and true positives/negatives, control samples are required to be tested. Samples known to amplify the target DNA (positive control) as well as no template controls (NTC) are required with each sample set processed. The PDC uses PCR in conjunction with other detection methods for the confirmation of several organisms, including viruses (Geminivirus) (34 ), fungi ( Phakospora spp .) (23 ) and bacteria ( Candidatus Liberibacter spp.) (39 ). Nested Polymerase Chain Reaction Nested polymerase chain reaction is a modifi ed conventional PCR. Nested PCR increases specificity by using two sequential amplification steps. Nested PCR consists of two or more consecutive PCR reactions using a dilution of th e first PCR product as the DNA template for the second PCR. The primers for the second PCR r eaction amplify a target region within the amplicon from the first reaction thus increasing sensitivity (21 ). The PDC uses the nested PCR technique to detect Leth al Yellowing phytoplasma (20 ) and Phytophthora ramorum (21 ). Multiplex Polymerase Chain Reaction Multiplex PCR enables multiple DNA sequences to be amplified in one reaction (36 ). The ability to detect more than one target DNA sequence in the same reaction enables the

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19 diagnostician to verify the presence and quality of the DNA by using an internal control as well as multiple organisms or species (33 ). Different sequences are dist inguished from one another in conventional PCR based on the size of the amp licon produced. Multiplex PCR requires the base pair length to be different enough to be able to distinguish th em readily on a gel. Special consideration is required when designing primers for a multiplex protocol. Guidelines for developing the primers included: 1) Nucleotide length of 21-30; 2) GC content between 40-60%; 3) Melt temperature of 60C to 68C with all primers having a similar value; 4) the sequence does not form secondary structures 5) avoid 3 or more Guanines (G) and/or Cytosines (C) at the 3 end; 6) Avoid complementary sequences within the primer sets; and 7) ensure the sequence is unique to the target DNA (33 ). The PDC uses the multiplex technique for the detection of Phytophthora ramorum (21 ). Real-Time Polymerase Chain Reaction Real-time PCR (also referred as quantitative PCR) can reduc e the amount of time needed to analyze a sample and eliminates th e need for running gel electrophoresis (13 ). Real-time PCR combines DNA amplification with detection using a reporter dye that is att ached to the 5 end of a short DNA sequence (approximately 15-20 base pair s) with a dark quencher attached to the 3end. When the DNA polymerase displaces the pr obe from the target DNA strand, the quencher is cleaved away from the reporter dye allowing it to fluoresce. The fluorescent signal increases with the target DNA exponentially with each cycle as in conven tional PCR. This fluorescence is measured at the end of each cycle and when the fluorescence achieves a preset threshold, the sample is considered positive. Quantification of DNA can be obtained mathematically by the cycle threshold value if a standard curve dilution series is used (35 ). Multiplex PCR is frequently used with real-time PCR by using reporter dyes that fluoresce at different wavelengths on different optical detection channels (13 ). Each channel is calibrated

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20 to read a range of wavelengths optimized for certain reporter dyes a nd quencher combinations. Special consideration is required when selec ting reporter dyes for multiplexing in a real-time PCR system. For optimum separation, each manufact urer of real-time PCR systems has a list of recommended reporter dyes and quencher pairings for optimal separati on of multiplex product (13 ). Plant Disease Clinic Protocol Before developing the PCR technique, Fusarium oxysporum isolates were determined to be the wilt-causing forma specialis based on a combination of host, symptom, and colony morphology. Kochs postulates were conducted on se lect isolates, but due to the difficulty of inoculating palms, amount of greenhouse space, cost of palms, and the amount of time for symptoms to appear (up to 18 months in some host s), routine inoculation te sts are not feasible as a diagnostic tool. Diagnoses were made on known palm hosts with the wilt symptoms if a F. oxysporum was isolated. Diagnoses made with this method can take up to three weeks to complete using culture morphology and may have had a high error rate. Palm samples submitted to the Plant Disease Clinic are processed following a standard operating procedure. Samples are examined visua lly and microscopically and then processed by culturing the symptomatic tissue. Initial cultures of Fusarium spp. are isolated from rachis, leaf, and leaflet. Root, bud, or trunk ti ssue is rarely used for the di agnosis. If roots, bud or trunk sections are the only tissue submitted for analysis to the clinic, the submitter is asked to resubmit a frond sample. Internal tissue samples are cut in to small pieces, rinsed in a five percent household bleach solution for one minute, and then rinsed in sterile de-ionized water. The surface sterilized tissue is plated on culture me dia. Three media commonly are used. Acidified potato dextrose agar (APDA) is made with 12.8 g Potato Dextrose Agar (Baltimore Biological Laboratories, BBL catalog #211550) in 500mL of de-ionized water. Seven drops of 50% lactic

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21 acid is added after autoclaving. On e quarter strength potato dextrose agar (QPDA) is made with 3.8g of Potato dextrose agar, 2.5g of granulated agar (BBL catalog # 214530), and 500mL of deionized water. Water agar (WA) is made with 2.5g of granulated agar an d 500mL of de-ionized water. A minimum of four pieces of surface-ster ilized tissue are placed on each type of media (APDA, QA, and WA) to get a representative sample of tissue. A larger piece (s) of non-sterilized plant tissue also is incubated in a moist chambe r. When fungal colonies are observed to be growing from the tissue, the plates are observed in situ using a compound microscope at 100X. If a Fusarium sp. is observed, a subculture is taken usi ng sterile technique an d single-spored to ensure a pure isolate. The smallest possible amount of the mycelium is transferred to another plate of APDA or Spezieller N hrstoffarmer Agar (SNA) (24 ). When the isolate is contaminated with other fungi or bacteria, it may be plated on a modified malachite green agar (MGA, 6 ) in order to obtain a pure culture. The formula for MGA was slightly modified from the one published in Castell et. al, 1996; rifampin (1mL of a 0.5g/100mL in ethanol per 500mL of agar) and ampicillin (0.25g per 500mL of agar) we re used instead of streptomycin and chloramphenicol. If more than one Fusarium colony is observed, they are plated separately. The Fusarium isolates are then single-sp ored onto APDA and given a letter designation after the sample number to distinguish them. These cult ures are incubated at room temperature on the laboratory bench for approximately 7-14 days. The Fusarium spp. isolates are then tested with the diagnostic PCR protocol and primer set HK66 and HK67 (31 ) and transferred to carnation leaf agar (CLA, water agar with 3-5 irradi ated carnation leaf pieces) where morphological characteristics are observed after approximately14 days. When cultures are submitted from other laboratories for analysis, the samples are analyz ed prior to single-sporing due to the time sensitive nature of the diagnostic clinic. If results from these cu ltures are found to be positive or

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22 suspect, samples are then single-spored and re-a nalyzed with PCR to confirm the results for addition to the Plant Disease Clinic culture collection.

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23 CHAPTER 2 REVISION OF THE PLYLER PCR PROTOCOL The standard operating procedure for Fusarium wilt diagnosis in the Florida Extension Plant Disease Clinic at the University of Fl orida was based on a PCR reaction with the HK66 and HK67 primers developed by Plyer et.al (30 ) The protocol resulted in a diagnostic amplicon of approximately 567 base pairs (bp) when culture s of FOC were tested. Amplicons also were found to be produced from Fusarium proliferatum cultures that are commonly isolated from palms (PDC records). Additional isolates of F. oxysporum from hosts other than Phoenix canariensis also have been found to produce the di agnostic amplicon. Although the degree of homology between the sequences of the F. proliferatum and non-host-Foc amplicons was unknown, these results call into question the protocols sp ecificity (PDC records, 19 ). Since F. proliferatum is a common saprophyte a nd because speciation of Fusarium fungi is based on attributes of colony morphology that can take a month or more to observe, these false positives preclude a rapid and reliable diagnosis using the PCR protocol. A second reason for reevaluation of the Plyler protocol is the availa bility of PCR kits, reagents, and real-time technol ogy (e.g. the Taq Polymerase in Storage Buffer B20mM TrisHCL at pH 8.0; 100mMKCl, 0.1mM EDTA, 1mM DTT, 50% glycerol, 0.5% tween 20 and 0.5% Nonidet-P40; Promega catalog #M1661, is no longe r available). The ob jectives of this research were: 1) to evaluate new reagents, kits, and technologies with the Plyler primers; 2) to develop new PCR primers and a protocol th at would better differentiate Foc and F. proliferatum ; and 3) to ultimately revise the Foc de tection protocol used by the PDC.

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24 Materials and Methods Fungal Isolates Eight isolates were used to compare and op timize the extraction and PCR protocols (Table 2.1). Two known isolates of F. oxysporum f. sp canariensis (Foc) included 703C and 2675A. Two additional isolates of F. oxysporum isolated from palms that were not f. sp. canariensis included 1072B and 1559C. Four isolates of F. proliferatum were used. Two of these were known to produce an amplicon with the HK66/67 primer set and PCR protocol (001B and 1118B), and two that did not (1550 and SP3A). Is olates were transfer red onto APDA and grown for approximately 7 days prior to DNA extraction. DNA Extraction The CTAB extraction pr otocol (Plyler Method) A thin 1cm2 piece of agar with abundant mycelia wa s removed from the leading edge of the culture and placed in a 1.5mL microcentrifug e tube with a conical bottom. Five hundred micrograms of 3% CTAB was added to the tube and heated to 65C for 15 minutes. Then 150L of TE buffer {10mM tris (hydroxymet hyl)aminomethane (TRIS) & 1mM ethylenediaminetetraacetic acid (EDTA) stock solution; 10L of stock solution in 1mL} was added to the tube and a sterile plastic pestle (USA Scientific catalog #1405-4390) attached to an electric screwdriver was used to disrupt the ce lls as much as possible. The tubes were then subjected to 3 or 4 freeze-thaw cycles using liqui d nitrogen and room temperature water. After the last cycle, the tubes were placed in a 65C waterbath for 15 minutes. Tubes were removed and centrifuged for 10 minutes at 14,000 x g Four hundred microliters of supernatant was removed to a new tube. One hundred and fifty micr oliters of chloroform:isoamyl alcohol (24:1) was added to the tube, vortexed, then centrifuged for 10 minutes at 14,000 x g One hundred microliters of supernatant was transferred to a new tube, 67L of 2-propanol was added and the

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25 tube was shaken. Tubes were then centrifuged for 6 minutes at 14,000 x g and the propanol carefully poured off. The pellet was then wash ed with 100L of 70% etha nol and centrifuged for 4 minutes at 14,000 x g The ethanol was then carefully poured off and the pellet was air-dried to ensure that there was no ethanol in the tube. After the pellet was dry, 100L of TE buffer was used to reconstitute the pellet. The sample D NA was then stored in a -20C freezer until PCR could be performed (22 ). Qiagen extraction protocol A thin 1cm2 block of agar with abundant mycelia gr owth is placed in a bead-beater tube (Biospec catalog # 10831) with two sterile 5mm glass beads (F isher catalog # 11-312C). The tubes are immersed into an ice bath for 30 s then removed for 30 s and returned three times. Tubes are placed on a bead-beater (Biospec Mini-Bead Beater) for 30 s at a rate of 2500 rpm or at approximately 1/3 power for the Bead-Beater 8. It was found that when liquid nitrogen was used, the beads would freeze in th e contents of the tubes such that cells were not properly disrupted. If a cell disrupter system was not av ailable, a 1.5mL microc entrifuge tube with a conical bottom and a sterile plastic pestle was us ed to grind the sample for approximately 1 min. The macerated sample was stored in a -20C freezer until DNA extraction. The DNA was extracted using the Qiagen DNeasy Mini-Plant Kit (cat # 69106) according to the protocol with one exception. In the second and final centrifuge step for the Buffer AW wash (supplied with the kit), the centrifuge time was extended from 2 to 4 min to ensure removal of all the ethanol from the colu mn. This method was compared to the original DNA extraction method given above. Two separate elutions with 100L AE buffer (supplied with the kit) were used in the last steps of the DNA extraction. The elution products were used directly in the PCR protocol. No dilu tion of the elution products was made.

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26 Sigma extraction protocol A second DNA extraction kit was tested and co mpared to the origin al extraction method. Sigma Extract-N-Amp Plant PCR Kit (Sig ma catalog #XNAP) or REDExtract-N-Amp (Sigma catalog #XNAP2E) takes approximately 20 minutes for a complete DNA Extraction. Mycelia are scraped off of a culture grown on APDA since aerial mycelia are usually abundant on this media. If cultures were pionnatal, a sterile 3 cm piece of filter paper (Whatman #1) was placed on APDA culture plates, and the isolate was tr ansferred to the center of the disc. After the colony covered the disc, a 1cm2-piece was used in the protocol in place of the mycelia scrape. A sterile plastic pestle was used to grind the sample before 100 L of the extraction solution was added. Tubes were placed in a Thermomi xer (Eppendorf catalog #0022670000) set at 95C for 10 minutes. The tubes were placed on ice, and 100L of dilution solution was added. Tubes were vortexed and placed in a -20 C freezer until needed. An aliquot of the sample was diluted 1:10 with water prior to PCR in accordance wi th the protocol supplied with the kit. Reaction Mix, Thermocycler Program, and Gel Visualization The Plyler PCR protocol used at the PDC calls for the fo llowing reaction mixture for each sample: 7.6L of Molecular Grade Water (Eppendorf, cat # 955-15-503-3) 4.4L of 10X buffer with 1.5mM MgCl2 (Plyler, 1997) 2.4L of 2.5mM (each) dNTPs (Promega cat #U1330) 1L of Primers (HK66 and HK67 5 M each) (Integrated DNA Technologies) 0.6L of Taq in Storage Buffer B (Promega M1661diluted with 40L of MG Water) 10L DNA template (sample DNA) The reaction mix is placed into a 0.2mL or 0.5 mL thin-walled PCR tube and placed in the thermocycler (Biometra T-3000) using the Foc program: Step1: 95C for 30 seconds Step 2: 94C for 60 seconds Step 3: 62C for 60 seconds

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27 Step 4: 72C for 120 seconds Loop of Steps 2-4 34 times Step 5: 72C for 10 minutes Step 6: 4C hold A 1.5% agarose solution (Gibco BRL, cat # 5510UB) in Tris-acetateethylenediaminetetraacetic acid (TAE) buffer wa s heated. For every 100 ml of gel, 2L of 25g/l ethidium bromide was added when the so lution had cooled to ap proximately 60C. The solution was poured into a form and allowed to ge l for at least 1 h. The gel was submerged in an electrophoresis tank in TAE buffer, and 10L of each PCR product was mixed with 2L of 6x Blue/Orange loading dye (Promega catalog #G1881) and loaded into a well in the gel. A 100bp molecular weight ladder (Promega catalog #G2101) was loaded into the first well of every gel and after approximately every ei ghth sample. The gel was subjected to electrophoresis at 75 volts for between 1h 15min to 1h 30min. The gel wa s illuminated with ultraviolet light and photographed with a Gel Doc System (BioRad). If there was a band of approximately 567 bp, the sample was recorded as positive. When the band was weak, or duplicates of the sample did not agree, the process was repeat ed to verify results. New Taq Polymerases Three different reaction mixes with distinct Taq polymerases were compared to the original Promega Taq and reaction mix. Invitrogen Taq Platinum PCR SuperMix (Invitrogen cat alog # 11306-016) was tested. The supermix contains a 1.1X PCR mixture consisting of 22U/mL Platinum taq polymerase, 22mM TRISHCl (pH8.4), 55mM KCl, 1.65mMMgCl2, 220M of each dNTP, and stabilizers. For each PCR reaction, 22.5L of supermix, 1.5 L of 2M HK66/HK67 primers, and 1.0L of DNA template was used.

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28 Platinum Taq DNA polymerase (Invitrogen catalog #10966-018) also was tested. The concentrations of the reagent mix were based on other PCR protocols used in the Plant Disease Clinic and then optimized for use with the HK 66 & 67 primers. The reaction mix consisted of 15.25L of molecular grade water, 0.25L of Platinum taq polymerase, 2.5L of 10x buffer supplied with the taq (200mM Tris-HCl/ pH8.4 and 500mM KCL without Mg), 0.5L of 10mM dNTP, 1.0L of 50mM MgCl2 supplied with the taq, 2.5L of 5M HK66 & HK67 primer mix, and 3.0L of DNA template. Sigma Taq Sigma REDExtract-N-Amp PCR Reaction mi xture (Sigma catalog #R4775, supplied in the kit) and Extract-n-Amp PCR mixture (Sig ma catalog #E3004) were evaluated next. The proprietary mixture contains a 2X concentration of all the reagents except primers and DNA template. The Extract-N-Amp mixture has the sa me components except the red dye used for gel electrophoresis to allow for products which are sensitive to the dye. For the PCR reaction, 10L of REDExtract-N-Amp (or Extract-nAmp), 4L of 5M each HK66 & HK67 primer mix, and 6L of DNA template were combined. Development of Fusarium proliferatum Primers A set of primers was developed from the tran slation elongation factor 1-alpha region of F. proliferatum from GenBank accession # AY337436.1 stra in MT-F141 to multiplex with the HK66 & 67 primers. Guidelines for developing the primers included: 1) Nucleotide length of 2130; 2) GC content between 40-60%; 3) Melt te mperature similar to HK67 and HK67; 4) the sequence does not form secondary structures 5) avoid 3 or more Guanines (G) and/or Cytosines (C) at the 3 end; 6) Avoid co mplementary sequences within the primer sets; and 7) ensure the sequence is unique to the target DNA (33). The forward primer, FP1 sequence, 5TGA GTA CTA CCC TGG ACG TTG A -3 has a length of 22 base pairs, GC content of 50%, melt

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29 temperature of 57.0C, and a molecular weig ht of 6750.4 g/mole. The reverse primer, FP2 sequence, 5TGA GGT TGT GGA CAG GAA AGG 3 has a length of 21base pairs, GC content of 52.4%, melt temperature of 57.0C, and a molecular weight of 6237.1 g/mole. The predicted amplicon is 348 bp. The primer seque nces were analyzed using Integrated DNA Technologies OligoAnalyzer software (http://idtdna.com/analyzer/Applications?OligoAnalyzer/ ) for secondary structures that might inhibit PCR. No secondary structures were found to occur with these sequences. The primers were then compared to sequences in GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi ) and were found to be unique to Fusarium proliferatum The Plyler PCR thermocycler setting was us ed with the Invitrogen Platinum taq PCR reaction mixture and the FP1 and FP2 primer s replacing the HK66 and HK67 primers. The following reaction mixture was used for each sample: 15.25L of Molecular Grade Water (Eppendorf, cat # 955-15-503-3) 2.5L of 10X buffer included w ith Invitrogen Platinum taq 1.0 L 50mM MgC2 included with Invitrogen Platinum taq 0.5L of 10mM dNTPs (Sigma cat #D-7295) 2.5L of Fp Primer mix (Fp-1 and Fp-2 ; 2M each) (Integrated DNA Technologies) 0.25L of Platinum taq (Invitrogen catalog #10966) 3L DNA template (sample DNA) The PCR product was then visualized on a 1.5% agarose solution (Gibco BRL, cat # 5510UB) in TAE buffer with Ethidium Bromid e. A 100bp molecular weight ladder (Promega catalog #G2101) was loaded into the first well of every gel and after appr oximately every eighth sample. The gel was subjected to electrophoresis at 75 volts for between 1h 15min to 1h 30min. The gel was illuminated with ultraviolet li ght and photographed with a Gel Doc System (BioRad) (Figure 2-3).

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30 Multiplex of Fusarium oxysporum f. sp. canariensis and Fusarium proliferatum Primers The HK 66 &67 primers were multiplexed with FP1 and FP2 primers. The Plyler PCR thermocycler setting was used with the Invitr ogen Platinum taq PCR reaction mixture for each sample: 12.75L of Molecular Grade Water (Eppendorf, cat # 955-15-503-3) 2.5L of 10X buffer included w ith Invitrogen Platinum taq 1.0 L 50mM MgC2 included with Invitrogen Platinum taq 0.5L of 10mM dNTPs (Sigma cat #D-7295) 2.5L of HK Primer mix (HK66 and HK67; 5M each) (Integrated DNA Technologies) 2.5L of Fp Primer mix (FP1 and FP2; 2M each) (Integrat ed DNA Technologies) 0.25L of Platinum taq (Invitrogen catalog #10966) 3L DNA template (sample DNA) The PCR product was then visualized on a 1.5% agarose solution (Gibco BRL, cat # 5510UB) in TAE buffer with Ethidium Bromid e. A 100bp molecular weight ladder (Promega catalog #G2101) was loaded into the first well of every gel and after appr oximately every eighth sample. The gel was subjected to electrophoresis at 75 volts for between 1h 15min to 1h 30min. The gel was illuminated with ultraviolet li ght and photographed with a Gel Doc System (BioRad) (Figure 2-4). Results DNA Extraction The CTAB DNA extraction method (Plyler), Qiag en DNEasy Plant Mini kit, and Sigma Extract-N-Amp Plant PCR kit were compared. Data showed that the PCR reactions using template from the Qiagen kit produced compar able bands to those produced from template extracted with the CTAB procedure (Figure 2-1). The Sigma extraction kit gave variable results depending on the type (i.e. pionnatal or with aerial) of growth of th e culture (data not shown).

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31 PCR Mix The Invitrogen Platinum PCR SuperMix did not amplify DNA. The Invitrogen Platinum Taq and Sigma REDExtract-N-Amp PCR mixture amplified template DNA in a manner consistent with the Promega taq in Storag e buffer B (Figure 2-2). The cultures identified morphologically as Fusarium proliferatum showed a slight shift in th e base pair length of the fragment with the Platinum taq and Sigma taq. Primer Development Primers FP1 and FP2 produced the predicte d 348 bp amplicon only from isolates of F. proliferatum (Figure 2-3). When multiplexed with the HK primer set, there was no cross reaction or inhibition. Two bands representing the two products of different lengths were visible for isolates of F. proliferatum that give false positives with the Plyler protocol (Figure 2-4). Conclusions Results of the Plyler protocol are not affected when the Qiag en DNEasy Plant Mini Kit is used for DNA extraction. When Sigma Extr act-N-Amp Plant PCR kit extraction method was used, results varied depending upon the growth stat e of the culture. Agar from culture plates inhibited the PCR reaction leading to inconsiste nt results. Qiagen DNEasy Plant Mini Kit includes all reagents needed w ith ethanol being the only reag ent supplied by the laboratory. Quality control and ease of use of these kits leads to less errors and higher yields of better quality DNA. The Invitrogen Platinum taq with PCR mi x and Sigma Extract-N-Amp Ready Mix are acceptable alternatives to the re agents called for in the Plyler protocol. Our results were consistent in multiple experiments comparing th e two. The Invitrogen Platinum SuperMix did not amplify the target DNA sequence when used with the Plyler protocol. It is not clear why this mix did not work, but since suitable alternativ es had been identified, the mix was not pursued.

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32 Fusarium oxysporum f. sp. canariensis and some F. proliferatum isolates, but not all, produce a DNA fragment of approximately the same base pair length with the Plyler protocol. Upon sequencing the amplicons, it was determined that the F. proliferatum sequence had a small deletion and several other small polymophisms. Th e size of the Fp amplicon is approximately 60 bp shorter than the amplicon produced by Foc; however, no suitable primer sets could be developed to take advantage of this divergen ce between the species (A ppendix C). Because the difference in length is relatively small, it woul d be difficult to disti nguish between the Foc amplicon and the Fp amplicon. Since F. proliferatum differs significantly in the TEF-1 region from other Fusarium species including F. oxysporum a set of primers was developed using the TEF-1 region of F. proliferatum Development of the primers used criteria in consideration for a multiplex reaction with the HK66 and HK67 primer set. A multiplex PCR reaction using the HK66 & HK67 and FP1 & FP2 primer sets distinguishes between Foc and F. proliferatum When multiplexed, isolates of Foc only produce th e diagnostic amplicon of 567 bp, but F. proliferatum isolates produce the 348 bp amplicons and th ose previously giving false positives also produced the 567 amplicon. The FP1 and FP2 primer set may be used as an individual conventional PCR for detection of Fusarium proliferatum Diagnosticians utilizing the multiplex method de scribed have four potential outcomes: A band at 567, a band at 348, two bands at 567 and 348, or no bands at all. A single band at 567 confirms the diagnosis of Fusarium wilt. A single band at 348 or a band at 348 and 567 both indicate the isolate is F. proliferatum so no Fusarium wilt diagnosis would be given. If no bands are produced, the isolate is not Foc, and no Fusarium wilt diagnosis would be given. If the isolate has characteristics of F. oxysporum but is not Foc, then Petio le blight is a potential

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33 diagnosis, because symptoms can be quite similar to foliar symptoms of Fusarium wilt. Additional research is needed to determine the efficiency of sampling and the implications of obtaining non-Foc isolates of Fusarium from palms with wilt symptoms. Table 2-1. Isolates used in optimization of protocol Isolate Host Morph. ID* TEF ** SP3A Phoenix canariensis F. proliferatum F. proliferatum 001B Cycas sp. F. proliferatum F. proliferatum 703C Dracaena marginata F. oxysporum F.o.c. 1072B Phoenix sylvestris F. oxysporum F. oxysporum f. sp dianthi 1118B Butia x Syagrus hybrid F. proliferatum F. proliferatum 1550 Syagrus romanzoffianum F. pr oliferatum F. proliferatum 1559C Phoenix canariensis F. oxysporum Fusarium sp. *** 2675A Phoenix canariensis F. oxysporum F.o.c. Fusarium speciation based on morphological characteristics on carnation leaf agar and PDA. ** Sequence homology of tef-1 alpha compared to known isolates in Fusarium database. *** Matched closest to Fusarium sp. cf dimerum at 93%

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34 Figure 2-1. Comparison of genomic DNA extracti on methods. This gel shows the results of using the Plyler DNA extraction method and Qiagen DNEasy Plant mini kit for DNA extraction. The results show that the Qiagen DNEasy Plant Mini extraction kit and the CTAB (Plyler) extr action method give the same results with no alteration in band patterns (top half compared to the bottom half of gel).

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35 Figure 2-2. Comparison of Ta q using HK66 and HK 67 primers The original Plyler PCR reaction mix using Promega Taq was compared to Invitrogen Platinum Taq and to the Sigma PCR ReadyMix. The PCR results (prese nce or absence of bands on gel) were consistent regardless of PCR reaction mix used.

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36 Figure 2-3. Fusarium proliferatum primers FP1 and 2. Isolates 703C, 2675A, 1072B, and 1559C were identified as Fusarium oxysporum and 001B, 1118B 1550, and SP3A were identified as F. proliferatum based on morphological characteristics and sequences of the TEF-1 alpha DNA. Isolates of F. proliferatum produced bands of 348 bp and F. oxysporum isolates did not.

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37 Figure 2-4. Multiplex of HK66 & 67 with Fp -1 & 2. Isolates 703C, 2675A, 1072B, and 1559C were identified as Fusarium oxysporum and 001B, 1118B, 1550, and SP3A were identified as Fusarium proliferatum using morphological characteristics (spores) and sequence of the TEF-1 alpha DNA. When multiplexed, both sets of primers produce the same amplicons (bands on the gel) for all of the isolates tested as when used in two separate reactions. .

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38 CHAPTER 3 DEVELOPMENT OF A REAL-TIME PCR PROTOCOL Real-time PCR (also referred as Quantitative P CR) is fast, easy and more sensitive than conventional PCR. Real-time PCR can reduce the amount of time needed to analyze a sample, because no gel electrophoresis steps are necessar y. A reporter dye combined with a quencher on a short DNA sequence (approximately 15-20 base pairs) is used in place of having to visualize the DNA with gel electrophoresis. The fluorescent signal from the reporter dye increases as the target DNA is amplified exponentially with each cycl e. This fluorescence is measured at the end of each cycle and when the fluorescence achieves a preset threshold, the sample is considered positive. Isolates of F. proliferatum produce false positives with the Pl yler protocol (see chapter 2). The objective of the research was to devel op a real-time PCR protocol for confirming Fusarium wilt diagnoses of palms. Two sets of primers and two probes were multiplexed. Materials and Methods Fusarium oxysporum f. sp. canariensis New forward and reverse primers were devel oped from the original reference sequence published in Plyler, 1999 (GenBank accessi on AF118442, Appendix A) for real-time PCR analysis. Initially the region of the polymorphism between the Foc and Fp HK sequences (bp 280 to 312, Appendix C) was considered for a new prim er thus eliminating the need for a multiplex PCR. The polymorphism region had a 13.3% G-C c ontent and the flanking regions also had low G-C content and low melt temperatures, thus maki ng an optimal primer set unrealistic. The ideal melt temperature for real-time PCR primers is approximately 60-70C. The probe melting temperature should be approximate ly 5-7 C higher than the pr imer pair. The target amplicon should have a length of appr oximately 80-120 base pairs (7 ).

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39 Primers and a probe were developed using the Integrated DNA Technologies DNA Primer Quest website (http://www.idtdna.com/Scitools/Applicat ions/Primerquest/Default.aspx). The primer FOC3 starts at pos ition 349 of the original seque nce and has a sequence of 5ACT TCT CCT TTG GGA AGT ACC GCA-3, a length of 24 base pairs, GC content of 50%, a melt temperature of 60.1C, and a molecular weight of 7303.8 g/mole. The reverse primer, FOC4 starts at position 438, has a sequence of 5' AGG GAT ATT GGT TCC GGT GGT GAA 3', a length of 24 base pairs, GC c ontent of 50%, a melt temperat ure of 60.1C, and a molecular weight of 7503.9 g/mole. The expected total DNA le ngth of the amplified region is 89 base pairs (Appendix A). A Taqman probe was developed for use with a real-time protocol using the Cepheid SmartCycler system(8 ). Probe FOC-1 was tagged at the 5 end with a FAM dye and a Black Hole Quencher-1 at the 3 end. The FOC pr obe-1 sequence is 5 6-FAM/AGC CTC CAC TAC ACG TTA GAG CAT TCT G/BHQ_1 3, a length of 28 base pairs, GC content of 50%, a melt temperature of 61.4C, and a molecular weig ht of 9600.5 g/mole. The sequence starts at nucleotide position 380 of the reference seque nce published in Plyer, 1999 (GenBank accession AF118442, Appendix A). The initial real-time PCR r eaction mix consisted of 13L of Sigma PCR ReadyMix, 3L of 5M primers (FOC3 & 4) 2L of 5M probe mix, 5 L of molecular grade water, and 3L of DNA template. Eight isolates previously used to optimize the conventional PCR were used to optimize the real-time PCR protocol (Table 3-1). DNA was extracted using the Qiagen DNEasy Plant Mini Kit. A two-step thermocycler protocol was used with the following stages: Stage 1: Hold 95C for 20 seconds Stage 2: 2-temperature repeat 36 times o 95C for 15 seconds o 60C for 40 seconds with optics on

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40 Fusarium proliferatum Primers and a probe were developed using the Integrated DNA Technologies DNA Primer Quest website (http://www.idtdna.com/Scitools/Applicat ions/Primerquest/Default.aspx). The same TEF-1 sequence (accession number AY337436.1) used to develop conventional PCR primers (FP1 and FP2, Chapter 2) was used to de velop primers suitable for the real-time protocol (Appendix A). The forward primer, FP3, starts at base pair104, has a sequence of 5TCG ACA AGC GAA CCA TCG AGA AGT-3, a length of 24 base pairs, GC content of 50%, a melt temperature of 60.2C and a molecular weight of 7379.9 g/mole. The reverse primer, FP4, has a starting position at base pair 208, has a se quence of 5GTA GCA GGC ACG TTT CAA ATC GCA-3, a length of 24 base pairs, GC conten t of 50%, a melt temperature of 60.3C, and a molecular weight of 7361.9 g/mole. The expected to tal length of the amplif ied region is 105 base pairs long (Appendix A). GenBank was searched using Blast for similar sequences to primers FP3 and FP4 and several matches were found to F. proliferatum and Gibberella fujikuroi A Taqman probe, FP probe-1, was developed and in itially tagged with a TET dye which crossfluoresced on the FAM channel, therefore making it unsuitable for a multiplex reaction. The probe was tagged with a Texas Red dye at the 5 end and Black Hole Quencher-2 at the 3 end. The sequence start position is at base pa ir 137, a sequence of 5-TexRd/TTA GTC ACT TTC CCT TCG ATC GCG CGT /BHQ_2-3, a length of 27 base pairs, content of 51.9%, a melt temperature of 63.4C, and a molecular weight of 9400 g/mole. The real-time PCR reaction mix consisted of 13L of Sigma PCR R eady Mix, 3L of 2M primer mix (FP3 & 4), 2 L of 2M probe, 5L of molecula r grade water, and 3L of DNA template. Eight isolates previously used to optimize the conventiona l PCR were used to

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41 optimize the real-time PCR protocol (Table 31). A two-step protocol was used with the following stages: Stage 1: Hold 95C for 20 seconds Stage 2: 2-temperature repeat 36 times o 95C for 15 seconds o 60C for 40 seconds with optics on Multiplex Real-Time PCR The real-time primers and probes for Foc a nd Fp were combined into a multiplex PCR reaction. The PCR reaction mix consisted of 13 L of Sigma ReadyMix, 3L of 2M FOC 3 and 4 primer mix, 2L of 5M FOC probe, 3L of 2M FP3 and 4 primer mix, 2L of 2M FP probe, and 3L of DNA template. Eight isolates previously used to optimize the conventional PCR and real-time PCR were used to optimize th e multiplex real-time PCR protocol (Table 3-1). A two-step protocol was used with the following stages: Stage 1: Hold 95C for 20 seconds Stage 2: 3-temperature repeat 36 times o 95C for 15 seconds o 60C for 40 seconds with optics on Isolates were also tested us ing Invitrogen Platinum Taq as an alternative PCR reaction mix. The Reaction mix consisted of: 7.5L of Molecular Grade Water (Eppendorf, cat # 955-15-503-3) 2.5L of 10X buffer included w ith Invitrogen Platinum taq 1.25 L 50mM MgC2 included with Invitrogen Platinum taq 0.5L of 10mM dNTPs (Sigma cat #D-7295) 3.0L of Foc Primer mix (FOC-3 and FOC4; 5M each) (Integrated DNA Technologies) 2.0L of Foc FAM-labeled probe (FOC pr obe; 2M) (Integrated DNA Technologies) 3.0L of Fp Primer mix (FP1 and FP2; 2M each) (Integrated DNA Technologies) 2.0L of Fp Texas Red-labeled probe (FP probe; 2M) (Integrated DNA Technologies) 0.25L of Platinum taq (Invitrogen catalog #10966) 2L DNA template (sample DNA)

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42 Results Fusarium oxysporum f. sp. canariensis Initial testing of the FOC 3 & 4 primers and the Sigma ReadyMix using conventional PCR illustrated that the primers were able to amplify target DNA of the F. oxysporum f. sp. canariensis and the F. proliferatum isolates that were previously positive with the HK primer set. (Figure 3.1). When the primers were combined with the FAM labeled FOC Probe 1 and assayed using the SmartCycler, the isolates identified as F. oxysporum f. sp. canariensis and F. proliferatum (positive for the HK 66 & 67 amplicon) cr ossed the cycle threshold (Ct) between 2530 cycles. The isolates which did not amplify using the HK 66 & 67 primers did not amplify using the FOC 3 & 4 with FOC probe 1 (Figure 3.2). Fusarium proliferatum Initial testing of the FP 3 & 4 primers and Sigma PCR ReadyMix using conventional PCR illustrated that the primers were able to amplify target DNA of F. proliferatum isolates (Figure 3.3). Isolates which amplified using the origin al HK 66 & 67 primers produced an amplicon of approximately 105bp. When the primers were combin ed with the Texas Red labeled FP Probe 1 and assayed using the SmartCycler, the isolates identified as F. proliferatum crossed the cycle threshold (Ct) between 2530 cycles. The isolates identified as F. oxysporum f. sp. canariensis and F. oxysporum did not amplify (Figure 3.4). Multiplex Reaction Fusarium oxysporum f. sp. canariensis isolates crossed the Ct on the FAM channel between 25 and 30 cycles and did not fluoresce on the Texas Red channel when the FOC and FP primers and probes were multiplexed. The F. oxysporum isolates did not fluoresce on either channel. The F. proliferatum isolates which amplified the HK amplicon crossed the Ct on both the FAM and Texas Red channels between 25 and 30 cycles. The F. proliferatum which did not

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43 amplify the HK amplicon did not fluoresce on the FAM channel, but crossed the Ct between 25 and 30 cycles on the Texas Red channel. There was no cross fluorescence with the multiplexing of these assays (Figure 3.5). An Invitrogen Platinum Taq PCR reaction mi x was tested with the multiplexed primers and probes. There was no difference in results between the optimized Invitrogen PCR reaction mix and the Sigma ReadyMix (results not shown). Conclusions Primers FOC 3 and FOC 4 were developed using the amplicon produced with the HK66 & 67 primer set and were designed for a real-tim e PCR assay. Primers FP3 & 4 were developed from the TEF-1 sequence for F. proliferatum Initial testing of the primer sets individually yielded results at the appropria te base pair lengths of 89 for FOC3 and FOC4, and 105 for FP3 and FP4. These results were consistent with the original HK66 & HK67 conventional PCR primer sets and morphological ch aracteristics (Table D-1). The FOC3 and FOC4 primers were tested with FAM Taqman /Black Hole Quencher-1 probe and analyzed using the Cepheid SmartCycle r. The FP3 and FP4 primers were originally coupled with a TET dye which cross-fluores ced with the FAM ch annel and therefore inappropriate for multiplexing with the FOC3 and FOC4 primers. A Texas Red/Black Hole Quencher-2 probe was then tested with the FP3 and FP4 primers and analyzed using the Cepheid SmartCycler. Results from both sets of primers and probes run individually were consistent with the morphological characteri stics, conventional PCR results, and TEF-1 sequencing results (Table D-1). Optimization of reagents and thermocycler settings yielded an optimum two-step thermocycler protocol with a re peat of 36 cycles using either Sigma ReadyMix or Invitrogen

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44 Platinum Taq. Results were consistent with morphological characteris tics, conventional PCR results, and TEF-1 sequencing results (Table D-1). The two primer sets were multiplexed using a FAM taqman probe and a Texas Red taqman probe. Results were consistent wi th conventional PCR results and morphological characteristics (Table D-1). When a cycle threshol d (Ct) value is 30 or higher on either the FAM or Texas Red channel, it is recommended that the sample be cross checked using conventional PCR to verify the results.

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45 Table 3-1. Isolates used in optimization of protocol Isolate Host Morph. ID* TEF ** SP3A Phoenix canariensis F. proliferatum F. proliferatum 001B Cycas sp. F. proliferatum F. proliferatum 703C Dracaena marginata F. oxysporum F.o.c. 1072B Phoenix sylvestris F. oxysporum F. oxysporum f. sp dianthi 1118B Butia x Syagrus hybrid F. proliferatum F. proliferatum 1550 Syagrus romanzoffianum F. pr oliferatum F. proliferatum 1559C Phoenix canariensis F. oxysporum Fusarium sp. *** 2675A Phoenix canariensis F. oxysporum F.o.c. Fusarium speciation based on morphological characteristics on carnation leaf agar and PDA. ** Sequence homology of tef-1 alpha compared to known isolates in Fusarium database. *** Matched closest to Fusarium sp. cf dimerum at 93%

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46 Figure 3-1. Gel electrophoresis of PCR amplicons using primers FOC3 and FOC4. Primers were diluted to 2, 5 and 10M to determine the optimal concentration for PCR. A Fusarium oxysporum f. sp. canariensis isolate (703C), a F. proliferatum negative for the HK amplicon isolate (1550), a positive F. proliferatum positive for the HK amplicon isolate (001B), and a no template control water blank were used to determine primer efficacy. A Promega 100bp ladder (catalog # G210A) was used to determine the base pair length. The sequence amplified was approximately 90 base pairs.

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47 Figure 3-2. Real-time PCR results using prim ers FOC3 and FOC4 with the FOC FAM probe. Graphic results of the real-time PCR prot ocol using primers FOC 3, FOC 4, and FOC Probe 1 for F. oxysporum isolates (A) and F. proliferatum isolates (B). The default threshold fluorescence of 30 units was us ed. If the fluorescence of the reaction exceeds the threshold before the 30th cycle the sample is pos itive. Foc isolates 703C and 2675a and Fp isolates 001b and 1118b were positive. A B

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48 Figure 3-3. Gel electrophoresis of amplicons ob tained with primers FP3 and FP4. Primers were diluted to 2, 5 and 10M to determine the best concentration to use for PCR. A Fusarium oxysporum positive control, a F. proliferatum negative control, a positive F. proliferatum and a no template control water bla nk were used to determine primer efficacy. A Promega 100bp ladder (catalog # G 210A) was used to determine the base pair length. The amplified sequence was approximately 105 base pairs.

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49 Figure 3-4. Real-time PCR results using primer s FP3 and FP4 with the FP Texas Red probe. Graph of initial real-time PCR using primer FP 3 and FP 4, and FP Probe 1 for F. oxysporum isolates show no amplification or cha nge in fluorescence (negative) (a). Isolates of F. proliferatum did amplify and fluorescence reached the threshold value between 25 and 30 cycles (positive) (b). A B

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50 Figure 3-5. Real-time multiplex PCR results-FAM channel. A) F. oxysporum isolates that are f. sp. canariensis amplified and fluoresced on the FAM channel, B) F. proliferatum isolates that were positive with HK prim ers also amplified and fluoresced on the FAM channel. A B

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51 Figure 3-6. Real-time multiplex PCR resultsTexas Red channel. A) F. oxysporum isolates did not amplify or fluoresce on th e Texas Red channel; B) all F. proliferatum isolates amplified and fluoresced on the Texas Red channel. A B

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52 CHAPTER 4 STANDARD OPERATING PROCEDURES Isolation Palm samples submitted to the Plant Disease Clinic are processed following a standard operating procedure. Samples are examined visua lly and microscopically and then processed by culturing the symptomatic tissue. Initial cultures of Fusarium spp. are isolated from rachis, leaf, and leaflet. Internal tissue samples are cut into small pieces, rinsed in a five percent household bleach solution for one minute, and then rinsed in sterile de-ionized water. The surface sterilized tissue is plated on culture media; Acidified potat o dextrose agar (APDA), quarter strength potato dextrose agar (QPDA), and Water agar (WA). A minimum of four pieces of surface-sterilized tissue are placed on a plate of each type of media. When fungal colonies are observed to be growing from the tissue, the plates are observed in situ using a compound microscope at 100X. If a Fusarium sp. is observed, a subculture is taken usi ng sterile technique an d single-spored to ensure a pure isolate. If more than one Fusarium colony is observed, The Fusarium isolates are given a letter designation after the sample number to distinguish them. These cultures are incubated at room temperature on the laborator y bench for approximately 7-14 days. Mycelia from approximately 1 cm2 are collected on a dissecting needle and transferred to a 2 ml conical bottom tube. Conventional Polymerase Chain Reaction The following outlines the steps should be used for detection of Fusarium oxysporum f. sp. canariensis using conventional PCR: 1. Cell disruption with plastic pestle or 2 glass beads and a Biospecs BeadBeater 2. Qiagens DNEasy Plant Mini kit for DNA extraction 3. PCR with the following reaction mix:

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53 a. Using Invitrogen Platinum taq 12.75L of Molecular Grade Water (Eppendorf, cat # 955-15-503-3) 2.5L of 10X buffer included w ith Invitrogen Platinum taq 1.0L 50mM MgC2 included with Invitrogen Platinum taq 0.5L of 10mM dNTPs (Sigma cat #D-7295) 2.5L of HK Primer mix (HK66 a nd HK67; 5M each) (Integrated DNA Technologies) 2.5L of Fp Primer mix (Fp-1 a nd Fp-2; 2M each) (Integrated DNA Technologies) 0.25L of Platinum taq (Invitrogen catalog #10966) 3L DNA template (sample DNA) b. Using Sigma ReadyMix (with or without the red dye) 10L of Sigma ReadyMix 2L of 10M each HK66 & HK67 primer mix 2L of 4M each FP1 & FP2 primer mix 6L of DNA Template 4. Thermocycler settings at: Step1: 95C for 30 seconds Step 2: 94C for 60 seconds Step 3: 62C for 60 seconds Step 4: 72C for 120 seconds Loop of steps 2-4 34 times Step 5: 72C for 10 minutes Step 6: 4C hold 5. Visualize the DNA using gel electrophoresis. Results should be interpreted in the following manner: One band only at the 567 bp position is identified as Fusarium oxysporum f. sp. canariensis Two bands, one at 567 bp and one at 348bp, or just one band at 348 bp, is identified as Fusarium proliferatum; Fusarium oxysporum f. sp. canariensis not detected; No bands presentFusarium oxysporum f. sp. canariensis and Fusarium proliferatum not detected; Weak or faint bands; or duplicate samp les that do not agree should be reanalyzed.

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54 Real-Time Polymerase Chain Reaction The following outlines the steps that should be used for detection of Fusarium oxysporum f. sp. canariensis using real-time PCR: 1. Cell disruption with plastic pestle or 2 glass beads and a Biospecs BeadBeater 2. Qiagens DNEasy Plant Mini kit for DNA extraction 3. PCR with the following reaction mix: a. Using Invitrogen Platinum taq 7.5L of Molecular Grade Water (Eppendorf, cat # 955-15-503-3) 2.5L of 10X buffer included w ith Invitrogen Platinum taq 1.25 L 50mM MgC2 included with Invitrogen Platinum taq 0.5L of 10mM dNTPs (Sigma cat #D-7295) 3.0L of Foc Primer mix (FOC-3 a nd FOC-4; 5M each) (Integrated DNA Technologies) 2.0L of Foc FAM-labeled probe (F OC probe; 2M) (Integrated DNA Technologies) 3.0L of Fp Primer mix (FP1 a nd FP2; 2M each) (Integrated DNA Technologies) 2.0L of Fp Texas Red-labeled prob e (FP probe; 2M) (Integrated DNA Technologies) 0.25L of Platinum taq (Invitrogen catalog #10966) 2L DNA template (sample DNA or controls) b. Using Sigma ReadyMix (with or without the red dye) 13L of Sigma ReadyMix 3L of 2M FOC 3 & 4 primer mix 2L of 5M FOC probe 1 3L of 2M FP 3 & 4 primer mix 2L of 2M FP probe 1 3L of DNA template (sample DNA or controls)

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55 4. Thermocycler settings at: Dye Set with both FAM and Te xas Red (FCTC25 or FTTC25) Stage 1: Hold 95C for 20 seconds Stage 2: 3-temperature repeat 36 times a. 95C for 15 seconds b. 60C for 40 seconds with optics on 5. Review the results: a. If samples fluoresced on FAM channel onl y, with a Ct value of 30 or less, sample is positive for F. oxysporum f. sp. canariensis b. If sample fluoresced on both the FA M channel and the Texas Red channel or the Texas Red channel only, with both Ct values of 30 or less, sample is negative for F. oxysporum f. sp. canariensis positive for F. proliferatum c. If sample did not fluoresce on either channel, sample is negative for F. oxysporum f. sp. canariensis and F. proliferatum d. Any sample which crossed the threshol d above 30 should be cross checked with the conventional PCR protocol to verify results.

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56 APPENDIX A PRIMER SEQUENCES 1 AAGAACTATC AGCGATCTTA CCGGGCCTAC AACAGAA TTC GTCCCATATG AAGAACTATC AGCGATCTTA CCGGG CCTAC GACAGAATTC GTCCCATATG 51 GATGGAAGCC TACCATCTTT GTCGACGGCA CCTTCGATCT GGTAGCTGGC GATGGAAGCC TACCATCTTT GTCGACGGCA CCTTCAATCT GGTAGCTGGC 101 TGTGCCAAAG CCCCAGATAA AGTCCT TGGG AAGCGACATG CTGAAAAACA TGTGCCAAAG CCCCAGATAA ATTCCT TGGG AAGCGACATG CTGAAAAATA 151 GATGAATAAA TAATAATAAT AATAGTAATA GTAATGATAG TAATAATAAT GATGAATGAA CAATAATGGT 201 AATAAAACTG ACTGGACTAA GAGCCTGATG AAAGTCGTTT ATGAAGGTAT AATAGAACGG ACTGGACTAG GAGCCTGGTG AAAGTCGTTT ATGGAGGTAT 251 CCCGTCACAG ACGGAACTTC CTTTTTTGAA TCTCCGAAAA CGTTCTTGAC CCCGTCACAG ACGGAACTTC C TTTTTTGAA TGTCCGAAAA CGTTCTTAAC 301 TGTAGGAACT TCTCCTTTG G GAAGTACCGC ATATTTAAGC CTCCACTACA TGTAGTAACT TCTTCTTTGG GAAGTACC GC ATATTTAAGC CTCCACTA A 351 CGTTAGAGCA TTCTGAGTAT G TTTCACCAC CGGAACCAAT ATCCCTTTTG CGTTAGAGCA TTCGGAGTAT GTTTT ACCAC CGGAACCAAT ATCTCTTCTG 401 GGAACACGGG GACTCAGCCA AATCACGCCT TGCTTGGATC TCGTCAGATG GGAACACGGG GACCCGGCCA AATCACG CCT TGCTTGGATC TCGTCAGATA 451 TAGCGGCCGT ATTCGACGCC TTGCACCGCC TT TAGCAGCCTT ATTCGACGGC TTCCACCGCC TCTT Figure A-1. Comparison of amp licons sequenced using HK66 and HK67 primer sequences from Fusarium oxysporum isolate 703A (top line) from a Dracaena marginata and F. proliferatum isolate 1118B (bottom line) from a Butia x Syagrus hybrid palm.

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57 Fp1 01 GACTNTGGCA AGTCGACCAC TGTG AGTACT ACCCTGGACG TTGAGCTTAT CTGAXACCGT TCAGCTGGTG ACACTCATGA TGGGACCTGC AACTCGAATA 51 NTGCCATCGT GATCCTGACC AAGATCTGGC GGGGTACATC TTGGAAGACA NACGGTAGCA CTAGGACTGG TTC TAGACCG CCCCATGTAG AACCTTCTGT 100 ACATGCTGAC ATCGCTTCAC AGA CCGGTCA CTTGATCTAC CAGTGCGGTG TGTACGACTG TAGCGAAGTG TCTGGCCAGT GAACTAGATG GTCACGCCAC 101 GTATCGACAA GCGAACCATC GAGA AGTTCG AGAAGGTTAG TCACTTTCCC CATAGCTGTT CGCTTGGTAG CTC TTCAAGC TCTTCCAATC AGTGAAAGGG 151 TTCGATCGCG CGTCCTCTGC CCACC GATTT CACTTGCGAT TTGAAACGTG AAGCTAGCGC GCAGGAGACG GGTGGCTAAA GTGAACGCTA AACTTTGCAC 201 CCTGCTACCC CGCTCGAGAC CAAAAA TTTT GCGATATGAC CGTAATTTTT GGACGATGGG GCGAGCTCTG G TTTTTAAAA CGCTATAC TG GCATTAAAAA 251 TTGGTGGGGC ATTT ACCCCG CCACTCGAGC GATGGGCGCG TTTTTGCCCT AACCACCCCG TAAATGGGGC GGTG AGCTCG CTACCCGCGC AAAAACGGGA 301 TTCCTGTCCA CAACCTCAAT GAGCGCATTG TCACGTGTCA AGCAGCGACT AAGGACAGGT GTTGGAGTTA CTCGCGTAAC AGTGCACAGT TCGTCGCTGA Fp2 351 AACCATTCGA CAATAGGAAG CCGCCGAGCT CGGTAAGG TTGGTAAGCT GTTATCCTTC GGCGGCTCGA GCCATTCC Figure A-2. Sequence and primer selection for Fusarium proliferatum primers FP1 and FP2 from Fusarium proliferatum sequence encoding the tran slation elongation factor (TEF-1 ) and complementary strand from GenBank accession # AY337436.1. New primers were developed using F. proliferatum strain MT-141 translation elongation factor EF-1 from GenBank accession number AY337436.1.

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58 HK66 01 CATCAGAAGT GCGTTCGTAG G AATTGCAGG CCACAACACC GGAAGAACTA GTAGTCTTCA CGCAAGCATC CTTAACGTCC GGTGTTGTGG CCTTCTTGAT 51 TCAGCGATCT TACCGGGCCT ACAACAGAAT TCGTCCCATA TGGATGGAAG AGTCTCTAGA ATGGCCCGGA TGTTGTCTTA AGCAGGTAT ACCTACCTTC 101 CCTACCATCT TTGTCGACGG CACCTTC GAT CTGGTAGCTG GCTGTGCCAA GGATGGTAGA AACAGCTGCC GTGGAAGCTA GACCATCGAC CGACACGGTT 151 AGCCCCAGAT AAGTCCTTGG GAAGCGAC AT GCTGAAAAAC AGATGAATAA TCGGGGTCTA TTCAGGAACC CTTCGCTGTA CGACTTTTTG TCTACTTATT 201 ATAATAATAA TAATAGTAAT AGTAATGATA GTAATAATAA TAATAAAACT TATTATTATT ATTATCATTA TCATTACTAT CATTATTATT ATTATTTTGA 251 GACTGGACTA AGAGCCTGAT GAA AGTCGTT TATGAAGGTA TCCCGTCACA CTGACCTGAT TCTCGGACTA CTTTC AGCAA ATACTTCCAT AGGGCAGTGT FOC3 301 GACGGAACTT CCTTTTTTGA ATCT CCGAAA ACGTTC TTGA CTGTAGGA AC CTGCCTTGAA GGAAAAAACT TAGAGGCTTT TGCAAGAACT GACATCCTTG FOC Probe 1 351 TTCTCCTTT GGGAAGTACC GC ATATTTAA GCCTCCA CTA CACGTTAGAG AAGAGGAAA CCCTTCATGG CGTATAAATT CGGAGGTGAT GTGCAATCTC 401 CATTCTGAGT ATGTTTCACC ACCGGAAC CA ATATCCCTTT TGGGAACACG GTAAGACTCA TACA AAGTGG TGGCCTTGGT TATAGGGA AA ACCCTTGTGC FOC4 451 GGGACTCAGC CAAATCACGC CTTG CTTGG ATCTCGTCAG ATGTAGCGGC CCCTGAGTCG GTTTAGTGCG GAAC GAACC TAGAGCAGTC TACATCGCCG 501 CGTATTCGAC GCCTTGCACC GCC TTCTGGG TCGCTACATG GAAAGGGAGT GCATAAGCTG CGGAAC GTGG CGGAAGCCCC AGCGATGTA C CTTTCCCT CA 551 GAGGCAAACC ATTACAACG (567 base pairs) CTCCGTTTGG TAATGTTGC HK67 Figure A-3. Sequence and pr imer/probe selection for Fusarium oxysporum f. sp canariensis primers FOC3 and FOC4 and FOC Probe 1; sequence of HK66 & HK67 amplicon showing the position of FOC3, FOC4 and FOC Probe 1.

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59 01 GACTNTGGCA AGTCGACCAC TGTG AGTACT ACCCTGGACG TTGAGCTTAT CTGAXACCGT TCAGCTGGTG ACACTCATGA TGGGACCTGC AACTCGAATA 51 NTGCCATCGT GATCCTGACC AAGATCTGGC GGGGTACATC TTGGAAGACA NACGGTAGCA CTAGGACTGG TTC TAGACCG CCCCATGTAG AACCTTCTGT 100 ACATGCTGAC ATCGCTTCAC AGA CCGGTCA CTTGATCTAC CAGTGCGGTG TGTACGACTG TAGCGAAGTG TCTGGCCAGT GAACTAGATG GTCACGCCAC Fp3 Probe 1 101 GTA TCGACAA GCGAACCATC GAGAAGT TCG AGAAGGTTAG TCACTTTCCC CATAGCTGTT CGCTTGGTAG CTC TTCAAGC TCTTCCAATC AGTGAAAGGG 151 TTCGATCGCG CGTCCTCTGC CCACC GATTT CACTTGCGAT TTGAAACGTG AAGCTAGCGC GCAGGAGACG GGTGGCTAAA GTGAACGCTA AACTTTGCAC 201 CCTGCTACCC CGCTCGAGAC CAAAAA TTTT GCGATATGAC CGTAATTTTT GGACGATGGG GCGAGCTCTG G TTTTTAAAA CGCTATAC TG GCATTAAAAA Fp4 251 TTGGTGGGGC ATTT ACCCCG CCACTCGAGC GATGGGCGCG TTTTTGCCCT AACCACCCCG TAAATGGGGC GGTG AGCTCG CTACCCGCGC AAAAACGGGA 301 TTCCTGTCCA CAACCTCAAT GAGCGCATTG TCACGTGTCA AGCAGCGACT AAGGACAGGT GTTGGAGTTA CTCGCGTAAC AGTGCACAGT TCGTCGCTGA 351 AACCATTCGA CAATAGGAAG CCGCCGAGCT CGGTAAGG TTGGTAAGCT GTTATCCTTC GGCGGCTCGA GCCATTCC Figure A-4. Sequence and pr imer/probe selection for Fusarium proliferatum primers FP3 and FP4 and FP Probe 1; TEF-1alpha Fusarium proliferatum sequence with its complementary strand. The newly developed primers and probe are highlighted in yellow.

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60 APPENDIX B SEQUENCING Amplicons of 8 Fusarium isolates produced with the HK66 and HK67 primers using the Qiagen extraction and Invitrogen Platinum Taq methods were purified using Qiagen Qiaquick PCR Purification kit (catalog # 2810 4) and were sequenced by the Interdiciplinary Center for Biotechnology Research (ICBR) at the University of Florida ( http://www.biotech.uf l.edu/services.html ). Two of the sequenced isolates were identified morphologically as F. proliferatum and six were F. oxysporum Host, isolate number, and county of origin of sequenced isolates are given in Appendix Tabl e F-1. Sequences were compared using Sequencher 4.6 (Gene C odes) and shown in Appendix B. The translation elongation fact or 1-alpha (TEF) region from one of the isolates obtained from Dracaena marginata identified as F. oxysporum by morphological criteria (703C) was sent to Kansas State University to be amplified and sequenced (18) to confirm the isolate was F. oxysporum f. sp. canariensis The sequence was compared to other similar sequences in the Fusarium Database ( http://fusarium.cbio.psu.edu ; Penn State). The TEF region was sequenced by the ICBR (UF) for isolates which were posit ive and negative for the HK66 and HK67 primers from the same host plant to verify the results (below). Sequencing results of isol ate 703C using the TEF 1 confirmed the isolate was F. oxysporum f. sp. canariensis with a 100% match with no gaps using the Fusarium Database (PSU). Sequencing results of the HK66 and HK67 amplicon determined the F. proliferatum had a deletion of approximately 60 bps compared to the F. oxysporum f.sp canariensis isolates. The region of the polymorphism between the Foc and Fp HK sequences located approximately base pairs 280 to 312 (below) was considered for a ne w primer thus eliminating the need for a multiplex PCR. The polymorphism region had a 13.3% G-C content and the flanking regions

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61 also had low G-C content and low melt temper atures, thus making an optimal primer set unrealistic.

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62 APPENDIX C HK SEQUENCE COMPARISONS

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63 1 50 001A__oxy_cycad_osceola .......... .......... .......... .......... .......... 001B_prolif_cycad_osceola .......... .......... .......... .......... .......... 1118B_prolif_butia_manatee .......... .......... .......... .......... .......... 1559B_oxy_CID_dade .......... .......... .......... .......... .......... 2675A_oxy_CID_sarasota .......... .......... .......... .......... .......... 3134D_oxy_CID_marion .......... .......... .......... .......... .......... 703A_oxy_Dracaena_hillsb .......... .......... .......... .......... .......... 703B_oxy_Dracaena_hillsb .......... .......... .......... .......... .......... HK_primer_sequence AAGCTTACCA AGAAATGGAA AATCGATACG CTCTGGCACC ATATTGTTTC 51 100 001A__oxy_cycad_osceola .......... .......... .......... .......... .......... 001B_prolif_cycad_osceola .......... .......... .......... .......... .......... 1118B_prolif_butia_manatee .......... .......... .......... .......... .......... 1559B_oxy_CID_dade .......... .......... .......... .......... .......... 2675A_oxy_CID_sarasota .......... .......... .......... .......... .......... 3134D_oxy_CID_marion .......... .......... .......... .......... .......... 703A_oxy_Dracaena_hillsb .......... .......... .......... .......... .......... 703B_oxy_Dracaena_hillsb .......... .......... .......... .......... .......... HK_primer_sequence AGCAAAGCGA TGTCATCAGA AGTGCGTTCG TAGGAATTGC AGGCCACAAC 101 150 001A__oxy_cycad_osceola .....AAGAA CTATCAGCGA TCTTACCGGG CCTACAACAG AATTCGTCCC 001B_prolif_cycad_osceola .....AAGAA CTATCAGCGA TCTTACCGGG CCTACGACAG AATTCGTCCC 1118B_prolif_butia_manatee .....AAGAA CTATCAGCGA TCTTACCGGG CCTACGACAG AATTCGTCCC 1559B_oxy_CID_dade ...GGAAGAA CTATCAGCGA TCTTACCGGG CCTACAACAG AATTCGTCCC 2675A_oxy_CID_sarasota ...GGAAGAA CTATCAGCGA TCTTACCGGG CCTACAACAG AATTCGTCCC 3134D_oxy_CID_marion ....GAAGAA CTATCAGCGA TCTTACCGGG CCTACAACAG AATTCGTCCC 703A_oxy_Dracaena_hillsb .....AAGAA CTATCAGCGA TCTTACCGGG CCTACAACAG AATTCGTCCC 703B_oxy_Dracaena_hillsb .....AAGAA CTATCAGCGA TCTTACCGGG CCTACAACAG AATTCGTCCC HK_primer_sequence ACCGGAAGAA CTATCAGCGA TCTTACCGGG CCTACAACAG AATTCGTCCC Consensus AAGAA CTATCAGCGA TCTTACCGGG CCTACAACAG AATTCGTCCC

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64 151 200 001A__oxy_cycad_osceola ATATGGATGG AAGCCTACCA TCTTTGTCGA CGGCACCTTC GATCTGGTAG 001B_prolif_cycad_osceola ATATGGATGG AAGCCTACCA TCTTTGTCGA CGGCACCTTC GATCTGGTAG 1118B_prolif_butia_manatee ATATGGATGG AAGCCTACCA TCTTTGTCGA CGGCACCTTC AATCTGGTAG 1559B_oxy_CID_dade ATATGGATGG AAGCCTACCA TCTTTGTCGA CGGCACCTTC GATCTGGTAG 2675A_oxy_CID_sarasota ATATGGATGG AAGCCTACCA TCTTTGTCGA CGGCACCTTC GATCTGGTAG 3134D_oxy_CID_marion ATATGGATGG AAGCCTACCA TCTTTGTCGA CGGCACCTTC GATCTGGTAG 703A_oxy_Dracaena_hillsb ATATGGATGG AAGCCTACCA TCTTTGTCGA CGGCACCTTC GATCTGGTAG 703B_oxy_Dracaena_hillsb ATATGGATGG AAGCCTACCA TCTTTGTCGA CGGCACCTTC GATCTGGTAG HK_primer_sequence ATATGGATGG AAGCCTACCA TCTTTGTCGA CGGCACCTTC GATCTGGTAG Consensus ATATGGATGG AAGCCTACCA TCTTTGTCGA CGGCACCTTC GATCTGGTAG 201 250 001A__oxy_cycad_osceola CTGGCTGTGC CAAAGCCCCA GATAAAGTCC TTGGGAAGCG ACATGCTGAA 001B_prolif_cycad_osceola CTGGCTGTGC CAAAGCCCCA GATAAATTCT TTAGGAAGCG ACATGCTGAA 1118B_prolif_butia_manatee CTGGCTGTGC CAAAGCCCCA GATAAATTCC TTGGGAAGCG ACATGCTGAA 1559B_oxy_CID_dade CTGGCTGTGC CAAAGCCCCA GATAAAGTCC TTGGGAAGCG ACATGCTGAA 2675A_oxy_CID_sarasota CTGGCTGTGC CAAAGCCCCA GATAAAGTCC TTGGGAAGCG ACATGCTGAA 3134D_oxy_CID_marion CTGGCTGTGC CAAAGCCCCA GATAAAGTCC TTGGGAAGCG ACATGCTGAA 703A_oxy_Dracaena_hillsb CTGGCTGTGC CAAAGCCCCA GATAAAGTCC TTGGGAAGCG ACATGCTGAA 703B_oxy_Dracaena_hillsb CTGGCTGTGC CAAAGCCCCA GATAAAGTCC TTGGGAAGCG ACATGCTGAA HK_primer_sequence CTGGCTGTGC CAAAGCCCCA GATAAAGTCC TTGGGAAGCG ACATGCTGAA Consensus CTGGCTGTGC CAAAGCCCCA GATAAAGTCC TTGGGAAGCG ACATGCTGAA 251 300 001A__oxy_cycad_osceola AAACAGATGA ATAAATAATA ATAATAATA. ..GTAATAGT AATGATAGTA 001B_prolif_cycad_osceola AAATAGATGA ATGAATAATA ATGGTAATA. .......... .......... 1118B_prolif_butia_manatee AAATAGATGA ATGAACAATA ATGGTAATA. .......... .......... 1559B_oxy_CID_dade AAACAGATGA ATAAATAATA ATAATAATAA TAGTAATAGT AATAATAGTA 2675A_oxy_CID_sarasota AAACAGATGA ATAAATAATA ATAATAATAA TAGTAATAGT AATAATAGTA 3134D_oxy_CID_marion AAACAGATGA ATAAATAATA ATAATAATA. ..GTAATAGT AATGATAGTA 703A_oxy_Dracaena_hillsb AAACAGATGA ATAAATAATA ATAATAATA. ..GTAATAGT AATGATAGTA 703B_oxy_Dracaena_hillsb AAACAGATGA ATAAATAATA ATAATAATA. ..GTAATAGT AATGATAGTA HK_primer_sequence AAACAGATGA ATAAATAATA ATAATAATA. ..GTAATAGT AATGATAGTA Consensus AAACAGATGA ATAAATAATA ATAATAATA GTAATAGT AATGATAGTA

PAGE 65

65 301 350 001A__oxy_cycad_osceola ATAATAATAA TAAAACTGAC TGGACTAAGA GCCTGATGAA AGTCGTTTAT 001B_prolif_cycad_osceola .......... ..GAACGGAC TGGACTAGGA GCCTGGTGAA AGTCGTTTAT 1118B_prolif_butia_manatee .......... ..GAACGGAC TGGACTAGGA GCCTGGTGAA AGTCGTTTAT 1559B_oxy_CID_dade ATAATAATAA TAAAACTGAC TGGACTAAGA GCCTGATGAA AGTCGTTTAT 2675A_oxy_CID_sarasota ATAATAATAA TAAAACTGAC TGGACTAAGA GCCTGATGAA AGTCGTTTAT 3134D_oxy_CID_marion ATAATAATAA TAAAACTGAC TGGACTAAGA GCCTGATGAA AGTCGTTTAT 703A_oxy_Dracaena_hillsb ATAATAATAA TAAAACTGAC TGGACTAAGA GCCTGATGAA AGTCGTTTAT 703B_oxy_Dracaena_hillsb ATAATAATAA TAAAACTGAC TGGACTAAGA GCCTGATGAA AGTCGTTTAT HK_primer_sequence ATAATAATAA TAAAACTGAC TGGACTAAGA GCCTGATGAA AGTCGTTTAT Consensus ATAATAATAA TAAAACTGAC TGGACTAAGA GCCTGATGAA AGTCGTTTAT 351 400 001A__oxy_cycad_osceola GAAGGTATCC CGTCACAGAC GGAACTTCCT TTTTTGAATC TCCGAAAACG 001B_prolif_cycad_osceola GAAGGTATCC CGTCACAGAC GGAACTTCCT TTTTTGAATG TCCGAAAACG 1118B_prolif_butia_manatee GGAGGTATCC CGTCACAGAC GGAACTTCCT TTTTTGAATG TCCGAAAACG 1559B_oxy_CID_dade GAAGGTATCC CGTCACAGAC GGAACTTCCT TTTTTGAATC TCCGAAAACG 2675A_oxy_CID_sarasota GAAGGTATCC CGTCACAGAC GGAACTTCCT TTTTTGAATC TCCGAAAACG 3134D_oxy_CID_marion GAAGGTATCC CGTCACAGAC GGAACTTCCT TTTTTGAATC TCCGAAAACG 703A_oxy_Dracaena_hillsb GAAGGTATCC CGTCACAGAC GGAACTTCCT TTTTTGAATC TCCGAAAACG 703B_oxy_Dracaena_hillsb GAAGGTATCC CGTCACAGAC GGAACTTCCT TTTTTGAATC TCCGAAAACG HK_primer_sequence GAAGGTATCC CGTCACAGAC GGAACTTCCT TTTTTGAATC TCCGAAAACG Consensus GAAGGTATCC CGTCACAGAC GGAACTTCCT TTTTTGAATC TCCGAAAACG 401 450 001A__oxy_cycad_osceola TTCTTGACTG TAGGAACTTC TCCTTTGGGA AGTACCGCAT ATTTAAGCCT 001B_prolif_cycad_osceola TTCTTAACTG TAGTAACTTC TCCTTTGGGA AGTACCGCAT ATTTAAGCCT 1118B_prolif_butia_manatee TTCTTAACTG TAGTAACTTC TTCTTTGGGA AGTACCGCAT ATTTAAGCCT 1559B_oxy_CID_dade TTCTTGACTG TAGGAACTTC TCCTTTGGGG AGGACCGCAT ATTTAAGCCT 2675A_oxy_CID_sarasota TTCTTGACTG TAGGAACTTC TCCTTTGGGG AGGACCGCAT ATTTAAGCCT 3134D_oxy_CID_marion TTCTTGACTG TAGGAACTTC TCCTTTGGGA AGTACCGCAT ATTTAAGCCT 703A_oxy_Dracaena_hillsb TTCTTGACTG TAGGAACTTC TCCTTTGGGA AGTACCGCAT ATTTAAGCCT 703B_oxy_Dracaena_hillsb TTCTTGACTG TAGGAACTTC TCCTTTGGGA AGTACCGCAT ATTTAAGCCT HK_primer_sequence TTCTTGACTG TAGGAACTTC TCCTTTGGGA AGTACCGCAT ATTTAAGCCT Consensus TTCTTGACTG TAGGAACTTC TCCTTTGGGA AGTACCGCAT ATTTAAGCCT

PAGE 66

66 451 500 001A__oxy_cycad_osceola CCACTACACG TTAGAGCATT CTGAGTATGT TTCACCACCG GAACCAATAT 001B_prolif_cycad_osceola CCACTA.ACG TTAGAGCATT CTGAGTATGT TTCACCACCG GAATAAATAT 1118B_prolif_butia_manatee CCACTA.ACG TTAGAGCATT CGGAGTATGT TTTACCACCG GAACCAATAT 1559B_oxy_CID_dade CCACTA.ACG TTAGAGCATT CTGAGTATGT TTCACCACCG GAACCAATAT 2675A_oxy_CID_sarasota CCACTA.ACG TTAGAGCATT CTGAGTATGT TTCACCACCG GAACCAATAT 3134D_oxy_CID_marion CCACTACACG TTAGAGCATT CTGAGTATGT TTCACCACCG GAACCAATAT 703A_oxy_Dracaena_hillsb CCACTACACG TTAGAGCATT CTGAGTATGT TTCACCACCG GAACCAATAT 703B_oxy_Dracaena_hillsb CCACTACACG TTAGAGCATT CTGAGTATGT TTCACCACCG GAACCAATAT HK_primer_sequence CCACTACACG TTAGAGCATT CTGAGTATGT TTCACCACCG GAACCAATAT Consensus CCACTACACG TTAGAGCATT CTGAGTATGT TTCACCACCG GAACCAATAT 501 550 001A__oxy_cycad_osceola CCCTTTTGGG AACACGGGGA CTCAGCCAAA TCACGCCTTG CTTGGATCTC 001B_prolif_cycad_osceola CTCTTTTGGG AACACGGGGA CCCGGCCAAA TCACGCCTTG ....GATCTC 1118B_prolif_butia_manatee CTCTTCTGGG AACACGGGGA CCCGGCCAAA TCACGCCTTG CTTGGATCTC 1559B_oxy_CID_dade TCCTTTTGGG AACAAGGGGA CTCAGCCAAA TCACGCCTTG CTTGGATCTC 2675A_oxy_CID_sarasota TCCTTTTGGG AACAAGGGGA CTCAGCCAAA TCACGCCTTG CTTGGATCTC 3134D_oxy_CID_marion CCCTTTTGGG AACACGGGGA CTCAGCCAAA TCACGCCTTG CTTGGATCTC 703A_oxy_Dracaena_hillsb CCCTTTTGGG AACACGGGGA CTCAGCCAAA TCACGCCTTG CTTGGATCTC 703B_oxy_Dracaena_hillsb CCCTTTTGGG AACACGGGGA CTCAGCCAAA TCACGCCTTG CTTGGATCTC HK_primer_sequence CCCTTTTGGG AACACGGGGA CTCAGCCAAA TCACGCCTTG CTTGGATCTC Consensus CCCTTTTGGG AACACGGGGA CTCAGCCAAA TCACGCCTTG CTTGGATCTC 551 600 001A__oxy_cycad_osceola GTCAGATGTA GCGGCCGTAT TCGACGCCTT GCACCGCCTT .......... 001B_prolif_cycad_osceola GTCAGATATA ACAGCCGTAT TCGACGCCTT CCACCGCCTC TT........ 1118B_prolif_butia_manatee GTCAGATATA GCAGCCTTAT TCGACGGCTT CCACCGCCTC TT........ 1559B_oxy_CID_dade GTCAGATGTA GCGGCCGTGT TCAACGCCTT CCACCGCCTT .......... 2675A_oxy_CID_sarasota GTCAGATGTA GCGGCCGTGT TCAACGCCTT CCACCGCCTT .......... 3134D_oxy_CID_marion GTCAGATGTA GCGGCCGTAT TCGACGCCTT GCACCGCCTT .......... 703A_oxy_Dracaena_hillsb GTCAGATGTA GCGGCCGTAT TCGACGCCTT GCACCGCCTT .......... 703B_oxy_Dracaena_hillsb GTCAGATGTA GCGGCCGTAT TCGACGCCTT GCACCGCCTT .......... HK_primer_sequence GTCAGATGTA GCGGCCGTAT TCGACGCCTT GCACCGCCTT CTGGGTCGCT Consensus GTCAGATGTA GCGGCCGTAT TCGACGCCTT GCACCGCCTT

PAGE 67

67 601 650 001A__oxy_cycad_osceola .......... .......... .......... .......... .......... 001B_prolif_cycad_osceola .......... .......... .......... .......... .......... 1118B_prolif_butia_manatee .......... .......... .......... .......... .......... 1559B_oxy_CID_dade .......... .......... .......... .......... .......... 2675A_oxy_CID_sarasota .......... .......... .......... .......... .......... 3134D_oxy_CID_marion .......... .......... .......... .......... .......... 703A_oxy_Dracaena_hillsb .......... .......... .......... .......... .......... 703B_oxy_Dracaena_hillsb .......... .......... .......... .......... .......... HK_primer_sequence ACATGGAAAG GGAGTGAGGC AAACCATTAC AACGCCAGAT TGAAAACGCT 651 689 001A__oxy_cycad_osceola .......... .......... .......... ......... 001B_prolif_cycad_osceola .......... .......... .......... ......... 1118B_prolif_butia_manatee .......... .......... .......... ......... 1559B_oxy_CID_dade .......... .......... .......... ......... 2675A_oxy_CID_sarasota .......... .......... .......... ......... 3134D_oxy_CID_marion .......... .......... .......... ......... 703A_oxy_Dracaena_hillsb .......... .......... .......... ......... 703B_oxy_Dracaena_hillsb .......... .......... .......... ......... HK_primer_sequence TAGTAATTCT GAGAGAAGAG AGTGACTAAA TAAAAGCTT

PAGE 68

68 APPENDIX D TRANSCRIBED ELONGATION FACTOR1 ALPHA SEQUENCES

PAGE 69

69 Name: Fp_001B Fusarium proliferatum isolate 001B Name: Fp_1118B Fusarium proliferatum isolate 1118B Name: Fp_1571E Fusarium proliferatum isolate 1571E Name: Fp_1571F Fusarium proliferatum isolate 1571F Name: Fp_1550 Fusarium proliferatum isolate 1550 Name: Fp_1571C Fusarium proliferatum isolate 1571C Name: Fp_SP3A Fusarium proliferatum isolate SP3A Name: Fp_EF1_22944 Fusarium proliferatum GenBank Accession # 22944 Name: Fs_1559A_pallidoroseum Fusarium pallidoroseum (syn. semitectum ) isolate 1559A Name: Fs_1571B_equiseti Fusarium equiseti isolate 1571B Name: Fs_1559C_dimerum Fusarium sp. identified closest to dimerum with 93% match isolate 1559C Name: Fo_1072B_dianthi Fusarium oxysporum f. sp. dianthi isolate 1072B Name: Foc_001A Fusarium oxysporum f. sp. canariensis isolate 001A Name: Foc_1559B Fusarium oxysporum f. sp. canariensis isolate 1559B Name: Foc_1571A Fusarium oxysporum f. sp. canariensis isolate 1571A Name: Foc_2675A Fusarium oxysporum f. sp. canariensis isolate 2675A Name: Foc_703A Fusarium oxysporum f. sp. canariensis isolate 703A Name: Foc_703B Fusarium oxysporum f. sp. canariensis isolate 703B Name: Foc_703C Fusarium oxysporum f. sp. canariensis isolate 703C Name: Foc_EF1_26035 Fusarium oxysporum f. sp. canariensis GenBank Accession # 26035

PAGE 70

70 1 50 Fp_001B .......... .......... .TCGGCCACG TCGACTC.TG GCAAGTCGAC Fp_1118B .......... .......... ATCGGCCACG TCGACTC.TG GCAAGTCGAC Fp_1571E .......... .......... .........G TCGACTC.TG GCAAGTCGAC Fp_1571F .......... .......... ...GGCCACG TCGACTC.TG GCAAGTCGAC Fp_1550 .......... .........C ATCGGCCACG TCGACTC.TG GCAAGTCGAC Fp_1571C .......... .......... ...GGCCACG TCGACTC.TG GCAAGTCGAC Fp_SP3A .......... .......... ATCGGCCACG TCGACTC.TG GCAAGTCGAC Fp_EF1_22944 .......... ..TCGTCGTC ATCGGCCACG TCGACTC.TG GCAAGTCGAC Fs_1559A_pallidoroseum .......... .......... .......... .....TC.TG G.AAGTCGAC Fs_1571B_equiseti .......... .......... .......... ...ACTCATG GCAAGTCGAC Fs_1559C_dimerum GCACCAAGCT AACCGATCGA AATAGCCACG TCGATTC.CG GCAAGTCTAC Fo_1072B_dianthi .......... .......... .TCGGCCACG TCGACTC.TG GCAAGTCGAC Foc_001A .......... .......... ATCGGCCACG TCGACTC.TG GCAAGTCGAC Foc_1559B .......... .......... ATCGGCCACG TCGACTC.TG GCAAGTCGAC Foc_1571A .......... .......... ATCGGCCACG TCGACTC.TG GCAAGTCGAC Foc_2675A .......... .......... .TCGGCCACG TCGACTC.TG GCAAGTCGAC Foc_703A .......... .......... .TCGGCCACG TCGACTC.TG GCAAGTCGAC Foc_703B .......... .......... ATCGGCCACG TCGACTC.TG GCAAGTCGAC Foc_703C .......... .......... ATCGGCCACG TCGACTC.TG GCAAGTCGAC Foc_EF1_26035 .......... ..TCGTCGTC ATCGGCCACG TCGACTC.TG GCAAGTCGAC

PAGE 71

71 51 100 Fp_001B CACTGTGAGT ACTACCCT.G GACGTTGAGC TTATCTGCCA TCGTGA.TCC Fp_1118B CACTGTGAGT ACTACCCT.G GACGTTGAGC TTATCTGCCA TCGTGA.TCC Fp_1571E CACTGTGAGT ACTACCCT.G GACGTTGAGC TTATCTGCCA TCGTGA.TCC Fp_1571F CACTGTGAGT ACTACCCT.G GACGTTGAGC TTATCTGCCA TCGTGA.TCC Fp_1550 CACTGTGAGT ACTACCCT.G GACGATGAGC TTATCTGCCA TCGTGA.TCC Fp_1571C CACTGTGAGT ACTACCCT.G GACGATGAGC TTATCTGCCA TCGTGA.TCC Fp_SP3A CACTGTGAGT ACTACCCT.G GACGATGAGC TTATCTGCCA TCGTGA.TCC Fp_EF1_22944 CACTGTGAGT ACTACCCT.G GACGTTGAGC TTATCTGCCA TCGTGA.TCC Fs_1559A_pallidoroseum CACTGTGAGT ACTACCCA.C GATGATTTGC TTATCAGCAG TCATCAACCC Fs_1571B_equiseti CACTGTGAGT ACTATCCT.C AATGACCTGC TTATCAGCAG TCATCAACCC Fs_1559C_dimerum CACCGTGAGT CCTCCCCTTC CGCGATGACA ATATCAGCT. ...TCTACCG Fo_1072B_dianthi CACTGTGAGT ACTCTCCT.C GACAATGAGC ATATCTGCCA TCGTCAATCC Foc_001A CACTGTGAGT ACTCTCCT.C GACAATGAGC TTATCTGCCA TCGTCAATCC Foc_1559B CACTGTGAGT ACTCTCCT.C GACAATGAGC TTATCTGCCA TCGTCAATCC Foc_1571A CACTGTGAGT ACTCTCCT.C GACAATGAGC TTATCTGCCA TCGTCAATCC Foc_2675A CACTGTGAGT ACTCTCCT.C GACAATGAGC TTATCTGCCA TCGTCAATCC Foc_703A CACTGTGAGT ACTCTCCT.C GACAATGAGC TTATCTGCCA TCGTCAATCC Foc_703B CACTGTGAGT ACTCTCCT.C GACAATGAGC TTATCTGCCA TCGTCAATCC Foc_703C CACTGTGAGT ACTCTCCT.C GACAATGAGC TTATCTGCCA TCGTCAATCC Foc_EF1_26035 CACTGTGAGT ACTCTCCT.C GACAATGAGC TTATCTGCCA TCGTCAATCC

PAGE 72

72 101 150 Fp_001B TGACCAAGAT CTGGCGGGGT ACATCTTGG. ...AAGACAA CATGCTGACA Fp_1118B TGACCAAGAT CTGGCGGGGT ACATCTTGG. ...AAGACAA CATGCTGACA Fp_1571E TGACCAAGAT CTGGCGGGGT ACATCTTGG. ...AAGACAA CATGCTGACA Fp_1571F TGACCAAGAT CTGGCGGGGT ACATCTTGG. ...AAGACAA CATGCTGACA Fp_1550 TGACCAAGAT CTGGCGGGGT ACATCTTGG. ...AAGACAA TATGCTGACA Fp_1571C TGACCAAGAT CTGGCGGGGT ACATCTTGG. ...AAGACAA TATGCTGACA Fp_SP3A TGACCAAGAT CTGGCGGGGT ACATCTTGG. ...AAGACAA TATGCTGACA Fp_EF1_22944 TGACCAAGAT CTGGCGGGGT ACATCTTGG. ...AAGACAA CATGCTGACA Fs_1559A_pallidoroseum CGCC..AGAT GTGGCGGGGT AATTTCAAC. ...TTGAATA TTTGCTGACA Fs_1571B_equiseti CGCC..ATAC GTGGTGGGGT AAATTCAAC. ...TTATACA TTTGCTGACA Fs_1559C_dimerum TGGCT.GCAT ATCATGCGAT CATTCCAGAC ATTTTAATCA GAAGCTAACA Fo_1072B_dianthi CGACCAAGAC CTGGCGGGGT ATTTCTCA.. ...AAGTCAA CATACTGACA Foc_001A CGACCAAGAC CTGGCGGGGT ACTTCTCA.. ...AAGGCAA CATACTGACA Foc_1559B CGACCAAGAC CTGGCGGGGT ACTTCTCA.. ...AAGGCAA CATACTGACA Foc_1571A CGACCAAGAC CTGGCGGGGT ACTTCTCA.. ...AAGGCAA CATACTGACA Foc_2675A CGACCAAGAC CTGGCGGGGT ACTTCTCA.. ...AAGGCAA CATACTGACA Foc_703A CGACCAAGAC CTGGCGGGGT ACTTCTCA.. ...AAGGCAA CATACTGACA Foc_703B CGACCAAGAC CTGGCGGGGT ACTTCTCA.. ...AAGGCAA CATACTGACA Foc_703C CGACCAAGAC CTGGCGGGGT ACTTCTCA.. ...AAGGCAA CATACTGACA Foc_EF1_26035 CGACCAAGAC CTGGCGGGGT ACTTCTCA.. ...AAGGCAA CATACTGACA

PAGE 73

73 151 200 Fp_001B TCGCTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Fp_1118B TCGCTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Fp_1571E TCGCTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Fp_1571F TCGCTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Fp_1550 TCGCTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Fp_1571C TCGCTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Fp_SP3A TCGCTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Fp_EF1_22944 TCGCTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Fs_1559A_pallidoroseum AGATTGCATA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Fs_1571B_equiseti AGATTGTATA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Fs_1559C_dimerum ACAC..GATA GACTGGTCAC TTGATCTACC AGTGCGGTGG TATTGACAAG Fo_1072B_dianthi TCGTTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Foc_001A TCGTTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Foc_1559B TCGTTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Foc_1571A TCGTTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Foc_2675A TCGTTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Foc_703A TCGTTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Foc_703B TCGTTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Foc_703C TCGTTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG Foc_EF1_26035 TCGTTTCACA GACCGGTCAC TTGATCTACC AGTGCGGTGG TATCGACAAG

PAGE 74

74 201 250 Fp_001B CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Fp_1118B CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Fp_1571E CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Fp_1571F CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Fp_1550 CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Fp_1571C CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Fp_SP3A CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Fp_EF1_22944 CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Fs_1559A_pallidoroseum CGAACCATCG AGAAGTTCGA GAAGGTTGGT TTCCATTTCC C..CGAT.CG Fs_1571B_equiseti CGAACCATCG AGAAGTTCGA GAAGGTTGGT TTCCATTTTC CT.CGAT.CG Fs_1559C_dimerum CGTACCATTG AGAAGTTCGA GAAGGTAAGA ACAGCCACTC CTTTGATACC Fo_1072B_dianthi CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCAAT.CG Foc_001A CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Foc_1559B CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Foc_1571A CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Foc_2675A CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Foc_703A CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Foc_703B CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Foc_703C CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG Foc_EF1_26035 CGAACCATCG AGAAGTTCGA GAAGGTTAGT CAC..TTTCC CTTCGAT.CG

PAGE 75

75 251 300 Fp_001B CGCGTCCT.C TGCCCACCGA TT..TCAC.. .......... TTGCGATTCG Fp_1118B CGCGTCCT.C TGCCCACCGA TT..TCAC.. .......... TTGCGATTCG Fp_1571E CGCGTCCT.C TGCCCACCGA TT..TCAC.. .......... TTGCGATTCG Fp_1571F CGCGTCCT.C TGCCCACCGA TT..TCAC.. .......... TTGCGATTCG Fp_1550 CGCGTCCT.C TGCCCACCGA TT..TCAC.. .......... TTGCGATTCG Fp_1571C CGCGTCCT.C TGCCCACCGA TT..TCAC.. .......... TTGCGATTCG Fp_SP3A CGCGTCCT.C TGCCCACCGA TT..TCAC.. .......... TTGCGATTCG Fp_EF1_22944 CGCGTCCT.C TGCCCACCGA TT..TCAC.. .......... TTGCGATTCG Fs_1559A_pallidoroseum CACGCCGT.C TACCCACCGA TCCATCAGTC GAATCAGTTA CGACGATTGA Fs_1571B_equiseti CACGCCCT.C TGCCCATCGA TCCATCACCC GAATCAGTCT CGACGACTGA Fs_1559C_dimerum CAGATCGTGC GGCGTCTCGC ATC.TCACAC .......... CTGGCATTC. Fo_1072B_dianthi CGCGTCCT.T TGCCCATCGA TT..TCCC.. .......... CTACGACTCG Foc_001A CGCGTCCT.T TGCCCATCGA CT..TCCC.. .......... CTACGACTCG Foc_1559B CGCGTCCT.T TGCCCATCGA CT..TCCC.. .......... CTACGACTCG Foc_1571A CGCGTCCT.T TGCCCATCGA CT..TCCC.. .......... CTACGACTCG Foc_2675A CGCGTCCT.T TGCCCATCGA CT..TCCC.. .......... CTACGACTCG Foc_703A CGCGTCCT.T TGCCCATCGA CT..TCCC.. .......... CTACGACTCG Foc_703B CGCGTCCT.T TGCCCATCGA CT..TCCC.. .......... CTACGACTCG Foc_703C CGCGTCCT.T TGCCCATCGA CT..TCCC.. .......... CTACGACTCG Foc_EF1_26035 CGCGTCCT.T TGCCCATCGA CT..TCCC.. .......... CTACGACTCG

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76 301 350 Fp_001B AAACGTGCCT GCTACCCCGC TCGAGACCAA AAATTTTGCG ATATGACCGT Fp_1118B AAACGTGCCT GCTACCCCGC TCGAGACCAA AAATTTTGCG ATATGACCGT Fp_1571E AAACGTGCCT GCTACCCCGC TCGAGACCAA AAATTTTGCG ATATGACCGT Fp_1571F AAACGTGCCT GCTACCCCGC TCGAGACCAA AAATTTTGCG ATATGACCGT Fp_1550 AAACGTGCCT GCTACCCCGC TCGAGACCAA AATTTTTGCG ATATGACCGT Fp_1571C AAACGTGCCT GCTACCCCGC TCGAGACCAA AAATTTTGCG ATATGACCGT Fp_SP3A AAACGTGCCT GCTACCCCGC TCGAGACCAA AAATTTTGCG ATATGACCGT Fp_EF1_22944 AAACGTGCCT GCTACCCCGC TCGAGACCAA AAATTTTGCG ATATGACCGT Fs_1559A_pallidoroseum ATATGCGCCT GTTACCCCGC TCGAGTACAA AA.TTTTGCG GTTCAACCGT Fs_1571B_equiseti ACATGCGCCT GTTACCCCGC TCGAGTACAA AA.TTTTGCG GTTCAATCGT Fs_1559C_dimerum .TGTGCCCCT CTTACCCCTC CTCAAAAATC AATTTTTTTT GTGGCCC... Fo_1072B_dianthi AAACGTGCCC GCTACCCCGC TCGAGACCAA AAATTTTGCA ATATGACCGT Foc_001A AAACGTGCCC GCTACCCCGC TCGAGACCAA AAATTTTGCA ATATGACCGT Foc_1559B AAACGTGCCC GCTACCCCGC TCGAGACCAA AAATTTTGCA ATATGACCGT Foc_1571A AAACGTGCCC GCTACCCCGC TCGAGACCAA AAATTTTGCA ATATGACCGT Foc_2675A AAACGTGCCC GCTACCCCGC TCGAGACCAA AAATTTTGCA ATATGACCGT Foc_703A AAACGTGCCC GCTACCCCGC TCGAGACCAA AAATTTTGCA ATATGACCGT Foc_703B AAACGTGCCC GCTACCCCGC TCGAGACCAA AAATTTTGCA ATATGACCGT Foc_703C AAACGTGCCC GCTACCCCGC TCGAGACCAA AAATTTTGCA ATATGACCGT Foc_EF1_26035 AAACGTGCCC GCTACCCCGC TCGAGACCAA AAATTTTGCA ATATGACCGT

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77 351 400 Fp_001B AATTTTTTTG GTGGGGCATT TACCCCGCCA CTCGAGCGAT GGGC.GCGGT Fp_1118B AATTTTTTTG GTGGGGCATT TACCCCGCCA CTCGAGCGAT GGGC.GCGTT Fp_1571E AATTTTTTTG GTGGGGCATT TACCCCGCCA CTCGAGCGAT GGGC.GCGTT Fp_1571F AATTTTTTTG GTGGGGCATT TACCCCGCCA CTCGAGCGAT GGGC.GCGTT Fp_1550 AATTTTTTTG GTGGGGCATT TACCCCGCCA CTCGAGCGAT GAGC.GCGTT Fp_1571C AATTTTTTTG GTGGGGCATT CACCCCGCCA CTCGAGCGAT GGGC.GCGTT Fp_SP3A AATTTTTTTG GTGGGGCATT CACCCCGCCA CTCGAGCGAT GGGC.GCGTT Fp_EF1_22944 AATTTTTTTG GTGGGGCATT TACCCCGCCA CTCGAGCGAT GGGC.GCGGT Fs_1559A_pallidoroseum AATTTTTTTG GTGGGGTTTC AACCCCGCTA CTCGAGCGAC AGAC...GTT Fs_1571B_equiseti AATTTTTT.G GTGGGGCTCA TACCCCGCTA CTCGAGTGAC AGGC...GCT Fs_1559C_dimerum ...T.TTTTA GTGGGGCCAC AACCCCGCCA ...GAG.... .......... Fo_1072B_dianthi AATTTTTTTG GTGGGGCACT TACCCCGCCA CTTGAGCGAA GGGA.GCGTT Foc_001A AATTTTTTTG GTGGGGCATT TACCCCGCCA CTTGAGCGAC GGGGCGCGTT Foc_1559B AATTTTTTTG GTGGGGCATT TACCCCGCCA CTTGAGCGAC GGGGCGCGTT Foc_1571A AATTTTTTTG GTGGGGCATT TACCCCGCCA CTTGAGCGAC GGGGCGCGTT Foc_2675A AATTTTTTTG GTGGGGCATT TACCCCGCCA CTTGAGCGAC GGGGCGCGTT Foc_703A AATTTTTTTG GTGGGGCATT TACCCCGCCA CTTGAGCGAC GGGGCGCGTT Foc_703B AATTTTTTTG GTGGGGCATT TACCCCGCCA CTTGAGCGAC GGGGCGCGTT Foc_703C AATTTTTTTG GTGGGGCATT TACCCCGCCA CTTGAGCGAC GGGGCGCGTT Foc_EF1_26035 AATTTTTTTG GTGGGGCATT TACCCCGCCA CTTGAGCGAC GGGGCGCGTT

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78 401 450 Fp_001B TTTGCCCTTT CCTGTC.CAC AACCTCAATG AGCGCATTGT CACGTGTCAA Fp_1118B TTTGCCCTTT CCTGTC.CAC AACCTCAATG AGCGCATTGT CACGTGTCAA Fp_1571E TTTGCCCTTT CCTGTC.CAC AACCTCAATG AGCGCATTGT CACGTGTCAA Fp_1571F TTTGCCCTTT CCTGTC.CAC AACCTCAATG AGCGCATTGT CACGTGTCAA Fp_1550 TTTGCCCTTT CCTGTC.CAC AACCTCAATG AGCGCATTGT CACGTGTCAA Fp_1571C TTTGCCCTCT CCTGTC.CAC AACCTCAATG AGCGCATTGT CACGTGTCAA Fp_SP3A TTTGCCCTCT CCTGTC.CAC AACCTCAATG AGCGCATTGT CACGTGTCAA Fp_EF1_22944 TTTGCCCTTT CCTGTC.CAC AACCTCAATG AGCGCATTGT CACGTGTCAA Fs_1559A_pallidoroseum TGCCCTCTTC CC......AC AAACTCATGT CTCGTG.CAT CACGTGTCCA Fs_1571B_equiseti TGCCCTCTTC CC......AC AAAATCA... CTTGCG.CAT CACGTGTCAA Fs_1559C_dimerum .......TTC TC........ .........G ATAGCA...T CTCAAGGAAG Fo_1072B_dianthi TGCCCTCTTA CCATTCTCAC AACCTCAATG AGTGCGTCGT CACGTGTGAA Foc_001A TGCCCTCTTA CCATT...AC AACCTCAATG AGTGCGTCGT CACGTGTCAA Foc_1559B TGCCCTCTTA CCATT...AC AACCTCAATG AGTGCGTCGT CACGTGTCAA Foc_1571A TGCCCTCTTA CCATT...AC AACCTCAATG AGTGCGTCGT CACGTGTCAA Foc_2675A TGCCCTCTTA CCATT...AC AACCTCAATG AGTGCGTCGT CACGTGTCAA Foc_703A TGCCCTCTTA CCATT...AC AACCTCAATG AGTGCGTCGT CACGTGTCAA Foc_703B TGCCCTCTTA CCATT...AC AACCTCAATG AGTGCGTCGT CACGTGTCAA Foc_703C TGCCCTCTTA CCATT...AC AACCTCAATG AGTGCGTCGT CACGTGTCAA Foc_EF1_26035 TGCCCTCTTA CCATT...AC AACCTCAATG AGTGCGTCGT CACGTGTCAA

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79 451 500 Fp_001B GCAGCGACTA ACCATTCGAC AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Fp_1118B GCAGCAACTA ACCATTCGAC AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Fp_1571E GCAGCAACTA ACCATTCGAC AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Fp_1571F GCAGCAACTA ACCATTCGAC AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Fp_1550 GCAGCGACTA ACCATTCGAC AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Fp_1571C GCAGCGACTA ACCATTCGAC AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Fp_SP3A GCAGCGACTA ACCATTCGAC AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Fp_EF1_22944 GCAGCGACTA ACCATTCGAC AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Fs_1559A_pallidoroseum TCAGCCACTA ACCACCCGAC AATAGGAAGC CGCCGAGCTC GGTAAGGGTT Fs_1571B_equiseti TCAGTCACTA ACCACCTGAC AATAGGAAGC CGCCGAGCTC GGTAAGGGTT Fs_1559C_dimerum GCACGCGCTG ACAGTCCCAA AATAGGAAGC CGCCGAACTC GGTAAGGGTT Fo_1072B_dianthi GCAGTCACTA ACCATTCAAC AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Foc_001A GCAGTCACTA ACCATTCAAT AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Foc_1559B GCAGTCACTA ACCATTCAAT AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Foc_1571A GCAGTCACTA ACCATTCAAT AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Foc_2675A GCAGTCACTA ACCATTCAAT AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Foc_703A GCAGTCACTA ACCATTCAAT AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Foc_703B GCAGTCACTA ACCATTCAAT AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Foc_703C GCAGTCACTA ACCATTCAAT AATAGGAAGC CGCTGAGCTC GGTAAGGGTT Foc_EF1_26035 GCAGTCACTA ACCATTCAAT AATAGGAAGC CGCTGAGCTC GGTAAGGGTT

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80 501 550 Fp_001B CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Fp_1118B CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Fp_1571E CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Fp_1571F CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Fp_1550 CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Fp_1571C CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Fp_SP3A CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Fp_EF1_22944 CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Fs_1559A_pallidoroseum CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCTGA GCGTGAGCGT Fs_1571B_equiseti CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Fs_1559C_dimerum CCTTCAAGTA CGCATGGGTC CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Fo_1072B_dianthi CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Foc_001A CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Foc_1559B CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Foc_1571A CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Foc_2675A CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Foc_703A CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Foc_703B CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Foc_703C CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT Foc_EF1_26035 CCTTCAAGTA CGCCTGGGTT CTTGACAAGC TCAAGGCCGA GCGTGAGCGT

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81 551 600 Fp_001B GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Fp_1118B GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Fp_1571E GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Fp_1571F GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Fp_1550 GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Fp_1571C GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Fp_SP3A GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Fp_EF1_22944 GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Fs_1559A_pallidoroseum GGTATCACCA TCGATATCGC CCTCTGGAAG TTCGAGACTC CTCGCTACTA Fs_1571B_equiseti GGTATCACCA TCGATATCGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Fs_1559C_dimerum GGTATCACCA TCGATATCGC CCTCTGGAAG TTCGAGACTC CCAAGTACCA Fo_1072B_dianthi GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Foc_001A GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Foc_1559B GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Foc_1571A GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Foc_2675A GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Foc_703A GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Foc_703B GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Foc_703C GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA Foc_EF1_26035 GGTATCACCA TCGATATTGC TCTCTGGAAG TTCGAGACTC CTCGCTACTA

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82 601 650 Fp_001B TGTCACCGTC ATTGGTATGT TGTCGCTCAT ACCTCATCCT ACTTC..... Fp_1118B TGTCACCGTC ATTGGTATGT TGTCGCTCAT ACCTCATCCT ACTTC..... Fp_1571E TGTCACCGTC ATTGGTATGT TGTCGCTCAT ACCTCATCCT ACTTC..... Fp_1571F TGTCACCGTC ATTGGTATGT TGTCGCTCAT ACCTCATCCT ACTTC..... Fp_1550 TGTCACCGTC ATTGGTATGT TGTCGCTCAT ACCTCATCCT ACTTC..... Fp_1571C TGTCACCGTC ATTGGTATGT TGTCGCTCAT ACCTCATCCT ACTTC..... Fp_SP3A TGTCACCGTC ATTGGTATGT TGTCGCTCAT ACCTCATCCT ACTTC..... Fp_EF1_22944 TGTCACCGTC ATTGGTATGT TGTCGCTCAT ACCTCATCCT ACTTC..... Fs_1559A_pallidoroseum TGTCACCGTC ATTGGTACGT TATCATCACT TACACTCAAT ACTTT..... Fs_1571B_equiseti TGTCACCGTC ATTGGTATGT TGTCACCACT TCCACTCATT ACCTT..... Fs_1559C_dimerum GGTCACCGTC ATTGGTACGT CATCGCACCT .CCGTTGTGT ATGTTGCAAA Fo_1072B_dianthi TGTCACCGTC ATTGGTATGT TGTCGCTCAT GCTTCATTCT ACTTCTC... Foc_001A TGTCACCGTC ATTGGTATGT TGTCGCTCAT GCTTCATTCT ACGTCTC... Foc_1559B TGTCACCGTC ATTGGTATGT TGTCGCTCAT GCTTCATTCT ACGTCTC... Foc_1571A TGTCACCGTC ATTGGTATGT TGTCGCTCAT GCTTCATTCT ACGTCTC... Foc_2675A TGTCACCGTC ATTGGTATGT TGTCGCTCAT GCTTCATTCT ACGTCTC... Foc_703A TGTCACCGTC ATTGGTATGT TGTCGCTCAT GCTTCATTCT ACGTCTC... Foc_703B TGTCACCGTC ATTGGTATGT TGTCGCTCAT GCTTCATTCT ACGTCTC... Foc_703C TGTCACCGTC ATTGGTATGT TGTCGCTCAT GCTTCATTCT ACGTCTC... Foc_EF1_26035 TGTCACCGTC ATTGGTATGT TGTCGCTCAT GCTTCATTCT ACGTCTC...

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83 651 700 Fp_001B CTCATA.... CTAAC..ACA TCATTCAGAC GCTCCCGGTC ACCGTGATTT Fp_1118B CTCATA.... CTAAC..ACA TCATTCAGAC GCTCCCGGTC ACCGTGATTT Fp_1571E CTCATA.... CTAAC..ACA TCATTCAGAC GCTCCCGGTC ACCGTGATTT Fp_1571F CTCATA.... CTAAC..ACA TCATTCAGAC GCTCCCGGTC ACCGTGATTT Fp_1550 CTCATA.... CTAAC..ACA TCATTCAGAC GCTCCCGGTC ACCGTGATTT Fp_1571C CTCATA.... CTAAC..ACA TCATTCAGAC GCTCCCGGTC ACCGTGATTT Fp_SP3A CTCATA.... CTAAC..ACA TCATTCAGAC GCTCCCGGTC ACCGTGATTT Fp_EF1_22944 CTACTC.... TTAAC..ACA TCATTCAGAC GCTCCCGGTC ACCGTG.... Fs_1559A_pallidoroseum CTCATG.... CTAAC..ATG TACTTCAGAC GCTCCCGGTC ACCGTGATTT Fs_1571B_equiseti CTCATG.... CTAAC..ATG TATTCCAGAC GCTCCCGGTC ACCGTGATTT Fs_1559C_dimerum GGCATGTTGA CTAACTGATA TATCACAGAC GCTCCCGGTC ACCGTGATTT Fo_1072B_dianthi TTCGTA.... CTAAC..ATA TCACTCAGAC GCTCCCGGTC ACCGTGATTT Foc_001A TTCGTA.... CTAAT..ATA TCACTCAGAC GCTCCCGGTC ACCGTGATTT Foc_1559B TTCGTA.... CTAAT..ATA TCACTCAGAC GCTCCCGGTC ACCGTGATTT Foc_1571A TTCGTA.... CTAAT..ATA TCACTCAGAC GCTCCCGGTC ACCGTGATTT Foc_2675A TTCGTA.... CTAAT..ATA TCACTCAGAC GCTCCCGGTC ACCGTGATTT Foc_703A TTCGTA.... CTAAT..ATA TCACTCAGAC GCTCCCGGTC ACCGTGATTT Foc_703B TTCGTA.... CTAAT..ATA TCACTCAGAC GCTCCCGGTC ACCGTGATTT Foc_703C TTCGTA.... CTAAT..ATA TCACTCAGAC GCTCCCGGTC ACCGTGATTT Foc_EF1_26035 TTCGTA.... CTAAT..ATA TCACTCAGAC GCTCCCGGTC ACCGTGG...

PAGE 84

84 701 729 Fp_001B CATCAAGAAC ATGATCA... ......... Fp_1118B CATCAAGAAC ATGATCA... ......... Fp_1571E CATCAAGAAC ATGATCACTG ......... Fp_1571F CATCAAGAAC ATGATCACTG ......... Fp_1550 CATCAAGAAC ATGATC.... ......... Fp_1571C CATCAAGAAC ATGATCACTG ......... Fp_SP3A CATCAAGAAC ATGATCACTG ......... Fp_EF1_22944 .......... .......... ......... Fs_1559A_pallidoroseum CATCAAGAAC ATGATCACTG GTACTTCCA Fs_1571B_equiseti CATCAAGAAC ATGATCACTG GTACTTCCA Fs_1559C_dimerum CATCAAGAAC ATGATCA... ......... Fo_1072B_dianthi CATCAAGAAC ATGATCACTG ......... Foc_001A CATCAAGAAC ATGATCACTG ......... Foc_1559B CATCAAGAAC ATGATCACTG ......... Foc_1571A CATCAAGAAC ATGATCA... ......... Foc_2675A CATCAAGAAC ATGATCA... ......... Foc_703A CATCAAGAAC ATGATCA... ......... Foc_703B CATCAAGAAC ATGATCA... ......... Foc_703C CATCAAGAAC ATGATCA... ......... Foc_EF1_26035 .......... .......... .........

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85 APPENDIX E KOCHS POSTULATES Material Preparation Kochs postulates were attempted on Dracaena marginata and Phoenix canariensis seedlings to determine if th e isolates obtained from a Dracaena marginata were pathogenic to these hosts. Dracaena marginata plants that had several leaves were obtained from Mid-Florida Research and Education Center a nd were potted in three-inch pl astic containers. Canary Island date (CID) palm seedlings were obtained from Whisper Palms Nursery. The seedlings had 1 to 3 leaves each. The CID palms were shipped without soil in a bundle wrapped in a wet paper towel to keep the roots moist duri ng shipment. Isolates of both Fusarium oxysporum and F. proliferatum from PDC samples were used in the inoc ulation study (Table F-1). Cultures were prepared for inoculation by tr ansferring each isolate onto 3 APDA plates. The plates were incubated at room temperature (approximately 20-25C) on the counter for two weeks prior to inoculation. All labo ratory materials used for the Kochs postulates procedures were autoclaved for 40 minutes at 121C and a pressure of 15 PSI prior to the inoculations. Three plants of each host were randomly selected for each treatment group. Treatment groups consisted of isolates 001B (Sago palm host, F. proliferatum morphologically, HK66&67 positive), 703A ( Dracaena marginata host, F. oxysporum morphologically, HK66&67 positive), 703B ( Dracaena marginata host, F. oxysporum morphologically, HK66&67 positive), 703C ( Dracaena marginata host, F. oxysporum morphologically, HK66&67 positive), 1072 (Silver date palm host, F. oxysporum morphologically, HK66&67 nega tive), and 1118B (Jelly and Queen Palm hybrid, F. proliferatum morphologically, HK66&67 posit ive), and a control group (sterile water only inoculation). Two plants were randomly chosen to determine if there was any

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86 Fusarium spp. on the plants prior to inoculation. The pl ants were analyzed using PDC protocol and were determined to be pathogen and Fusarium free. Inoculation Procedure The Dracaena marginata plants were not watered for 24 h before inoculation to increase absorption of the isolates by the plants. Plants were labeled with the isolate treatment numbers and then grouped together and photographed for future reference. The plants were then placed in a Rubbermaid bin to collect any runoff and to ease the transfer to a moist chamber. A thin layer of sterile water was poured onto one of the three isolate plates. A cell spreader (Fisher cat. # 05541-10) was gently rubbed across the surface of the plate to dislodge spores. The spore suspension was then poured into a funnel lined with two layers of cheesecloth si tting in a 100mL bottle with graduated marks. This was repeated w ith each plate for the isolate until the water ran clear. It was then brought up to vol ume with more sterile water so the concentration of the spores was approximately 106 measured by using a Spec 20. Each individual plant was inoculated using 40mL of the spore suspension. The plants were drenched with the cell suspension using a disposable Pasteur pipette to we t the entire surf ace of the leaves, especially around the base. The Rubbermaid containers containing the inoculated plants were then placed in a plastic bag to increase humidity. The moist chambers were left on the counter in the bags for 24 h and then the plants were removed from the bags and were moved to a shaded greenhouse. Each plant was watered with 10-15mL of tap water approximate ly 48 h after inoculation. The plants were transplanted into 3-in. clay pot s with a standard soilless potti ng mixture72 h after inoculation. The plants were watered as needed. The procedur es for inoculating the palm seedlings were the same for the Dracaena marginata inoculation, but seedlings of each treatment were transplanted into a single 3-inch clay pot.

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87 Results Dracaena marginata Leaf spots and tip-dieback were observed two weeks post-inocul ation on one plant, but all plants had a flush of new leaves and were growing rapidly. The l eaf spots and tip-dieback did not spread or worsen. All of the plants were doing well after 5 months. The Dracaena marginata were transplanted into 6-in. clay pots due to being potbound. Dead leaves were removed periodically from the plants Although 3 isolates origin ally were isolated from Dracaena marginata no wilt symptoms were observed. Analysis of the leaves and roots of the Dracaena marginata inoculated with isolate 703C following standard Plant Disease Clinic protocol concluded that the plants were Fusarium free and Kochs postulates were not fulfilled. Phoenix canariensis Inoculations of isolates 001B (o riginal PDC number 5795), and 703A onto Phoenix canariensis seedlings in addition to isolates of Fusarium oxysporum f. sp. canariensis from California obtained from Phoenix canariensis palms were made by Dr James Downer at the University of California in Davis, CA. Dr. Down er was unable to complete Kochs postulates with any of the isolates. The Palm seedlings inoc ulated at the University of Florida have shown no symptoms of disease and have grown 1-2 ne w leaves and extensive root systems since inoculation. Kochs postulates were not fulf illed with any isolate on either plant host.

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88 Figure E-1. Inoculated pl ants pre-inoculation A) Dracaena marginata plants and B) Phoenix canariensis seedlings immediately before inoculation. A B

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89 Figure E-2. Inoculated plants 5-6 months post-inoculation A) Dracaena marginata plants 6 months post-inoculation in 6-inch pots and B) Phoenix canariensis seedlings 5 months post inoculation in 3-in. clay pots. A B

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90 APPENDIX F LIST OF ISOLATES

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91 Table F-1. Isolates used in Kochs Postulate and Sequencing Sample Number Host Growth Environment County Fusarium s p. Morphologic Identification PCR results from HK 66 & 67 primers Fusarium sp. TEF Sequencing Test(s) performend SP3A Phoenix canariensis Research culture Hillsborough proliferatum No Amplification Fusarium proliferatum Protocol optimization; TEF sequencing 001A (5795A) Cycas sp. Resort Hotel Landscape Osceola oxysporum Amplification Fusarium oxysporum f. sp canariensis Inoculation; Sequence of HK 66& 67 amplicon; Protocol optimization; TEF seqencing 001B (5795B) Cycas sp. Resort Hotel Landscape Osceola proliferatum Amplification Fusarium proliferatum Inoculation; Sequence of HK 66& 67 amplicon; Protocol optimization; TEF sequencing 703A Dracaena marginata Nursery Hillsborough oxysporum Amplification Fusarium oxysporum f. sp canariensis Inoculation & Sequence of HK 66& 67 amplicon; TEF sequencing 703B Dracaena marginata Nursery Hillsborough oxysporum Amplification Fusarium oxysporum f. sp canariensis Inoculation & Sequence of HK 66& 67 amplicon; TEF sequencing 703C Dracaena marginata Nursery Hillsborough oxysporum Amplification Fusarium oxysporum f. sp canariensis Inoculation; Sequence of HK 66& 67 amplicon; Protocol optimization: TEF sequencing 1072B Phoenix sylvestris Nursery Hardee oxysporum No Amplification Fusarium oxysporum f. sp dianthi Inoculation; Protocol optimization; TEF sequencing 1118B Butia x Syagrus hybrid Nursery Manatee proliferatum Amplification Fusarium proliferatum Inoculation & Sequence of HK 66& 67 amplicon; TEF sequencing 1550 Syagrus romanzoffi anum Research culture Broward proliferatum No Amplification Fusarium proliferatum Protocol optimization; TEF sequencing

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92 Table F-1. Continued Sample Number Host Growth Environment County Fusarium s p. Morphologic Identification PCR results from HK 66 & 67 primers Fusarium sp. TEF Sequencing Test(s) performend 1559A Phoenix canariensis Resort Hotel Landscape Miami-Dade oxysporum No Amplification Fusarium sp. TEF sequencing 1559B Phoenix canariensis Resort Hotel Landscape Miami-Dade oxysporum Amplification Fusarium oxysporum f. sp canariensis Sequence of HK 66& 67 amplicon; Protocol optimization; TEF sequencing 1559C Phoenix canariensis Resort Hotel Landscape Miami-Dade oxysporum No Amplification Fusarium sp. TEF sequencing 1571A Phoenix dactylifera Nursery Orange oxysporum Amplification Fusarium oxysporum f. sp canariensis TEF sequencing 1571B Phoenix dactylifera Nursery Orange equiseti No Amplification Fusarium equiseti TEF sequencing 1571C Phoenix dactylifera Nursery Orange proliferatum Amplification Fusarium proliferatum TEF sequencing 1571E Phoenix dactylifera Nursery Orange proliferatum Amplification Fusarium proliferatum TEF sequencing 1571F Phoenix dactylifera Nursery Orange proliferatum Amplification Fusarium proliferatum TEF sequencing 2675A Phoenix canariensis Commercial Landscape Sarasota oxysporum Amplification Fusarium oxysporum f. sp canariensis Sequence of HK 66& 67 amplicon; TEF sequencing

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93 LIST OF REFERENCES 1. Abdalla, M.Y., Al-Rokibah, A., Moretti, A. and Mul, G. 2000. Pathogenicity of toxigenic Fusarium proliferatum from date palm in Saudi Arabia. Plant Disease. 84:321324. 2. Armengol, J., Moretti, A., Perrone, G., Vicent, A., Bengoechea, J.A., and GarciaJimnez, J. 2005. Identification, in cidence and characterization of Fusarium proliferatum on ornamental palms in Spain. European Journal of Plant Pathology 112:123-131. DOI:10.1007/s10658-005-2552-6 3. Arai, K. and Yamamoto, A. 1977. New Fusarium disease of Canary Island date palm in Japan. Bulletin of the Faculty of Ag riculture. Kagoshima Univ. 27:31-37. 4. Blomquist, C., Irving, T., Osterbauer, N., and Reeser, P. 2005. Phytophthora hibernalis : A new pathogen on Rhododendron and evidence of cross amplification with two PCR detection assays for Phytophthora ramorum Online. Plant Health Progress. Doi:10.1094/PHP-2005-0728-01-HN 5. Booth, C. 1975. The present status of Fusarium taxonomy. Annual Review of Phytopathology. 13:83-93. 6. Castell, G., Bragulat, M.R ., Rubiales, M.V., and Cabaes, F.J. 1996. Malachite green agar, a new selective medium for Fusarium spp. Mycopathologia 137: 173-178. 7. Cepheid Technical Support. Cepheid SmartNote 6.1 : Designing real-time assays on the SmartCycler II System. Sunnyvale, CA. 8. Cepheid Technical Support. Cepheid SmartNote 6.3 : Dye-Quencher considerations for the SmartCycler II System. Sunnyvale, CA 9. DiPietro, A., Madrid, M.P., Caracuel, Z., Delgado-Jarana, J., and Roncero, M.I.G. 2003. Fusarium oxysporum : exploring the molecular arse nal of a vascular wilt fungus. Molecular Plant Pathology 4:315-325. 10. Elena, K. 2005. Fusarium wilt of Phoenix canariensis : first report in Greece. Plant Pathology 54:244. DOI: 10.1111/j.1356-3059.2005.01170.x 11. Elliott, M. 2006. Fusarium wilt of canary is land date palm. University of Florida, Gainesville, FL. EDIS PP139 12. Elliott, M., Broschat, T.K., Uchida, J.Y ., and Simone, G.W. 2004. Compendium of Ornamental Palm Diseases and Disorder s. APS Press, St Paul, MN. Pp 18-22. 13. Espy, M.J., Uhl, J.R., Sloan, L.M., Buckwalter, S.P., Jones, M.F., Vetter, E.A., Yao, J.D.C., Wengenack, N.L., Rosenblatt, J.E., Cockerill, F.R., and Smith, T.F. 2006. Realtime PCR in clinical microbiol ogy; Applications for routine laboratory testing. Clinical Microbiology Reviews. 19:165-256.

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95 26. Migheli, Q. and Balmas, V. 2005. First report of Fusarium oxysporum f. sp. canariensis causing Fusarium wilt on Phoenix canariensis in Sardinia, Italy. Plant Disease. 89:773. DOI: 10.1094/PD-89-0773A 27. Nelson, P.E., Toussoun, T.A., and Marasas, W.F.O. 1983. Fusarium Species: An Illustrated Manual for Identification Pennsylvania State University Press, University Park, PA. 28. Nelson, P.E., Dignani, C.M., and Anaissie, E.J. 1994. Taxonomy, biology, and clinical aspects of Fusarium species. Clinical Microbiology Reviews. pp. 479-504. 29. Neumann, M.J., Backhouse, D., Carter, D.A., Summerell, B.A., and Burgess, L.W. 2004. Genetic structure of populations of Fusarium proliferatum in soils associated with Livistona mariae palms in Little Palm Creek, Northe rn Territory, Australia. Australian Journal of Botany 52:543-550. 30. Plyler, T.R. 1997. Genetic Diversity Studies on Fusarium oxysporum f.sp. canariensis and the Development of a Polymerase Chain Reaction Technique for its Detection University of Florida, Gainesville Florida. 31. Plyler, T.R., Simone, G. W., Fernandez, D., and Kistler, H.C. 1999. Rapid detection of the Fusarium oxysporum lineage containing the Canary Island Date Palm wilt pathogen. Phytopathology 89:407-413. DOI: 10.1094/PHYTO.1999.89.5.407. 32. Polizzi, G. and Vitale, A. 2003. First report of Fusarium blight on majesty palm caused by Fusarium proliferatum in Italy. Plant Disease. 87:1149. 33. Qiagen Technical Support. Qiagen Multiplex PCR Handbook 2008, Valencia, CA. 34. Rojas, M.R., Gilbertson, R.L. Russell, D.R., and Maxwell, D.P. 1993. Use of degenerate primers in the polymerase chain reaction to detect whitefly-transmitted geminiviruses. Plant Disease. 77:340-347. 35. Schena, L., Nigro, F., Ippolito, A., and Gallite lli, D. 2004. Real-time quantitative PCR: A new technology to detect a nd study phytopathogenic and an tagonistic fungi. European Journal of Plant Pathology. 110:893-908. 36. Sen, K. 2005. Development of a rapid identification method for Aeromonas species by multiplex-PCR. Canadian Journal of Microbiology. 51: 957-966. 37. Summerell, B.A. and Gunn, L.V. 2001. First record of Fusarium wilt of Phoenix canariensis in South Australia. Austra lasian Plant Pathology 30:75. 38. Summerell, B.A., Smith, D.I., Gunn, L.V. Smith, I.W., and Pascoe, I.G. 2006. Fusarium wilt of Phoenix canariensis in Victoria. Australasi an Plant Pathology 35:289-290. 39. Tatineni, S., Sagaram, U.S., Gowda, S., Robertson, C.J., Dawson, W.O., Iwanami, T., and Wang, N. 2008. In planta distribution of Candidatus Liberibacter asiaticus as

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BIOGRAPHICAL SKETCH Anne Benner Vitoreli received he r Bachelor of Science in microbiology from the College of Agriculture at The University of Florida in 1992. She married Odenis Vitoreli in 2002 and has 2 Rottweilers named Barnie and Bettie, and cat named Tiberius. Her work history includes working in the environmental field as an Environmental Scientist and Laboratory Technicia n, in the health professions with diabetes research and in hospice care, in food science in research and in a food safety dia gnostics laboratory. She currently works for the University of Florida in the Florida Extension Plant Disease Clinic. Her responsibilities include serological and molecular diagnostic techniques, as well as general laboratory work. She is provisionally approved by the USDA for Phytophthora ramorum and Citrus Greening. She is the SPDN repres entative on the NPDN La boratory Accreditation subcommittee, the Database Committee and the Regional lab representative on the SPDN Infrastructure committee. When she has time, Annes hobbies include gardening, traveling, pl aying with her dogs and cat, and reading.