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Population Structure and Pathogenicity of Colletotrichum gloeosporioides from Strawberry and Noncultivated Hosts in Florida

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POPULATION STRUCTURE AND PATHOGENICITY OF Colletotrichum gloeosporioides FROM STRAWBERRY AND NONCULTIVATED HOSTS IN FLORIDA By STEVEN JOHN MACKENZIE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Steven John MacKenzie

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iii ACKNOWLEDGMENTS Most of all, I would like to thank my wi fe, Enas, for her support during the years I spent in graduate school. I w ould also like to thank the staff and faculty at the Gulf Coast Research and Education Center fo r their help. In particular, I appreciate the assistance of Teresa Seijo for collecting oa k and grape leaves, conducting C. gloeosporioides isolations, PCR reactions and pathogenicity assays for me when I was in Gainesville, Florida; of Jim Mertely for providing single-ascospore isolat es from perithecia he found on strawberry, helping with isolate identific ations, and assisting with pathogenicity assays; of Chang Xiao for doi ng the initial collection of C. gloeosporioides from noncultivated hosts; of Natalia Peres for use of her laboratory and for providing assistance and helpful discussions ; and of Jim Sumler for his help in identifying wild plants and doing crosses. I would also lik e to thank the plant pa thology faculty at the University of Florida in Gainesville. Tha nks to all of the members of my graduate committee for their efforts and use of their equipment, especially Pete Timmer, who accepted me as a graduate student when I needed a major professor, and Dan Legard for his continuing interest in the st udies designed under his guidance.

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iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES.............................................................................................................vi LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ..x CHAPTER 1 INTRODUCTION........................................................................................................1 2 SEXUAL RECOMBINATION AND PATHOGENIC VARIATION AMONG ISOLATES OF Colletotrichum gloeosporioides ON STRAWBERRY.....................8 Introduction...................................................................................................................8 Materials and Methods...............................................................................................10 Perithecium Production on Strawberry Petioles..................................................10 Imaging and Morphology....................................................................................11 Fungal Isolates and Analyses..............................................................................11 Laboratory Crosses..............................................................................................12 DNA Isolation.....................................................................................................13 Marker Analysis..................................................................................................14 Contour-clamped Homogeneous Elec tric Field Gel Electrophoresis..................17 Pathogenicity Tests..............................................................................................17 Statistical Analysis..............................................................................................18 Results........................................................................................................................ .19 Perithecial Morphology.......................................................................................19 AT-rich DNA Analysis........................................................................................19 Characterization of Natura lly Occurring Isolates................................................20 Laboratory Crosses..............................................................................................21 Discussion...................................................................................................................22 3 GENETIC AND PATHOGENIC ANALYSIS OF Colletotrichum gloeosporioides ISOLATES FROM STRAWBERRY AND NONCULTIVATED HOSTS..............41 Introduction.................................................................................................................41 Materials and Methods...............................................................................................43

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v Fungal Isolate Collections...................................................................................43 Extraction of Fungal DNA..................................................................................45 Species Identification..........................................................................................46 Pathogenicity Tests..............................................................................................47 Randomly Amplified Polymorphic DNA Markers.............................................47 Statistical Analyses..............................................................................................48 Results........................................................................................................................ .49 Identification of Isolates......................................................................................49 Pathogenicity Tests..............................................................................................49 Randomly Amplified Polymorphic DNA Analyses............................................50 Discussion...................................................................................................................52 4 SELECTION FOR PATHOGENICITY TO STRAWBERRY IN POPULATIONS OF Colletotrichum gloeosporioides FROM NATIVE PLANTS...............................65 Introduction.................................................................................................................65 Materials and Methods...............................................................................................67 Sampling Strategy and Isolate Codes..................................................................67 Fungal Isolation...................................................................................................68 DNA Extraction and PCR Amplifications..........................................................69 Pathogenicity Tests..............................................................................................71 Statistical Analyses..............................................................................................72 Results........................................................................................................................ .73 Species Identification an d Population Structure..................................................73 Pathogenicity.......................................................................................................75 Discussion...................................................................................................................76 5 RESISTANCE OF STRAWBERRY CU LTIVARS TO CROWN ROT CAUSED BY Colletotrichum gloeosporioides ...........................................................................91 Introduction.................................................................................................................91 Materials and Methods...............................................................................................92 Plant Materials and Cultivation...........................................................................92 Fungal Isolates and Inoculation Procedure..........................................................94 Experimental Design and Statistical Analysis.....................................................95 Results........................................................................................................................ .97 Disease Progression in Cultivar Isolate Experiments......................................97 Cultivar and Isolate Evaluation...........................................................................98 Inheritance of Crown Rot Resistance................................................................102 Discussion.................................................................................................................103 6 CONCLUSION.........................................................................................................122 LIST OF REFERENCES.................................................................................................127 BIOGRAPHICAL SKETCH...........................................................................................136

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vi LIST OF TABLES Table page 2-1 Glomerella cingulata/Colle totrichum gloeosporioides isolates from Dover, FL used to compare AT-rich DNA banding patt erns and for recombination studies.....32 2-2 Crown rot pathogenicity phenotype of single-ascospore isolates from eight perithecia from naturally infected strawberry petioles.............................................33 2-3 Segregation of mitochondrial DNA, fungicide sensitivity, RAPD bands, and (CAT)5 bands from a cross between pat hogenic crown isolate 97-15A and nonpathogenic ascospore isolate P3-8......................................................................34 3-1 Collection sites and host species for Colletotrichum spp.........................................57 3-2 Isolates of Colletotrichum spp. collected from nonc ultivated hosts summarized according to site and pathogenicity on strawberry plants.........................................60 3-3 Isolates of Colletotrichum spp. collected from nonc ultivated hosts summarized according to host species and pathogenicity on strawberry plants...........................61 3-4 Frequencies of RAPD bands for Colletotrichum gloeosporioides isolates from strawberry and noncultivated hosts...........................................................................62 4-1 Frequencies of randomly amp lified polymorphic DNA bands from Colletotrichum gloeosporioides (C.g.) and Colletotrichum fragariae (C.f.) isolates.......................................................................................................................83 4-2 Estimates of for pairwise comparisons of Colletotrichum gloeosporioides isolates from oak and grape hosts at four sites.........................................................85 4-3 Pairwise estimates of (above diagonal) for Colletotrichum gloeosporioides populations at four sites and the es timated 90% confidence interval for (below diagonal)...................................................................................................................86 4-4 Percentage of isolates pat hogenic on strawberry from oak ( Quercus spp.) and grape ( Vitis spp.) lesions at four sites.......................................................................87 4-5 Likelihood ratio statistics examining th e effect of local strawberry production, specific sampling site and native host sp ecies on the proportion of native host isolates pathogenic to strawberry..............................................................................88

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vii 5-1 Description of Colletotrichum isolates used in inoculation experiments conducted over three seasons in Dover, Florida.....................................................112 5-2 Analysis of variance for three experime nts evaluating incidence of crown rot in relation to strawberry cu ltivar and isolate of Colletotrichum gloeosporioides .......113 5-3 Analysis of variance for 2003-2004 expe riment evaluating incidence of crown rot in relation to strawberry cultivar and isolate of Colletotrichum fragariae alone or in comparison to C. gloeosporioides ........................................................114 5-4 Mean percent plant collapse of cultivars inoculated with Colletotrichum gloeosporioides or C. fragariae during three seasons in Dover, Florida................115 5-5 Mean percent plant collapse for Colletotrichum gloeosporioides or Colletotrichum fragariae isolates used to inoculat e strawberry cultivars during three seasons in Dover, Florida...............................................................................116

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viii LIST OF FIGURES Figure page 2-1 Glomerella cingulata from strawberry petiole.........................................................35 2-2 AT-rich DNA banding pattern produced by digestion of total DNA with the restriction enzyme HaeIII.........................................................................................36 2-3 Randomly amplified polymorphic DNA banding patterns of single-ascospore isolates from eight Glomerella cingulata perithecia removed from strawberry petioles.....................................................................................................................37 2-4 Randomly amplified polymorphic DNA banding patterns of two pathogenic and two nonpathogenic isolates to strawb erry and RAPD banding patterns of progeny from crosses using these four isolates as parents.......................................38 2-5 Molecular markers for 97-15A, an isol ate pathogenic on strawberry, and isolate P3-8, an ascospore isolate nonpathogenic to strawberry..........................................39 2-6 Chromosomes ranging in size fr om 300 kb to 1100 kb for pathogenic crown isolate 97-15A, nonpathogenic ascospore is olate P3-8, and progeny from a cross between these isolates..............................................................................................40 3-1 A phenogram using unweighted pair group method with arit hmetric averages showing similarity (Dice) between Colletotrichum gloeosporioides and C. acutatum isolates from noncultivated plants strawberry crowns, citrus, and mango.......................................................................................................................64 4-1 Map showing the three locations where Colletotrichum gloeosporioides isolates were sampled from lesions on oak and grape leaves................................................89 4-2 Unweighted pair group method with arithmetic averages phenogram showing genotypic similarity between Colletotrichum isolates.............................................90 5-1 Mean percent mortality for strawberry cultivars calculated at weekly intervals over the course of the growing season...................................................................117 5-2 Cultivar isolate means for arc-sine-square root transformed disease incidences calculated on a specific date duri ng or at the end of the season.............................118

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ix 5-3 Cultivar species means for arc-sine-square root transformed disease incidences calculated 125 days af ter inoculation with Colletotrichum fragariae or C. gloeosporioides during the 2003-2004 season.......................................................119 5-4 Distribution of transformed cumula tive temperature until 50% plant death for progeny inoculated with Colletotrichum gloeosporioides .....................................120 5-5 Distribution of percent plant mort ality 42 days after inoculation for progeny inoculated with Colletotrichum gloeosporioides ...................................................121

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x Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy POPULATION STRUCTURE AND PATHOGENICITY OF Colletotrichum gloeosporioides FROM STRAWBERRY AND NONCULTIVATED HOSTS IN FLORIDA By Steven John MacKenzie December 2005 Chair: Lavern W. Timmer Major Department: Plant Pathology Colletotrichum crown rot, caused by Colletotrichum gloeosporioides, limits strawberry transplant production in Florida summer nurseries and causes moderate plant losses during the winter season. Marker data have shown that C. gloeosporioides crown isolates are diverse and recombinati on contributes to this diversity. Glomerella cingulata the teleomorph of C. gloeosporioides, has been observed on petioles, but the role of the meiotic cycle in crown rot disease is unknown. Ther e is little evidence that the primary inoculum for infections comes from imported transplants or from debris from past seasons. Colletotrichum gloeosporioides has a broad host range and hosts other than strawberry could contribute inoculum. In the current study, isolat es from strawberry crowns, noncultivated hosts, and perithecia we re characterized using randomly amplified polymorphic DNA markers, AT-rich DNA banding patterns, pathogenicity assays, and laboratory crosses. Genetic data indicated the population from crowns produced

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xi recombinant ascospores from perithecia a nd was found on noncultiva ted hosts close to fields. Pathogenicity to st rawberry was variable among pe rithecia and noncultivated host isolates, but laboratory crosse s indicated that sexual reco mbination occurred between isolates with different pathoge nicity phenotypes. The same C. gloeosporioides population on strawberry was also found on two native hosts at sites distant from strawberry fields. A low frequency of isolat es pathogenic to strawberry at these sites relative to sites close to st rawberry fields suggests th at there was selection for pathogenicity. Although strawberry transplants for winter production are rarely propagated in Florida, crown rot resistant cultivars could ma ke this desirable. To identify resistant cultivars and to determine if resistance is is olate specific, plant mortality was evaluated for cultivars following inoculation with an isolat e differential. Repeatable differences in resistance were observed among cultivars, but no cultivar isolate inte ractions. The lack of a cultivar isolate interaction sugges ted limited biotrophic interaction between pathogen and host. Crosses using a cultivar with superior resistance and a susceptible cultivar as parents indicated that major gene s contributing to resist ance could be useful for breeding. In conclusion, it is unlikely that crown rot introductions into nurseries or fields can be prevented in Florida, but resistant cultivars could help make propagation of transplants between seasons possible.

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1 CHAPTER 1 INTRODUCTION At least three distinct species of Colletotrichum are responsible for anthracnose diseases of strawberry ( Fragaria ananassa Duchesne): Colletotrichum acutatum J. H. Simmonds (80), C. gloeosporiodes (Penz.) Penz. & Sacc. (teleomorph Glomerella cingulata (Stoneman) Spauld. & H. Schrenck) (52) and C. fragariae A. N. Brooks (9). All three of these Colletotrichum species have been reported to cause fruit rot (49,52,80), although in Florida and strawberry -producing regions around the world, C. acutatum appears to be responsible for most fruit ro t epidemics (30,92). Colletotrichum crown rot, another economically important an thracnose disease, is caused by C. gloeosporioides and C. fragariae (10,52). Both species produce a reddishbrown necrosis in crown tissue that eventually causes plants to coll apse (49). Infection of roots by C. acutatum may also cause plants to collapse, but this pathogen does not appear to col onize the crown tissue (37). Colletotrichum fragariae or C. gloeosporioides infection can also cause black leaf spot (51), lesions on petioles, and lesions on runners (9,27). The first report of C. fragariae causing crown rot was nearly 50 years ago in 1935 (10). In 1984 isolates described as G. cingulata the teleomorph of C. gloeosporioides, were reported to cause crow n rot (52). These isolates were referred to by their teleomorphic name based on their ability to fo rm perithecia when gr own alone in culture and were distinguished from C. fragariae isolates by the color of their conidia in addition to the ability to form perithecia (52). Although G. cingulata was reported from strawberry plants in Florida, the plants a ppeared to have been infected in Arkansas,

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2 Tennessee or North Carolina nurseries (53). Glomerella cingulata has also been isolated from strawberry plants in Europe and Ja pan (30). In 1992, it was noted that, among isolates classified as C. fragariae, a subset lacked pointed conidia and failed to produce phialidic setae and these isol ates were reclassified as C. gloeosporioides (43). Molecular studies have confirmed that these isolates ar e distinct from those initially described as C. fragariae (38). The reason that these populations were not distinguished earlier may have been due to the belief that C. fragariae was simply C. gloeosporioides from strawberry (51), since they have been considered synonymous (97). Although never explicitly stated in the literature, mo lecular marker data from self-fertile G. cingulata isolates and self-sterile C. gloeosporioides isolates indicate that they are from genetically distinct populations (38,43). Currently, the self-sterile population from strawberry appears to be prevalent in Fl orida, although genotypes of th ese isolates have not been compared to reference homothallic isolates Unfortunately, isolates often used in molecular studies to represent the G. cingulata / C. gloeosporioides population from strawberry were self-fertile and were not representative of the population in Florida, where crown rot caused by C. gloeosporioides is a problem (13,30). A population study examining variation of randomly amplified polymorphic DNA (RAPD) marker data among C. gloeosporioides isolates on strawberry in Florida found that the C. gloeosporioides population was diverse and recombining, as most markers were in linkage equilibrium (92). Colletotrichum gloeosporioides also forms quiescent infections on petioles that can be detected after petiole tissue se nesces and the fungus sporulates (66). Some of the isolates deri ved from these infecti ons are pathogenic to strawberry in crown inoculation tests, whereas others are not. Occasionally perithecia

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3 with morphology consistent with that of G. cingulata are found among C. gloeosporioides acervuli. Single-ascospore isolates fr om these perithecia are self-sterile in culture, suggesting that they may be fr om the same population responsible for crown rot. However, the genetic relationship betw een ascospore isolates and isolates from diseased crown tissue has not been examined. In addition, the ability of these isolates to cause crown rot has not been confirme d and recombination among progeny from perithecia has not been validated. In southeastern states such as Florida a nd Louisiana, strawberries are grown as an annual crop. Plants are established from late September to early November onto raised, methyl-bromide-fumigated beds covered with polyethylene film. Harvest usually begins during the month of December in Florida. In this region, Colletotrichum crown rot is a serious disease and is especia lly devastating if growers pr oduce their own transplants in summer nurseries (53), because both C. fragariae and C. gloeosporioides grow and reproduce best under moist conditio ns at temperatures exceeding 25oC (81). Along with providing chilling to induce fl oral bud initiation, the high incidence of crown rot in summer nurseries in Florida is one of the primary reasons why transplant production for the winter season has moved to northern states and provinces of Ca nada (53). Although the movement of summer nurseries out of Fl orida has dramatically reduced the incidence of crown rot in production fields in this state, a portion of pl ants still become infected. Analysis of the Colletotrichum species infecting crowns reveal ed that they were primarily C. gloeosporioides (92). In contrast, C. fragariae accounted for most of the mortality due to crown rot in Louisian a production fields (65).

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4 In Florida, C. gloeosporioides does not appear to have the capability of surviving over the summer between seasons on plant debris (93). Colletotrichum gloeoporioides cannot be isolated from petio les of transplants shipped fr om northern latitudes into Florida, although it can be obtained from petiole tissue after plants are set in the field (66). Depending on the nursery source, C. acutatum can be isolated from petiole tissue of transplants. Because C. gloeosporioides does not appear to be introduced into fields each season on transplants or plant debris from th e previous season, a lternative host species may play a role in providing primary inocul um for crown rot epidemics. Although there is no genetic or pathogenicity data to confirm this hypothesi s, several characteristics of the C. gleoesporioides species assemblage indicate that this may be the case. Colletotrichum gloeosporioides has a broad host range (69). Cross-inoculations indicate that isolates can produce disease symptoms on hosts other than those from which they were isolated (1). Although use of rapidly evolving genetic markers has identified hostspecific subpopulations within C. gloeosporioides (47,92), some isolates appear to lack any host specificity (1). Colletotrichum gloeosporioides is also a common endophyte on numerous tropical forest plants and there does not appear to be any discernible host specificity among isolates from these plants (60). The best way to control crow n rot in Florida production fields is to eliminate it from nurseries where plants are propagated (53). Movement of transplant production from Florida to temperate regions has effec tively done this. If Colletotrichum crown rot is observed during the production season, it is us ually at either the beginning or end of the season when temperatures are high and generally no more than a few percent of plants in a field die. Because th e conditions for growth and spread of Colletotrichum

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5 gloeosporioides are not ideal during the relatively c ool weather that coincides with the production season, it is not likely to be the fo cus of chemical control programs. Weekly fungicide applications are gene rally designed to control gray mold, anthracnose fruit rot, and powdery mildew. However, both preventi ve and systemic fungicides used in these programs have activity against C. gloeosporioides Prior to the migration of nurseries to cooler climates, management of soil fertilit y, fungicide applications host resistance, and sanitary measures were all employed to contro l the disease. Measur es included reduction of fertilizer to maintenance le vels during July and August, tw o fungicide applications per week and after rain events, careful selection of runners to be used in nurseries, and use of resistant cultivars such as Dover ( 53). Fungicides with activity against C. gloeosporioides include captan, benzimidazoles, and QoI fungicides (J. Mertely, personal communication ). Resistant cultivars can be successf ully propagated in Florida and local production of transplants is one means of lowering costs to growers, but cultivars such as Dover that are relatively resistant to crow n rot are not grown on a large scale because they lack desired yield and fr uit characteristics. Levels of resistance to crown rot in cultivars currently used for strawberry produc tion in Florida have not been documented and it is conceivable that one or more of thes e cultivars could be sufficiently resistant to crown rot to justify attempts at local propagation. Identification of cultivars with resistance in a genetic background with desi rable fruit and yield properties might also help future breeding efforts. The resistance of cultivars to multiple C. fragariae isolates has been investigated (28,48,81). The resi stance of cultivars to self-sterile C. gloeosporioides isolates that are responsible for most of the crown rot observed in Florida

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6 has not been. This specie s is more variable than C. fragariae and isolate specific resistance could be more prevalent. This dissertation was designed to address some of the fundamental questions that remain regarding crown rot caused by C. gloeosporioides in Florida. Original research is presented in the following four chapters. Chapter 2, the first chapter following this introduction, examines the genetic relati onship of single-ascospore isolates from perithecia collected from senescent petioles to isolates known to produce crown rot on strawberry. Both pathogenic ity and mating compatibility of isolates are examined to determine if the perithecia observed on stra wberry petioles represent the teleomorph of the self-sterile popula tion that causes crown rot. Chapter 3 examines the genetic relationship between isolates from strawbe rry crown and isolates from noncultivated hosts growing close to strawberry fields. In this chapter, marker frequencies, as opposed to the ability of isolates to mate, are examined along with the occurrence of the pathogenic phenotype to determine if noncu ltivated hosts can provide inoculum for crown rot epidemics. In chapter 4, C. gloeosporioides from two native host species are examined at sites distant from strawberry production and compared to those close to strawberry production. Because the strawberry industry in Florida is highly concentrated (5) and strawberry plants are not native to Florida (26), sampling host populations away from strawberry fields may provide informa tion regarding the origin of the pathogen and whether selection for pathogenicity on strawber ry might occur in areas where it is grown in abundance. In chapter 5, resi stance of cultivars to a group of C. gloeosporioides isolates is examined to determin e the mechanism of resistance to C. gloeoporioides and to identify cultivars with genes that could be used in future breeding programs. In

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7 chapters 2 and 4, data regardi ng the inheritance of genes c ontrolling pathogenicity for a cross between C. gloeosporioides isolates and resistance to cr own rot for a cross between strawberry cultivars are presented.

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8 CHAPTER 2 SEXUAL RECOMBINATION AND PATHOGENIC VARIATION AMONG ISOLATES OF Colletotrichum gloeosporioides ON STRAWBERRY Introduction Crown rot of strawberry caused by self-fertile Glomerella cingulata strains was first reported in Florida in 1984 (52). In additi on to the ability of these strains to produce fertile perithecia when grown alone in culture, they were distinguished from Colletotrichum fragariae by the production of white rather than salmon-colored conidia. A more recent evaluation of Colletotrichum species responsible for strawberry diseases indicated that isolates pr eviously characterized as C. fragariae consisted of two morphologically distinct groups of isolates (43). One group of isolates conformed to the initial description of C. fragariae and the other group was reclassified as C. gloeosporioides Isolates in the second group produ ced more oblong conidia that were rounded at both ends and no conidia were form ed on the setae. Unlike isolates previously classified as G. cingulata fertile perithecia were not formed when these isolates were grown singly in culture. However, thes e isolates formed fertile perithecia morphologically similar to G. cingulata when paired in culture. No teleomorph has been described for isolates classified as C. fragariae Molecular analysis using AT-rich DNA band patterns, arbitrarily primed polymeras e chain reaction (PCR), and sequence data from the internally transcribed spacer 1 region of the rDNA repeat revealed that isolates classified as C. fragariae were similar to each other, whereas G. cingulata / C. gloeosporioides isolates fell into two groups with a high level of similarity among

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9 isolates within each group (38,39,83). These two G. cingulata / C. gloeosporioides groups were designated Cgl-1 and Cgl-2 (3 8,39). Isolates found to form the G. cingulata state when grown singly fell in the Cgl-1 group whereas those isolates that reproduce only clonally or outcross were in group Cgl-2 (39,43). In Florida, C. gloeosporioides is the Colletotrichum species most frequently isolated from the crowns of wilted strawberry plants (92). Self-f ertility within this C. gloeosporioides population is rare. Analysis of RAPD marker data revealed a high level of diversity among isolates and a low leve l of linkage disequilibrium among markers (92). Both findings are consistent with ge netic recombination w ithin this population, although there is no direct evidence. Colletotrichum gloeosporioides also infects strawberry petioles producing asymptomatic quiescent infections in Florida (66). Acervuli are produced after the petiole tissue ha s senesced. Some isolates from petiole tissue are capable of causing crow n rot and plant collapse and others are not. In addition to acervuli of C. gloeosporioides perithecia morphologically similar to G. cingulata are occasionally observed on senescent petioles (6 6). The relationship of isolates produced from ascospores from these perithecia to isolates from crown tissue has not been determined. It is also not certain whether th e perithecia on petioles are from a self-fertile strain or the result of re combination between two hetero thallic strains nor whether ascospore isolates vary in pathogenic ity as do isolates from acervuli. This study examines the relationship between isolates of G. cingulata from perithecia on strawberry petioles to C. gloeosporioides isolates known to cause disease on strawberry. AT-rich DNA banding pattern s of single-ascospore isolates from perithecia were compared to those from the two genotypically distinct G. cingulata/C.

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10 gloeosporioides groups known to cause disease on strawberry. Recombination among progeny was evaluated by comparing RAPD markers of isolates from the same perithecium. The ability of crown rot is olates, pathogenic isolates from single ascospores, and nonpathogenic isolates from single ascospores to recombine was evaluated in laboratory crosses. In addition to studies condu cted to define biological species boundaries, segregation analysis of marker data a nd pathogenicity phenotype was conducted for a cross between a nonpathogenic asco spore isolate and a crown rot isolate. Materials and Methods Perithecium Production on Strawberry Petioles Perithecia were collected from senescent petioles during a stu dy to evaluate the effectiveness of freezing tissue to detect latent infections of Colletotrichum spp. on strawberry (66). In the study, healthy petioles with no visibl e lesions were removed from field-grown plants in pl ots untreated with any fungicides, cut to lengths of 5 to 7 cm and frozen for 1 to 2 h at C. After thawing, petioles were surface sterilized for 1 min in 0.5% NaOCl plus 20 l/L Tween 20, rinsed with sterile water, and placed on moistened filter paper inside petri dishes. Petri dish es were placed in clear plastic boxes and incubated on a laboratory bench at 23-25 C under continuous fluores cent light. Petioles were monitored for perithecia production for 21 days. When detected, a single perithecium was transferred with a scalpel to a microscope slide and gently crushed in a drop of sterile water between the slide and a co ver slip. If ascospor es were present in a perithecium and Colletotrichum conidia were not observed, as much vegetative tissue as possible was removed and the suspension contai ning a cluster of asci and ascospores was transferred to 0.75 mL sterile water in a test tube. Ascospore release from asci was

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11 stimulated by repeatedly pipetting the susp ension. The suspension was then spread on semi-selective media (16 g potato dextrose broth, 14 g agar, 250 mg ampicillin, 150 mg streptomycin sulfate, 5 mg iprodione, 100 l tergitol, and deionized water to 1 L) and incubated overnight. Germinating ascospores were subsequently transferred from the semi-selective media with a sterile scalpel and transferred onto potato dextrose agar (PDA). Imaging and Morphology Perithecia from field samples were examin ed using a Zeiss Stemi SV-6 stereo dissecting scope (Carl-Zeiss-Stiftung, Ober kochen, Germany) and asci with a Zeiss Axiolab microscope using brightfield optics at 400 magnification. Microscopic images were captured using a Spot digital camera sy stem (Diagnostic Instruments, Inc., Sterling Heights, MI). Dimensions and morphology of conidia were based on measurements taken on 75 conidia from three isolates. Fungal Isolates and Analyses Eight perithecia (P1-P8) were recovered from strawberry cultivars Camarosa, Strawberry Festival, and Rosa Lind a during the 1998-1999 and 2000-2001 growing seasons at the University of Florida Gulf Coast Research and Education Center (GCREC) in Dover, Florida. Seven to ten ascospore isolates were obtained from each perithecium. Pathogenicity to strawberry was determined for all ascospore isolates. The AT-rich DNA banding pattern was determined for one isol ate from each perithecium and compared to banding patterns from Cgl-1 and Cgl-2 genot ype isolates known to be pathogenic on strawberry. To determine if ascospores from perithecia on petioles were produced by recombination between two or more fungal strains, RAPD DNA ba nds were amplified

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12 from a subset of four to six isolates from each perithecium. In addition to progeny from perithecia found on petiole tissue, RAPD ba nds from progeny of laboratory crosses between two pathogenic ascospore isolates, two nonpathogenic ascos pore isolates and a cross using pathogenic and nonpathogenic ascosp ore isolates as parents were analyzed for recombination. Pathogenic ascospore is olates P1-9 and P8-1 and nonpathogenic ascospore isolates P2-6 and P3-3 were used as parents in these crosses. For quick reference regarding the perithecium and pathoge nicity phenotype of these isolates, they are also referred to as P1-path, P8-path, P2-nonpath and P3-nonpath respectively. Isolates 97-15A and 99-51, representative of C. gloeosporioides genotype Cgl-2 came from crown rot samples submitted to the G CREC diagnostic clinic by local growers in 1997 and 1999. Cgl-1 genotype isolates 311 and 329 were collected by C. M. Howard in Florida. These isolates were characterized previous ly with respect to morphological traits and AT-rich banding pattern (38,43). Crown isol ate 97-15A was also used as a parent in a cross with nonpathogenic ascospore isolate P38. A description of all isolates used in this study and specific analysis in which th ey were employed is given in table 2-1. Laboratory Crosses Laboratory crosses were performed in 9 cm diameter plastic petri plates on sucrosefree Czapek-Dox medium (2 g/L NaNO3, 1 g/L K2HPO4, 0.5 g/L MgSO4H2O, 0.5 g/L KCl, and 0.01 g/L FeSO4H2O) containing 1.5% agar overl aying a piece of Whatman No. 3 filter paper (Whatman International, Maidstone, UK) (21). Isolates were inoculated on opposite sides of a plate approxi mately 7 cm apart and the plates incubated under fluorescent light at 24C. Perithecia formed a line at the point of contact between the two isolates. Few, if any, acervuli were present along the line of intersection between

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13 isolates, greatly reducing the likelihood of conidial contam ination. Single-ascospore isolates were obtained from fertile perithecia as described above for isolations from petiole tissue. DNA Isolation Mycelia was collected from 2to 4-dayold cultures grown in 100 mL of Emerson media (4g/L yeast extract, 15 g/L Soluble Starch, 1g/L K2HPO4, and 0.5 g/L MgSO4) by vacuum filtration through Whatman No. 3 filter paper. After transfer to 15 mL tubes, mycelia was dried overnight in a centrifugal evaporator. Sixty mg of the dried mycelia were then suspended in 750 l of DNA extraction buffer consisting of 700 mM NaCl, 50 mM Tris(pH 8.0), 10 mM EDTA (pH 8.0), 1% CTAB, and 1% -mercaptoethanol and heated to 65C for 2 h with periodic shaking. Particulat e material was pelleted by centrifugation at 12,000 g for 10 min, the supernatant removed, and extracted once with chloroform:isoamyl alcohol (24:1). Two vol umes of 100% ethanol were added to the aqueous extract and the mixture incubated at room temperature for 10 min. Nucleic acids were pelleted from the ethanol solution by centrifugation at 12,000 g for 10 min. Subsequently the pellet was washed w ith 100% ethanol and resuspended in 400 l 1 TE buffer containing 10 g/mL RNase for 1 h at 37C. Ribonuclease was removed from the nucleic acid solution by extraction with 400 l phenol/chloroform/isoamyl alcohol (25:24:1). To the aqueous extract, 1/10 vol ume 3 M sodium acetate and 2.5 volumes of ethanol were added to precipitate DNA. Th is solution was incubate d at C for 1 h and the DNA pelleted at 12,000 g for 10 min. The DNA pellet was washed once with 1 mL 80% ethanol, dried, suspended in 100 l 1 TE buffer, and stored at C.

PAGE 25

14 Marker Analysis AT-rich DNA bands were identified by di gesting 5 to 10 g genomic DNA with the restriction enzymes HaeIII or MspI (103) Digested DNA was separated on a 1% agarose gel in 1TBE buffer for 24 h at 50 V and subsequently stained with ethidium bromide. Successful differentiation of Colletotrichum species and subpopulations using this technique has previously been demonstrated (38). Randomly amplified DNA fragments were obtai ned using the tetranucleotide repeat primers (ACTG)4 and (GACA)4, the trinucleotide repeat primer (TCC)5, and two short oligonucleotides 5-GTGAGGCGTC-3 and 5GATGACCGCC-3 referred to as OPC-2 and OPC-5 (Operon Technologies, Alameda, CA ). Amplifications were carried out under mineral oil in a 20l volume containing 1 reaction buffer (50 mM Tris(pH 8.3), 0.25 mg/mL BSA, 2 mM MgCl2, 0.5% Ficoll, and 1 mM Tartrazine), 200 M dNTP, 1 unit Taq polymerase and 20 mol primer/reaction [primers (ACTG)4, (GACA)4, and (TCC)5] or 8 mol primer/reaction [primers OPC-2 and OPC-5]. Cycling parameters for the PCR reactions consisted of a 5-min denatu ring step at 95C followed by 30 cycles of 1 min at 95C, 1 min at 48C, a nd 2 min at 72C for primers (ACTG)4 and (GACA)4; 34 cycles of 1 min at 95C, 1 min at 46 C, and 1.5 min at 72C for primer (TCC)5 or 38 cycles of 1 min at 95C, 1 min at 35C, and 2 min at 72C for primers OPC-2 and OPC5. The amplified products were separa ted by electrophoresis through a 1.5% high resolution blend agarose (3 :1) gel or 2% analytical grade agarose gel in 1 TAE buffer. Gels were photographed on a UV transilluminator after staining with ethidium bromide. DNA fingerprints were obtai ned for progeny from the cross between crown rot isolate 97-15A and nonpathogenic perithecium is olate P3-8 using a digoxigenin-labeled

PAGE 26

15 (CAT)5 oligonucleotide fingerprintin g probe. For this analysis, 5 to 10 g of PstI digested DNA were separated on a 1% gel in 1 TAE for 16 to 24 h at 25 to 35 V. Gels were incubated subsequently for 30 min in 1.5 M NaCl/0.5 N NaOH buffer followed by 30-min and 15-min incubations in 1 M Tris (p H 7.4)/1.5 M NaCl. Capi llary transfer of DNA to nylon membrane was conducted with 10 SSC buffer overnight. DNA was UV cross-linked to the membrane prior to prehybr idization in 5 SSC buffer, 0.1% N-laurylsarcosine, 0.02% SDS, and 1% blocking r eagent (Roche Applied Science, Mannheim, Germany) at 30C for 4 h. Hybridization wa s carried out in the same buffer with 10 mol/mL oligonucleotide probe at 30C overn ight. Following hybridization, blots were washed twice with 2 SSC/0.1 % SDS for 5 min at room temperature and twice with 0.5 SSC/0.1 % SDS at 30C for 5 min. The probe was detected with an antidigoxigenin Fab fragment c onjugated to alkaline phosphata se and NBT/BCIP color substrate according to instructions supplied wi th a DIG nucleic acid detection kit (Roche Applied Science, Mannheim, Germany). Resistance to benomyl among progeny of th e cross between isolates 97-15A and P3-8 was determined by growing fungi on PDA amended with 5 g/mL benomyl. Susceptible isolates did not grow at this concentration, whereas growth of resistant isolates was not affected. Benomyl resistance was found to segregate at a 1:1 ratio in crosses with Glomerella graminicola and a point mutation in a -tubulin gene was shown to confer benomyl resistance in C. gloeosporioides f. sp. aeschynomene (14,95). A polymorphism observed among AT-rich DNA bands was identified with the restriction enzyme MspI and used to determ ine mitochondrial inhe ritance among progeny of the cross between 97-15A and P3-8. Th at the polymorphic MspI band was comprised

PAGE 27

16 of mitochondrial DNA was determined by comparison to MspI-digested DNA from purified mitochondria. Purified mitochondria l DNA was isolated from approximately 6 g wet weight mycelia collected onto a filte r disk using a modification of the method described for isolation of Epichlo typhina mitochondrial DNA (77). Mycelia was ground in 30 mL buffer containing 15% sucr ose, 10 mM Tris-HCl(pH 7.5), and 0.2 mM EDTA(pH 7.5) at 4C. Nuclei and cellular debris was removed by centrifugation at 1,000 g for 10 min and the supernatant was saved. The pellet was re-extracted in 20 mL buffer, ground, and debris removed by centrifugation at 1,000 g for 10 min. The supernatant from both centrifugations was pooled and centrifuged at 15,000 g for 15 min. The pellet was suspended in 10 mL 20% sucrose, 10 mM Tris-HCl(pH 7.5), and 0.01 mM EDTA(pH 7.5) a nd centrifuged at 15,000 g for 15 min. Following centrifugation, the pellet was resuspended in 5 mL buffer containing 1.75 M sucrose, 10 mM Tris-HCl(pH 7.5), 5 mM EDTA(pH 7.5), 12 mM MgCl2, 100 g/mL DNase, and 50 g/mL RNase and incubated for 1.5 h at 4C. Mitochondria were pelleted from this solution at 20,000 g for 10 min. The pellet was suspe nded in 5 mL of the same buffer without MgCl2 or nuclease and pelleted a second time at 20,000 g. The pellet was then suspended in 0.5 mL lysis buffer (0.44 M sucros e, 1% SDS) and incubated at 37C for 30 min. Lysis buffer was extracted once with phenol:chloroform:isoamyl alcohol (25:24:1) followed by an extraction with chloroform:isoa myl alcohol. DNA was precipitated by addition of 1/10 volume 3 M sodium acet ate and 2.5 volumes ethanol. DNA was subsequently washed with 70% ethanol, dried, and resuspended in 1 TE.

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17 Contour-clamped Homogeneous El ectric Field Gel Electrophoresis High molecular weight DNA was prepared from agarose embedded conidia using a modification of the method described for C. lindemuthianum (72). Conidia from 5to 7day-old cultures were harvested in steril e water and the concentration of conidia determined using a haematocytometer. Conidi a were pelleted and suspended in SCE (1 M sorbitol, 50 mM sodium citrate, 50 mM EDTA, pH 5.7, and 20 M DTT) containing 75 mg/mL glucanex at a concentration of 8 108 conidia/mL. An equal volume of 1.2% low melting point agarose in SCE was then adde d to the conidial suspension at 42C and the mixture cast into 10 mm 5 mm 0.5 mm we lls. Once solidified, plugs (1 to 4 each) were incubated in 0.5 mL SCE buffer at 37C ov ernight. The plugs were then rinsed with 1 mL of EST (0.5 M EDTA, 0.1 M Tris-HCl, and 1% lauryl sarcosyl, pH 9.5) and incubated in 1 mL EST containing 2 mg/mL proteinase K at 50 C for 48 h with one change of solution. After the proteinase di gestion was complete, the buffer was removed and plugs incubated 0.5 h in 1 mL 50 mM ED TA (pH 8.0) at room temperature. This incubation was repeated twice with fresh buffer. Plugs were then stored at 4C in 50 mM EDTA (pH 8.0) until used. Chromosomes we re separated on a CHEF-DR II pulsed field electrophoresis system (Bio-Rad, Hercules, CA) at 200 V in 0.5 TBE buffer using a run time of 24 h and 60 to 120 s ramped sw itch time. This run condition resolves chromosomes less than 1200 kb in length. Pathogenicity Tests To determine if isolates were pathogenic on strawberry, approximately 0.1 mL of 1 106 conidia/mL inoculation solution was in jected directly into crown tissue of greenhouse grown strawberry plants (cv. Cama rosa) as described previously (66).

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18 Conidial suspensions were prepared in st erile water from 7-day-old cultures grown on PDA. Plants injected with sterile water only served as controls. Plants were monitored weekly over 28 days for symptoms of crown rot (wilting and collapse of plants). For single-ascospore isolates from petiole tissue, three plants were inoculated with each isolate on two dates. Isolates that caused the collapse of two or more plants for both inoculation dates were classi fied as pathogenic and those which failed to cause collapse of at least two plants for both inoculations we re classified as nonpathoge nic. If an isolate caused collapse of two or more plants after one inoculation date a nd not the other, the assay was repeated a third time and data from this experiment used to classify the isolate. For progeny from a cross between the crown rot isolate 97-15A and nonpathogenic perithecium isolate P3-8, pathogenicity was de termined from inocula tion of three plants on a single date. Statistical Analysis A hierarchal analysis using theta () was employed to determine if there was population subdivision between pe rithecia that yielded pathogen ic isolates and perithecia that had only nonpathogenic isolates. The pe rithecium that an isolate was obtained from defined the subpopulation. Values of were calculated using the method of Weir and Cockerham (99) with confidence intervals ge nerated by bootstrapping over loci. Theta measures the correlation of alleles from indi viduals in the same popul ation relative to all populations and is desc ribed by the equation ( Q q )/(1 q ), where Q is the probability that two randomly sampled genes within a population are the same allele and q is the probability that genes randomly selected from different populations are the same allele (22). A hierarchal analysis was required due to repeated sampling w ithin perithecia. All

PAGE 30

19 values of were calculated using the software program TFPGA (Utah State University, Logan, UT). Chi-square analyses and a Wilcoxon-Mann-Whitney test were done using the SPSS 8.0 statistics package (SPSS Inc., Chicago, IL). Results Perithecial Morphology The proportion of petioles with at least one perithecium ranged from 0% to 10% of those examined, depending on the sample da te. Perithecia were typically globose, approximately 100 to 400 m in diameter, dark, covered with gray mycelia, and found among acervuli of C. gloeosporioides (Fig. 2-1A). Each perith ecium contained tens of asci holding three to eight ascospores each (Fig 2-1B). Asci were cylindrical to clavate and contained ascospores in both linear and alternately biserrate arrangements. Ostioles were not discernible. Ascospore shape was va riable both within and among perithecia. Allantoid, ellipsoid, and oblong ascospores we re observed in squashes. No fertile perithecia were observed in cultures of singl e-ascospore progeny, indi cating that isolates were not homothallic when cultured on artificial substrate. The size and shape of conidia from isolates grown in culture were consistent with C. gloeosporioides from both strawberry and nonstrawberry hosts. Average conidial length was 16.8 0.32 m and width 5.07 0.037 m. Ninety-five percent were oblong with obtuse ends. AT-rich DNA Analysis AT-rich DNA banding patterns for the two Cgl1 genotype isolates from strawberry (isolates 311 and 329) were id entical to one another and th e two isolates with a Cgl-2 genotype (isolates 97-15A and 99-51) were iden tical to one another (Fig. 2-2). Dice similarity between the two genotypes was 0.33. Isolates from six of the eight naturally

PAGE 31

20 occurring perithecia (P 1, P3, P4, P5, P6, P8) had AT-ri ch DNA banding patterns identical to the pattern of the Cgl-2 genotype isolates (Similarity = 1.0). The banding patterns of the two unique perithecial isolates (P2 and P7 ) were more similar to the Cgl-2 genotype than to the Cgl-1 genotype (0.83 vs 0.33 and 0.5) with the gain or loss of a single restriction site able to account for the differe nce between the pattern for these isolates and those for Cgl-2 isolates from strawberry. Characterization of Naturally Occurring Isolates RAPD markers were used to determ ine if segregation was occurring among isolates from the same perithecium. Usi ng five RAPD primers, eleven polymorphic bands consistently amplified from the te mplate DNA of single-ascospore isolates collected from perithecia on petioles. Bands at 2.15 kb and 1.15 kb for the primer (ACTG)4; 0.9 kb for primer (GACA)4; 2.0 kb and 1.9 kb for primer (TCC)5; 1.9 kb for primer OPC-2; and bands at 2.15 kb, 2.0 kb, 1.8 kb, 1.6 kb, and 1.2 kb for primer OPC-5 segregated among progeny from at least one peri thecium (Fig 2-3). For each of the eight perithecia, between one and five of the ba nds were different am ong progeny. Four of eight perithecia (P2, P3, P4, P6) contained only nonpathogenic isolates as determined by the greenhouse bioassay and tw o perithecia contained only pathogenic isolates (P5 and P8) (Table 2-2). Of the two perithecia w ith progeny that segregated for pathogenicity, only perithecium 1 had multiple representati ves of each phenotype. Of the 68 isolates tested, the pathogenicity phenotype was the same in repeated experiments for 62 isolates. Of the six isolates in which there was a c onflict in scoring, two were classified as pathogens and four as nonpathogens based on a third assay. Three of the four isolates classified as nonpathogens came from perithecia with progeny that were both pathogenic

PAGE 32

21 and nonpathogenic. Based on RAPD marker s, there was no evidence for population subdivision between perithecia with progeny pathogenic on strawberry and those with only nonpathogenic progeny ( = 0.037, 90% C. I. -0.078 to 0.149), although there was strong support for correlation of RAPD ba nds among progeny from the same perithecium ( = 0.515, 90% C. I. 0.393 to 0.634). Laboratory Crosses Thirty-six of 80 crosses attempted between single-ascospore isolates or between single-ascospore isolates and isolates from di seased crown tissue yielded perithecia with mature ascospores. Some isolates successf ully mated with both parents that were compatible with one another in a third cross, indicating that mating compatibility was not determined by a single mating type locus with two alleles. Viab le ascospore progeny were obtained from crosses in which both parents were nonpathogenic on strawberry, both parents were pathogenic on strawberry, as well as with parent s that had different pathogenicity phenotypes. Recombination among progeny from one cross for each of these parent combinations was examined using RAPD markers. Of four progeny examined from each cross, at least three we re recombinants (Fig 2-4), indicating that recombination had occurred in these crosses. Forty-six progeny from the cross betw een isolate 97-15A, from a crown-rot affected plant and isolate P3-8, a nonpathoge nic ascospore isolate, were evaluated for pathogenicity using the greenhouse bioassa y. Of these 46 isolates, two were nonpathogenic and 44 were pathogenic on strawberry. A 2.5-kb MspI band observed only in mitochondrial DNA of isolate P3-8 wa s inherited by all progeny (Fig 2-5A, Table 2-3). Putative genomic DNA markers include d benomyl sensitivity, three RAPD bands,

PAGE 33

22 and three (CAT)5 repeat bands (Fig 2-5B, 2-5C a nd 2-5D). Segregation of the 1.6-kb OPC-5 RAPD band, the 0.9-kb (GACA)4 band, and two of the three bands identified by the (CAT)5 repeat probe diverged from the e xpected 1:1 ratio assumed under the hypothesis that these markers randomly assort and identify a single site within the genome ( P < 0.05) (Table 2-3). Segregation of the 1.8-kb OPC-5 RAPD band, benomyl resistance, and one of the (CAT)5 repeat bands did not diffe r significantly from a 1:1 ratio. Of 25 progeny scored for all seven ma rkers, three isolates inherited all of the dominant markers examined. It is un likely that progeny we re derived from contaminating parental material since only one of the progeny had a phenotype that was identical to either parent. The genotypes of the two isolates that were not pathogenic on strawberry were distinct from either parent. Chromosomes in the size range of 220 to 1100 kb were very different between the parental isolates (Fig 2-6). Isolate 9715A had four chromosomes ranging from 730 to 940 kb, whereas isolate P3-8 had five chro mosomes ranging from 350 kb to 640 kb. The sum of sizes in this range was greater for isolate 97-15A than it was for isolate P3-8 (3310 kb vs. 2240 kb). Chromosome numbers for progeny ranged from three to six. Most progeny had either a different number of chromo somes or different sized chromosomes from either parent. The tota l length of chromosome DNA for the two isolates that failed to cause crown rot on strawberry was less than it was for ten isolates pathogenic on strawberry (1875 kb vs 3411 kb, P = 0.03, Mann-Whitney-Wilcoxon test). Discussion Perithecia with morphology consistent with G. cingulata were observed on strawberry petioles in a previous study, howev er the genetic relationship between singleascospore isolates from these perithecia and isolates known to infect crown tissue was not

PAGE 34

23 investigated (66). Self-sterile C. gloeosporioides isolates are most commonly isolated from crown tissue in Flor ida, although homothallic G. cingulata strains have also been isolated from strawberry in this state. It was conceivable th at the population that produced the perithecia obser ved on petiole tissue came fr om either one of these populations or a population unrelat ed to those responsible for crown rot. In addition to differences in the ability of is olates to self-fertilize, the G. cingulata/C. gloeosporioides populations on strawberry can be distinguished from one another based on AT-rich DNA banding patterns. Self-fertile isolates ha ve a Cgl-1 genotype banding pattern and selfsterile isolates have a Cg l-2 genotype banding pattern. The AT-rich DNA banding patterns observed for perithecial isolates indicate that they are from the same population or are closely related to is olates with the Cgl-2 genotype, which are most frequently isolated from crowns in Florida. Sexual reproduction has been s uggested as playing a role in the reproduction of this population, si nce it is comprised of genetically diverse isolates and linkage disequilibrium among RAPD bands is not observed (92). The occurrence of ascospore isolates with iden tical or similar AT-rich DNA banding patterns to isolates from crown tissue supports the hypot hesis that isolates responsible for crown rot reproduce sexually on strawberry. Furt her evidence for the occurrence of sexual recombination in this populat ion comes from the analysis of RAPD banding patterns. From each perithecium examined, at least two unique banding patterns were observed among progeny indicating that ascsopores were produced by recombination of parental strains. Because there is no information on parental genotypes for perithecia collected from field material, it is more difficult to be certa in that unique banding patterns do not result

PAGE 35

24 from poor reproducibility of RAPD bands. Evidence that RAPD genotype differences were representative of true genetic differe nces comes from the ab ility to reproducibly amplify scored bands from the same isolat e and the observation th at bands polymorphic among progeny from laboratory crosses were al so different between parents. Further evidence that band differences were real and not artifacts of the P CR reaction comes from the correlation of marker data among isolat es from the same perithecium. As measured by the correlation of marker data among isol ates from the same perithecium was 0.515. Assuming that only one male parent gives rise to as cogenous hyphae, RAPD bands segregate among progeny in a 1:1 ratio, a nd that there is random mating among individuals within a populat ion, the expected value of for a heterothallic fungus would be 0.5. This value is well within th e 90% confidence interval calculated for The expected value of would be 1 for completely self-fer tile isolates, and 0, if ascospores from the same perithecium are unrelate d. Assuming that a fungal population is heterothallic, processes such as nonmendelian inheritance of marker s would tend to bias measurements of towards 1 and multiple male parents toward 0. Although no attempt was made to determine the effect thes e processes might have on estimates of in this study, segregation of markers from crosses conducted in the laboratory indicate that nonmendelian inheritance mi ght bias the estimate of upwards, whereas analysis of ascospore isolates collected from the field for other fungi indicate that multiple male parents could bias downward (34). Both pathogenic and nonpathogenic singleascospore isolates were obtained from perithecia. Most of the peri thecia yielded ascospores with only one phenotype or the other. Although three of four perithecia yielding pathogeni c ascospore isolates had AT-

PAGE 36

25 rich DNA genotypes identical to three of the perithecia yielding only nonpathogenic ascospore isolates, it is concei vable that isolates with diffe rent pathogenicity phenotypes derived from different populations. This would occur if the mutation rate in mitochondrial DNA, which accounts for the bulk of AT-rich bands, was not fast enough to produce detectable polymorphisms to dist inghuish the two groups. Several additional lines of evidence, however, support the hypot hesis that pathogenic and nonpathogenic isolates derive from the same population. These include the ability of pathogenic and nonpathogenic isolates to produce recombinan t offspring when crossed on agar, the occurrence of both phenotypes among isolates from perithecia 1 and 7, and there was no evidence for population subdivision based on RAPD band frequencies, although this analysis probably suffered from reduced st atistical power as pr ogeny from the four perithecia in each group only represented eight parental genotypes. The most frequently occurring AT-rich DNA banding pattern from perithecia isolates matched that for isolates from stra wberry crown, indicating that they are both derived from the same population. Howeve r, as noted above, mitochondrial DNA may fail to evolve fast enough to identify a recent divergence between the two groups. Additional evidence that crow n and perithecia isolates are from the same population comes from the ability of strawb erry crown isolates to succes sfully cross with perithecial isolates on agar. RAPD markers were not am plified from a population of crown isolates in this study. However, in the next ch apter band frequencies from a population of strawberry crown isolates are reported. A comparison of these frequencies to those observed among perithecia provided no ev idence for population subdivision (data not reported).

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26 The cross between a crown isolate and a nonpathogenic perithecium isolate yielded a highly skewed distribution of progeny pathogenic on strawberry, suggesting that pathogenicity on strawberry is determined by genes at more than one locus or nonmendelian segregation of pathogenicity de terminants encoded at a single locus. Plasmids and double-stranded RNA viruses lo calized within mitochondria have been shown to induce hypovirulence in several species of f ungi (29,68). Mitochondrial restriction fragment length polymorphisms (RFLP) also correlate with host preference in Mycosphaerella graminicola populations (105). These stud ies suggest that mitochondrial inheritance of a pat hogenicity or hypovirulence factor could account for the skewed distribution of pathogenicity among progeny. However, all progeny examined inherited the 1.6-kb MspI mitochondrial DNA fragment found in nonpathogenic isolate P3-8. Given that inheritance was skewed toward the pathogenic phenotype it does not appear that factors associated with the mitochondr ia affect pathogenic ity. Inheritance of assumed genomic markers and chromosomes indicate nonmendelian mechanisms govern inheritance in portions of the genome. In mo st studies examining segregation of markers in fungal crosses, a subset of markers typically deviate from expected ratios (36,76). However, in the cross between 97-15A and P3 -8, the number of markers that deviated from a 1:1 segregation ratio was greater than th at typically observed. Use of markers that require hybridization to simple sequence repeat s might account for the skewed ratios, as a high proportion of bands identified with the (CAT)5 probe fail to segregate at expected ratios in other fungi (32). However, simple sequence repeat loci do not segregate in a nonmendelian fashion at a higher freque ncy than restriction fragment length polymorphisms in plants (82). The occurren ce of dominant markers at more than one

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27 locus, inheritance of more than one homol ogous chromosome within progeny, or simply preferential inheritance of one or more chromosomes with limited recombination could also account for the skewed ratios. All of the skewed ratios result from a larger proportion of progeny inheriting the dominant marker, sugges ting inheritance of more than one copy of the marker or more than one copy of homologous chromosomes. If a significant number of progeny we re heterokaryons, one would expect skewed inheritance of dominant markers among progeny, but hete rokaryon formation could be excluded for all but three of 25 isolates examined. The meiotic events producing the skewed ratios could also have arisen from genetic incompatibili ty of the isolates used in the cross as has been observed with geographically isolated strains of Uromyces appendiculatus (62) or alternatively they may be a nor mal occurrence in crosses of C. gloeosporioides Segregation of markers in crosses between peri thecial isolates was de tected at a high rate by sampling only four progeny, indicating that segregation of markers in these crosses probably did not deviate substa ntially from mendelian ratios This suggests that the diseased crown isolate and the ascospore isolat e might have lacked genetic compatibility. In crosses of C. gloeosporioides from jointvetch and pecan most genomic markers displayed normal mendelian segregation ratios on agar, although use of jointvetch as substrate tended to skew inheritance of ma rkers (21). Ultimately, segregation of codominant markers using more than one comb ination of parents will be required to determine normal mechanisms of segregati on among isolates of this species from strawberry. Distinct electrophoretic karyot ypes have been observed for C. gloeosporioides isolates from Stylosanthes sp., with all of the variation w ithin biotypes due to differences

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28 in the size and number of small chromoso mes that range from 0.27 Mb to 1.2 Mb in length and comprise approximately 15% or less of the total genome (63). In this size range, there were also differences in th e size and number of chromosomes between isolates 97-15A and P3-8. Most proge ny from the cross of these isolates had chromosome banding patterns distinct from each other and both parents. Although recombination appears to have produced some chromosomes that migrate at very different rates from those of the parental isolates, assortment of chromosomes produced most of the differences in banding patterns. This conclusion is based on the observation that most chromosomes inherited by progeny were identical or close to the size of chromosomes from parent isolates. No speci fic probes were generated for chromosomes, so it is not possible to determine with any certainty which ones might be homologous or if one isolate has coding sequence absent in the other. However, for isolate 97-15A, the sum of chromosome lengths was substantia lly greater than it was for isolate P3-8, indicating that this is olate may possess genes not found in isolate P3-8. Also, based on the size of progeny chromosomes it appears th at chromosomes from parent 97-15A were inherited preferentially. Thus inheritance of pathogenicity determinants on these chromosomes might account for the high proportion of offspring pathogenic to strawberry. There are a number of examples of genes encoded on chromosomes less than 2.0 Mb in length that are required for virule nce on specific hosts, but dispensable for saprophytic growth (24,45). Often the small, dispensable chromosome s that encode these genes display nonmendelian inhe ritance in crosses (24). The occurrence of progeny pathogenic to no rthern jointvetch seedlings has been reported from a cross between a pathogenic isolate from northern jointvetch and a

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29 nonpathogenic isolate from pecan (19). None of the progeny from this cross consistently killed northern jointvetch seedlings. Because there was little evidence from marker data to suggest that nonmendelian mechanisms cont ributed to the skewed ratio of pathogenic to nonpathogenic progeny, it was suggested th at multiple avirulence genes or multiple pathogenicity genes differentiate isolates pathogenic to jointv etch from those that are not and that the genetic requirements for successful infection of jointvetch are complex. In the cross between pathogenic crown isolat e 97-15A and nonpathogenic ascospore isolate P3-8 results opposite to those observed for north ern jointvetch were obtained, as most of the progeny were pathogenic to strawberr y. Discounting the role that nonmendelian inheritance might play in the inheritance of pathogenicity to strawberry, it appears that there may be more plasticity with respect to genetic requirements for causing crown rot on strawberry. However, the nonpathogenic isolate in the current experiment was obtained from a latent infection on strawbe rry, not a distantly re lated host, and could already have met most of the physiological requirements for pa thogenicity. Also of note, laboratory crosses done in the current study di d not support that a si ngle locus with two alleles regulated mating compatibility, a findi ng consistent with studies examining the mating system of C. gloeosporioides and other Colletotrichum species (20,94). The bioassay used to determine pathogeni city phenotype was highly reproducible for most isolates, although several isolates from perithecium 1 were difficult to categorize. This perithecium yielded both pathogenic and nonpathogenic isolates. In field experiments examining plant mortality in response to inoculation with multiple C. gloeosporioides isolates presented in chapter 5, the isolates display quantitative differences in aggressiveness. This suggests that multiple genes affect pathogenicity on

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30 strawberry and that the crown injection assay may identify isolates as pathogens only if they possess physiological capabilities that exceed a threshold. Since perithecium 1 yielded both pathogens and nonpathogens, the disease-causing ability of isolates from this source would lie close to the threshold. Also, given that many of the isolates from this perithecium are weak pathogens, one w ould expect environmental variation between experiments to have a greater effect on the outcome of pathogenicity tests using these isolates. Differences in temperature acro ss experiments is one variable which may account for the conflicting results, as temperat ure has been shown to alter final mortality of plants inoculated with G. cingulata (70) Colletotrichum gloeosporioides has been reported from a wide range of host species (69). Although reproducti vely isolated subgroups with narrow host ranges likely exist within the C. gloeosporioides species aggregate, th ere are populations of C. gloeosporioides in which isolates from different hos ts are not genetical ly distinct from one another and isolates from different hos ts can successfully recombine in culture (19,60). The acquisition of si ngle-ascospore isolates that are able to interbreed and display variation in pathogenic ability on strawb erry will benefit future studies examining the specific genetic re quirements for pathogenicity and how they are distributed within the population on strawberry and other plant sp ecies that the population from strawberry might colonize. In summary, it appears that C. gloeosporioides from strawberry is part of a recombining population that consists of st rains both pathogenic and nonpathogenic to strawberry. The mechanism by which the pat hogenicity phenotype is inherited in this population remains unclear. It is apparent that a gene or cluster of genes at a single

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31 mendelian-segregating locus does not determine pathogenicity. However, based on the data derived from this study no conclusions could be made regarding wether pathogenicity is determined at a nonmendeliansegregating locus or th at it is affected by multiple unlinked genes each having a qua ntitative effect on pathogenicity.

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32Table 2-1. Glomerella cingulata/Colle totrichum gloeosporioides isolates from Dover, FL used to compare AT-rich DNA banding patterns and for recombination studies Analysisa Isolates Description Tissue AT-rich DNA Recomb ination on petiole Parent agar crosses P1-1 to P1-10b Perithecium 1 ascospore Petiole P1-9 P1-3 to P1-4, P1-7 to P1-10 P1-9 (P1-Path)c P2-1 to P2-10 Perithecium 2 ascospore Petio le P2-6 P2-7 to P2-10 P2-6 (P2-Nonpath) P3-1 to P3-10 Perithecium 3 ascospore Petiole P3-3 P3-7 to P3-10 P3-3 (P3-Nonpath), P3-8 P4-1 to P4-10 Perithecium 4 ascospore Petio le P4-7 P4-5 to P4-7, P1-9 to P1-10 P5-1 to P5-7 Perithecium 5 ascospor e Petiole P5-3 P5-1 to P5-4 P6-1 to P6-7 Perithecium 6 ascospor e Petiole P6-3 P6-1 to P6-4 P7-1 to P7-7 Perithecium 7 ascospor e Petiole P7-3 P7-1 to P7-4 P8-1 to P8-7 Perithecium 8 ascospore Pe tiole P8-1 P8-1 to P8-4 P8-1 (P8-Path) 311 Cgl-1 self-fertile ? Yes 329 Cgl-1 self-fertile ? Yes 97-15A Cgl-2 conidial Crown Yes Yes 99-51 Cgl-2 conidial Crown Yes aSubsets of isolates used to compare AT-rich DNA banding patte rns, to compare RAPD bands among progeny from perithecia on petioles, and as parents for crosses on agar. bPrefixes P followed by numbers 1 through 8 represent the individual perithecium sa mpled. Numbers following - represent specific ascospore isolates sampled from each perithecium, with only the range of these designati ons given in column 1. All isolates in column 1 were tested for pathogenicity to strawberry. cIsolate codes in parentheses represent alternate names for isolates to the left. Alternate names identify the perithecium and pathogenicity phenotype on strawberry of the isolate.

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33 Table 2-2. Crown rot pathogenicity phenot ype of single-ascospore isolates from eight perithecia from naturally infected strawberry petioles Phenotype Perithecium Pathogenic Nonpathogenic No. of ascospore isolates evaluated 1 6 4 10 2 0 10 10 3 0 10 10 4 0 10 10 5 7 0 7 6 0 7 7 7 6 1 7 8 7 0 7

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34 Table 2-3. Segregation of mitochondria l DNA, fungicide sensitivity, RAPD bands, and (CAT)5 bands from a cross between pat hogenic crown isolate 97-15A and nonpathogenic ascospore isolate P3-8 97-15A genotype P3-8 genotype P > 2 2.5-kb Msp I mitochondrial band 0 39 Benomyl sensitivity 29 22 0.33 1.8-kb OPC-5 band 11 14 0.55 1.6-kb OPC-5 band 22 3 0.001 0.9-kb (GACA)4 band 7 18 0.04 9.9-kb (CAT)5 band 1 27 0.001 7.7kb (CAT)5 band 17 11 0.26 3.5-kb (CAT)5 band 20 8 0.02

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35 Fig. 2-1. Glomerella cingulata from strawberry petiole (A) Perithecia of G. cingulata growing on a field-infected strawberry peti ole after the petiole was freeze-killed and incubated at room temperature 2-3 wk. Bar = 1000 m. (B) Squash showing asci and ascospores of a perithecium collected fr om a strawberry petiole. Bar = 50 m.

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36 Fig 2-2. AT-rich DNA banding pattern produced by digestion of total DNA with the restriction enzyme HaeIII. Isolates 311 and 329 are Cgl-1 genotype isolates from strawberry. Isolates 97-15A and 99-51 are Cg l-2 genotype isolates from strawberry. Isolates P1 P8 are perithecium is olates from strawberry petioles.

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37 Fig 2-3. Randomly amplified polymorphic DNA ba nding patterns of single-ascospore isolates from eight Glomerella cingulata perithecia removed from strawberry petioles. Frames within columns labeled P1-P8 contain banding patterns of isolates from the same perithecium amplified using primers (ACTG)4, (GACA)4, (TCC)5, OPC-2, or OPC-5. Arrows to the left of the frame show the position of ba nds that were polymorphic among isolates from the perithecium identified by the label at the top of the column.

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38 Fig 2-4. Randomly amplified polymorphic DNA bandi ng patterns of two pathogenic and two nonpathogenic isolates to strawberry and RAPD banding patterns of progeny from crosses using these four isolates as parents. The first colu mn of five frames contains bands amplified from the four parents using primers (ACTG)4, (GACA)4, (TCC)5, OPC2, and OPC-5. The arrows to the left of these frames show the positions of bands polymorphic among parents. The next two columns show bands amplified using the same primers from four progeny of crosse s using two nonpathogeni c and two pathogenic isolates as parents. The final column show s bands from progeny of a cross using isolates of each phenotype as parents. Arrows to the left of frames show the position of bands polymorphic among progeny. Progeny with ba nding patterns distinct from both parents are identified by the letter R, those that had banding patterns identical to a parent are identified by the parent perithecium.

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39 Fig 2-5. Molecular markers for 97-15A, an isolate pathogenic on strawberry and isolate P3-8, an ascospore isolate nonpathogenic to strawberry that were used to examine mitochondrial and genomic inheritance am ong progeny from a cross between these isolates. (A) The frame on the left is 700g mitochondrial DNA digested with MspI. The frame at the right is 10 g of total DNA digested with MspI, which was used to identify mitochondrial inheritance among proge ny. (B) Bands amplified with the primer OPC-5. (C) Bands amplified with the primer (GACA)4. (D) PstI-digested DNA probed with (CAT)5. Arrows indicate bands used to ex amine inheritance of mitochondria and recombination among progeny.

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40 Fig 2-6. Chromosomes ranging in size from 300 kb to 1100 kb for pathogenic crown isolate 97-15A, nonpathogenic ascospore isolat e P3-8, and progeny from a cross between these isolates. The only two progeny that we re nonpathogenic on stra wberry are in lanes 3 and 4 of frame 1. Ten repres entative isolates of those pa thogenic on strawberry are in lanes 5 through 10 of frame 1 and lanes 3 through 6 of frame 2.

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41 CHAPTER 3 GENETIC AND PATHOGENIC ANALYSIS OF Colletotrichum gloeosporioides ISOLATES FROM STRAWBERRY AND NONCULTIVATED HOSTS Introduction Under west central Florida climatic conditions, C. gloeosporioides does not appear to have the capacity to survive on strawb erry plant debris in soil during the summer (53,93). In addition, soil is fumigated with a mixture of methyl bromide and chloropicrin before bare-root transplants ar e set in the fall (3), making tr ansfer of inoculum between plants from different production seasons even less likely. Due to the low probability that infections are transfe rred directly between plants grown in different seasons, inoculum for the initiation of epidemics in Florida mu st come from sources outside of production fields. Infected transplants are one po tential source and there are some apparent correlations between crown rot epidemics in Florida production fields with the nursery providing transplants (59). Anot her potential source of inocul um is alternate host species growing in the vicinity of stra wberry fields. In Florida, st rawberry fields are generally surrounded by noncultivated trees, shrubs, and he rbaceous plants and in some fields it Most of the material in this chapter is reprinted w ith permission from Xiao CL, MacKenzie SJ, and Legard DE. 2004. Genetic and pathogenic analyses of Colletotrichum gloeosporioides from strawberry and noncultivated hosts. Phytopathology 94: 446-453. Additional permission to reproduce this material came from The American Phytopathological So ciety. Chang L. Xiao and Daniel E. Legard originally conceived of the experiments presented here. Chang L. Xiao and Steven J. MacKenzie performed pathogenicity assays. Steven J. MacKenzie collected the genetic data, analyzed all data, and wrote the manuscript submitted to Phytopathology under the guidance of Daniel E. Legard. Both Daniel E. Legard and Chang L Xiao agree that it is appr opriate for this material to be included in Steven J. MacKenzies dissertation.

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42 appears as though strawberry plants with Colletotrichum crown rot symptoms are aggregated near the edge of the field adjacent to these noncultivated plants. Colletotrichum gloeosporioides has been isolated from numerous host species suggesting that it is a physiologically adapta ble fungus (69). This is supported by cross infection experiments using strains of C. gloeosporioides from different cultivated plants, the occurrence of crop pathogens on nonculti vated plants, and genetic analysis of C. gloeosporioides populations from different hosts. Colletotrichum gloeosporioides from noncultivated host species have been shown to be pathogenic on black locust (102). Isolates of C. gloeosporioides recovered from plant material of seven tropical fruits tended to be more pathogenic on leaves of th e plant species from which they are isolated, but they still produced symptoms on alternativ e host leaves and mo lecular data did not indicate that isolates from different hosts were derived from genetically distinct populations (1). Among C. gloeosporioides living as endophytes on tropical forest trees no host specificity could be detected using molecular fingerprints (60). Colletotrichum fragariae closely related to C. gloeosporioides has been reported on a noncultivated host (50) and was shown to produce disease sy mptoms on an alternative host (100). In addition to being physiologically adaptive, the C. gloeosporioides taxonomic group probably includes more than one biological species as well as clonally reproducing lineages with specific host requirements. Based upon ribosomal DNA sequence analysis, C. gloeosporioides isolates cluster into at least two distinct gr oups (55,78). In addition, RAPD, RFLP, and isozyme markers have been used to identify genetically distinct subgroups on hosts such as citrus and coffee (84,93).

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43 The goal of the study presented in this chapter was to determine whether noncultivated hosts adjacent to strawberry fields serve as po tential sources of C. gloeosporioides inoculum for infection of strawb erry crown. The specific objectives were to collect and identify isolates of Colletotrichum spp. from noncultivated plant species adjacent to strawberry fields, evaluate C. gloeosporioides isolates from noncultivated hosts for their pathogenicity on strawberry, and determine the genetic relatedness of C. gloeosporioides isolates from noncultivated plant species to isolates recovered from diseased strawberry plants. Materials and Methods Fungal Isolate Collections From 1995 to 1998, isolates of Colletotrichum spp. with morphology consistent with descriptions for C. gloeosporioides (43,81) were collected by staff at the University of Florida, Gulf Coast Research and Edu cation Center in Dover, FL from various noncultivated hosts growing adjacent to strawb erry production fields and from diseased strawberry plants in west-central Florida (Table 3-1). Isolations were made from diseased tissues, such as foliar and fruit lesi ons of noncultivated host plants and diseased crowns of strawberry plants. Diseased pl ant tissues were surface disinfested for 5 min with 0.525% sodium hypochlorite and plated onto either potato dextrose agar (PDA) amended with neomycin sulfate (20 mg/L), chloramphenicol (6.5 mg/L), tetracycline hydrochloride (25 mg/L), and erythromycin (7.5 mg/L) or a semi-selective medium for Collectotrichum (16 g of Difco potato dextrose br oth, 14 g of Difco agar, 250 mg of ampicillin, 150 mg of streptomycin sulfate, 5 mg of iprodione, 100 l of Tergitol, and 1 L deionized water). Isolation plates were incubated under c ontinuous fluorescent light at

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44 room temperature (~22C) or in an incuba tor at 24C for 3 to 7 days. Cultures of Colletotrichum spp. were single-spored and stor ed in 20% glycerol at C. A total of 53 Colletotrichum spp. isolates were obtained between 1996 and 1997 from noncultivated plants growing on land adjace nt to strawberry fields at five locations (Table 3-1). Among these sites, ther e were 16 known and two unknown noncultivated hosts from which Colletotrichum spp. were isolated. A Colletotrichum species was found growing on both foliar tissue a nd fruit of one host species, Smilax rotundifoli Isolates coming from strawberry or noncultiva ted hosts can be identified by the letter S for strawberry or NC for noncultivated at th e beginning of the isolate code (Table 3-1). Approximately half of the isolates in cluded in the noncultivated population were collected from site NC1. This site wa s sampled once in 1996 and again in 1997. The population of Colletotrichum spp. obtained from infected st rawberry crowns consisted of 42 isolates collected from 17 sites between 1995 and 1998 (Table 3-1). Site S2 was sampled in both 1995 and 1997. Approximately half of the strawberry crown rot isolates evaluated in the study were from this site. If isolates were collected from a site more than once, the site number is followed by a slash and the year of collection. For isolates from noncultivated hosts, the host species are in dicated by the letters sp followed by a number referring to the particular host spec ies (Table 3-1 lists species corresponding to the code number). Three C. gloeosporioides isolates from mango, four C. gloeosporioides isolates from citrus, and one C. acutatum isolate from citrus were used as representative outgroup populations for genetic comparisons. These isolates are believed to be from or have previously been shown to be distinct from populations on strawberry (47,92). One C. acutatum isolate from strawberry fruit wa s also included as an outgroup

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45 in the genetic analysis and used to confirm the identity of any C. acutatum isolates that may have been isolated from crown tissue. Extraction of Fungal DNA Total fungal DNA was extracted from myce lia obtained from cultures grown in 100 mL of Emerson Media (4 g/L yeast extract, 15 g/L soluble starch, 1g/L K2HPO4 and 0.5 g/L MgSO4) for 2 to 4 days at room temperature (~22 C). Mycelium was harvested from the liquid cultures by vacuum filt ration through Whatman no. 3 filter paper and transferred into a 15 mL tube. Mycelia we re then dried overni ght in a centrifugal evaporator and subsequently ground into a fine powder using a sterile glass rod. Sixty milligrams of the dried powder for each isolate was suspended in 750 l of DNA extraction buffer consisting of 700 mM Na Cl, 50 mM Tris(pH 8.0), 10 mM EDTA(pH 8.0), 1% cetyltrimethylammonium bromide, and 1% -mercaptoethanol for 2 h with periodic shaking at 65C. Particulate material was pell eted by centrifugation at 12,000 g for 10 min, the supernatent removed and extracted once with chloroform:isoamyl alcohol (24:1). Two volumes of 100% ethanol were added to the aqueous extract and the mixture incubated at room temperature for 10 min. Nucleic acids were pelleted from the ethanol solution by centrifugation at 12,000 g for 10 min. The pellet was washed with 100% ethanol and suspended in 400 l 1 TE buffer containing 10 g/mL RNase for 1 h at 37C. Ribonuclease was removed from th e nucleic acid solution by extraction with 400 l phenol/chloroform/isoamyl alcohol (25: 24:1). One-tenth volume 3 M sodium acetate and 2.5 volumes of ethanol were added to the aqueous extract to precipitate the DNA. This solution was incubated at C for 1 h and the DNA was pelleted at

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46 12,000 g for 10 min. The DNA pellet was washed once with 1 mL 80% ethanol, dried, suspended in 50 to 500 l 1 TE buffer, and stored at C. Species Identification A species-specific internal transcribe d spacer region 1 (ITS1) primer and the conserved universal primer ITS4 (5-T CCTCCGCTTATTGATATGC-3) encoded in the 28S ribosomal subunit were used in pairs to iden tify isolates to species (101). The ITS1 primers used were either the C. gloeosporioides specific ITS primer 5-GACCCTCCCGGCCTCCCGCC-3 or the C. acutatum specific ITS primer 5-GGGGAAGCCTCTCGCGG-3 (83,85) Isolates where assi gned to the species group for which a positive amplification with a specific ITS1 primer was obtained. Amplifications were carried out under mineral oil in a 20l volume containing 1 reaction buffer (10 mM Tris(pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 0.001% Gelatin), 200 M dNTP, 1 unit Taq polymerase and 10 mol of each primer/reaction. The reaction buffer for the C. acutatum specific primer also contained 5% glycerol. Temperature cycling parameters for the C. gloeosporioides specific/ITS4 pair c onsisted of a denaturing step for 5 min at 94C, followed by 26 cycl es at 94C for 1 min, 60C for 2 min, and 72C for 2 min. Temperature cycling parameters for the C. acutatum specific/ITS4 pair consisted of a denaturing step for 5 min at 94C followed by 32 cycles at 94C for 1 min, 60C for 2 min, and 72C for 2 min. Th e amplified products were separated by electrophoresis through a 2% agarose gel c ontaining 1 TAE buffer. Gels were photographed on a UV transilluminator after ethidium bromide staining.

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47 Pathogenicity Tests Pathogenicity of the 53 Colletotrichum isolates recovered from noncultivated plants was evaluated on the susceptible strawberry cv. Camarosa in a greenhouse. Conidial suspensions were made from 7to 10day-old PDA cultures grown at 24C under continuous fluorescent light and adjusted to 1 106 conidia per mL in sterile deionized water. Infections were begun by injecting ap proximately 100 l of a conidial suspension into the crown of mature transplants with a 25G1 syringe needle. Strawberry plants were evaluated weekly for the development of Colletotrichum crown rot symptoms (i.e., wilting and collapse of plant). An isolate was considered to be pathogenic to strawberry if at least 2 of 3 inoculated plants collapse d within the 4 weeks af ter inoculation. Each isolate was tested at least tw ice in separate inoculation ex periments. In addition, ten, three, and four isolates of C. gloeosporioides recovered from diseas ed strawberry plants, mango, and citrus, respectively, we re also tested for pathogeni city on strawberry in the same manner as described above. Randomly Amplified Polymorphic DNA Markers Five primers, including two tetr anucleotide repeat primers (ACTG)4 and (GACA)4, the trinucleotide repeat primer (TCC)5, and two short oligonucleotides 5-GTGAGGCGTC-3 (OPC-2) and 5 -GATGACCGCC-3 (OPC-5) (Operon Technologies, Alameda, CA), were selected for the population studies based on their ability to consistently amplif y bands that demonstrated a high level of fluorescence under UV light. DNA amplifications were car ried out under mineral oil in a 20 l volume containing 1 reaction buffe r [50 mM Tris (pH 8.3), 0.25 mg/mL BSA, 2 mM MgCl2, 0.5% Ficoll, and 1 mM Tartrazine], 200 M dNTP, 1 unit Taq polymerase, and 20 mol

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48 primer/reaction [primers (ACTG)4, (GACA)4, and (TCC)5] or 8 mol primer/reaction (primers OPC-2 and OPC-5). Cycling paramete rs for the PCR reactions consisted of a 5 min denaturing step at 95C followed by 30 cycl es of 1 min at 95C, 1 min at 48C, and 2 min at 72C for primers (ACTG)4 and (GACA)4; 34 cycles of 1 min at 95C, 1 min at 46C, and 1.5 min at 72oC for primer (TCC)5 or 38 cycles of 1 min at 95C, 1 min at 35C, and 2 min at 72C for primers OPC-2 and OPC-5. The amplified products were separated by electrophoresis through a 1.5% high resoluti on blend agarose (3:1) gel containing 1 TAE buffer. Gels were photographed on a UV transilluminator after ethidium bromide staining. Statistical Analyses The SPSS 8.0 statistics package (SPSS Inc., Chicago, IL) was used to perform Fishers exact tests to determine the associa tion between the pathogeni city of isolates and the site or noncultivated host species from which the isolates were recovered. The genetic relationship of isolates to one another was summarized in a phenogram constructed from dice similarity coeffici ents using the unweighted pair group method with arithmetic averages (U PGMA) clustering algorithm (NTSYS, PC version 2.0, Exeter software, Setauket, NY). Statistical support for branches was based on 1,000 bootstrapped samples using Winboot (35,71). Th e probability of obtaining identical genotypes among strains in the sample populat ion assuming a random distribution of alleles was determined using a custom written QBASIC program which shuffles alleles among strains at each locus to mimic recomb ination and subsequently determines the frequency of the most common genotype obser ved in the shuffled data set (86,89). Probabilities of obtaining cl one frequencies were based on analysis of 10,000 randomized

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49 data sets. Population differentiation was examined using an ex act test for population differentiation at each locus and Fishers combin ed probability test to obtain a probability estimate over all loci (75). Tests for populat ion differentiation were calculated using the software program TFPGA (Utah St ate University, Logan). Results Identification of Isolates Of the 53 isolates recovered from noncultivated hosts, 52 produced a characteristic PCR product when the C. gloeosporioides specific primer was used and they were therefore identified as C. gloeosporioides One isolate produced an amplification product when the C. acutatum species specific primer was used (Table 3-1). The C. acutatum isolate obtained from the noncultivated host came from the fruit of Callicarpa americana It was genetically distinct from any of the C. acutatum isolates from strawberry (Fig. 3-1) and did not produ ce either crown rot (T able 3-2) or fruit lesions when inoculated on strawberry (data not shown). Of the 42 isolates recovered from diseased strawberry cr owns, 39 were identified as C. gloeosporioides and three as C. acutatum The species identities of Colletotrichum isolates from mango and citrus (Table 3-1) were also confirmed with C. gloeosporioides or C. acutatum specific primers. Pathogenicity Tests Of the 52 C. gloeosporioides isolates from noncultivated hosts, 18 produced typical symptoms of Colletotrichum crown rot (i.e. w ilting and collapse of plants) on inoculated plants. Therefore, these isolates were consider ed to be pathogenic to strawberry crowns. The 18 pathogenic isolates were recovered fr om nine different nonc ultivated host species from three separate sites (Tables 3-2 and 3-3). There was a significant association between the pathogenicity of the isolates a nd the site from which the isolates were

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50 collected (Table 3-2, Fishers exact test P = 0.02). Of the 18 pathogenic isolates, 16 came from a single site (NC1). Half of the pathogenic isolates from this site were collected in 1996 and the other half in 1997. There was no significant association between the pathogenicity of isolates and the noncultivated host species from which the isolates were recovered (Tab le 3-3, Fishers exact test P = 0.23). Of ten isolates that were recovered from diseased strawb erry plants, nine produced typical crown rot symptoms on inoculated stra wberry plants. Two of the three isolates from mango and none of the four citrus is olates were pathogenic to strawberry. Randomly Amplified Polymorphic DNA Analyses RAPD amplifications using five primers yi elded 60 scorable bands from all isolates examined. Forty-one scorable bands were amplified from C. gloeosporioides isolates recovered from strawberry and noncultivated ho sts. When isolates from citrus and mango were included, 44 scorable bands were obtained. Thirty scorable bands were amplified from C. acutatum isolates. Isolates identified as C. gloeosporioides grouped into three clusters with between cluster simila rities less than 0.50 (F ig. 3-1). All of the C. gloeosporioides isolates from citrus had a level of similarity to one another greater than 0.75 and formed a cluster in 93% of boot strapped phenograms. A second cluster consisted of two clonal isolates from different noncultivated host species at site NC3 and occurred in 100% of bootstrapped trees. The two isolates in this cluster were homothallic (data not shown). Self-fert ility was not observed for any other isolates, although it is possible that low numbers of fertile perithecia may have been overlooked. A third cluster contained C. gloeosporioides from noncultivated hosts, strawberry and mango. This cluster occurred in onl y 41% of bootstrapped trees, giving weak

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51 support for a monophyletic origin of this cluste r. A large amount of the genetic variation within this cluster can be at tributed to the mango isolates. The three mango isolates were distinct from most strawbe rry and noncultivated host isolat es based on Dice similarity coefficients, but isolates from this host di d not compromise a gr oup of closely related organisms. When mango isolates are exclude d from the analysis the cluster containing strawberry and nonhomothallic, noncultivated host isolates occurred in 59% of bootstrapped samples. Bootstrapping did not provide support for strawberry isolates forming an evolutionary lineage distinct fr om noncultivated isolates, as strawberry isolates were interspersed among these isolates in the phenogram. However, clustering did occur among subsets of strawberry and noncultivated host isolates. Severa l of these clusters were supported by relatively high bootstrap values (Fig. 3-1), although the bootstrap values may not be reliable as there are few in formative sites separating the clusters from other isolates (46). Seven clusters of nonhomothallic isol ates contained two or more clonal individuals based on RAPD profiles. Of these seven clonal lineages, two consisted of isolates from strawberry and five containe d isolates from noncul tivated hosts. Two of the clonal groups contained three or more is olates. One clonal genotype on strawberry was found at two sites (four isolates at site S2/97; two isolates at S14). Of the five clonal genotypes found on noncultivated hosts, four cont ained only isolates from the same field and in only one of these fields were all of the isolates from the same host species. Pathogenicity phenotypes were the same am ong all individuals possessing identical genotypes. Based on probabilities obtained by re peated shuffling of the data set, two or more individuals having the same genotype occurred in 4.39% of the randomized data

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52 sets and three or more individuals with th e same genotype did not occur in any of the 10,000 randomized data sets examined. Allele frequencies were significantly ( P < 0.05) different between strawberry isolates and nonhomothallic isolates from nonc ultivated hosts at only four of 36 (11 %) polymorphic loci based on exact tests with the level of type I error, equal to 0.05 (Table 3-4). A test for population differentia tion combining all polymorphic loci was not significant ( P = 0.29). The 1.6 kb band amplified with primer OPC-5, one of four loci displaying allele frequency differences between these isolates, also displayed frequency differences between pathogenic and nonpathoge nic isolates when only isolates from noncultivated hosts were compared (freque ncy = 0.50 among pathogens and 0.06 among nonpathogens). There were no significant di fferences in allele frequencies between pathogenic and nonpathogenic isolates from nonc ultivated hosts at th e three other loci. Discussion In this study we found that a pproximately onethird of the C. gloeosporioides isolates recovered from noncultivated hosts grown in the areas adjacent to strawberry fields were pathogenic to strawberry in greenhouse tests. Phylogenetic analysis of RAPD data and tests for genetic differentiation between C. gloeosporioides from noncultivated hosts and those from diseased strawberry crowns sugge st that they were from a single population. These results indicate that nonc ultivated hosts growing adjacent to strawberry fields may serve as a source of inoculum for epid emics of strawberry crown rot caused by C. gloeosporioides. Isolates of C. gloeosporioides from a wide range of te mperate, subtropical, and tropical fruits have shown cross infection pot ential (1,40). However, pathogenicity tests

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53 in artificial inoculation expe riments are not conclusive ev idence to support that cross infection occurs under natural conditions. Fo r this reason, in addi tion to pathogenicity tests, the genetic relationship between isolates from strawberry and different noncultivated hosts was determined in the present study. Based upon bootstrap analysis of a phenogram constructed using RAPD markers, isolates of C. gloeosporioides from noncultivated hosts fell into two genetically distinct populations. The largest population consisted of 50 isolates and the smalle r population consisted of two genotypically identical isolates. The popul ations from noncultivated hosts also could be distinguished from one another based upon the presence of homothallism in isolates from the smaller population. Homothallic isolates of C. gloeosporioides from strawberry appear to be genetically distinct from in terbreeding heterothallic isol ates (38,43). However, the homothallic isolates from stra wberry do not appear to be re lated to those isolated from noncultivated hosts (data not shown). Isolates of C. gloeosporioides from strawberry crowns ha d a high level of diversity and were not genetically distin ct from isolates from noncultivated hosts that were not homothallic in culture when all polymorphic loci were included in the analysis. Tests for differences in allele frequencies at single loci revealed a 1.6-kb OPC-5 amplification product that occurred at a higher frequency in the strawberry populat ion relative to the noncultivated host population and also occurr ed at a higher frequency among pathogenic isolates from the noncultivated host population re lative to nonpathogenic isolates. Allele frequencies from this locus do not provide strong evidence fo r population subdivision, given that the frequency of the vast majority of allelic marker s, assumed to be neutral, are not different between populations. The pos itive correlation between the 1.6-kb OPC-5

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54 product and pathogenicity on strawberry does, ho wever, suggest that it may be linked to a genetic factor conferring pathogenicity on st rawberry. In the previous chapter, this marker also displayed highly skewed nonme ndelian segregation in a cross between a pathogenic and nonpathogenic isolate. Although isolates with the same genotype occurred on either strawberry or noncultivated hosts, no identical genot ypes were found on both strawberry and noncultivated hosts. Assuming a randomly mating population, the occurrence of even two isolates with identical ge notypes would be a relatively rare event given the sample size and polymorphic loci examined in this study. In total, there were seven genotypes detected more than once in isolates from the nonhomothallic strawberry and noncultivated host population. This overrepr esentation of specific genotypes is consistent with the important role of clonal reproduction in this sp ecies (33). Genetic bottlenecks created by recent co lonizing events in spatially subdivided populations can also result in overrepresentation of genotypes at specific sites. However, this is unlikely as the sites from where clonal genotypes were collected cont ained a substantial amount of genetic diversity. Genetically isolated pa thogen subpopulations can also arise relatively rapidly from interbreeding fungal populations due to ase xual reproduction and may serve as a mechanism to preserve particularly virule nt gene combinations on specific hosts (8). The data provided from this study does not support the hypothesis that this has occurred with C. gloeosporioides from either strawberry or noncul tivated hosts as clonal isolates tended to occur at specific sites and not on specific hosts. There was, however, one genotype that was isolated from six different strawberry crowns at two separate sites (S2/97 and S14). This was the most co mmon genotype observed and suggests that

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55 selection may be preserving some combinations of genes for pathogencity on strawberry. Alternatively, the occurrence of this genotype may result from selection for a clone at a nursery supplying transplants to farms in the area investigated. Although a defined group of species harboring pathogeni c isolates could not be identified among the nonpathogenic hosts sampled, there was a strong correlation between sampling site and pathogenicity on strawberry. Because not all of the host species were present at each sample site, there may be some bias in the tests for association. This bias might make it difficu lt to discern whether or not isolates from particular hosts or particular sites differed in pathogenici ty. However, eleven of 19 isolates from site NC1 that had hosts id entical to those found at sites NC2, NC3, NC4 and NC5 were pathogenic to strawberry, wh ereas only two of the 22 isolates from the four other sites were pathogenic on strawberr y. This result indicates that the analyses were correct in that pathogeni city correlated primarily with the site from which isolates were recovered and not the host species from which they were isolated. Variation in levels of pathogenicity among noncultivated host sampling sites may result from different levels of migration from strawberry fields, where selection for virulence on strawberry would likely occur. Three C. gloeosporioides isolates from mango and four from citrus were included in the phenogram (Fig. 3-1). The mango isolates examined in this study did not appear to form a subgroup genetically distinct from C. gloeosporioides isolated from strawberry or noncultivated hosts. These findings we re not expected because a study of C. gloeosporioides isolates from mangos at locations around the world found the isolates to be relatively homogeneous and genetically dis tinct from those recovered from other fruit

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56 species (47). However, that study only in cluded one mango isolate from Florida. The Florida isolate had a slightly smaller rDNA size compared to the isolates from other sites. Repeated sampling of C. gloeosporioides from mango in Sri Lanka also revealed a greater amount of diversity in rDNA a nd mtDNA restriction fragment length polymorphisms than was previously thought to exist in that populat ion (2). Also of interest is that the two mango isolates pathogenic on strawberry in greenhouse inoculation tests were more clos ely related to strawberry isolates than they were to the third mango isolate that was not pathogenic on strawberry. The citrus isolates used in this study were previously demonstrated to be genetically distinct from strawberry isolates (92). Whether or not this genetic divergence is due to geographic isolation or sexual incompatibility was not examined, but te st crosses of citrus isolates to apple reference strains have been unsuccessful (23). A C. acutatum isolate was also obtained from a noncultivated host in this study. The isolate was not pathogenic on strawberry and was genetically distinct from strawberry crown isolates, indicat ing this strain of C. acutatum is not responsible for diseases on strawberry.

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57Table 3-1. Collection sites and host species for Colletotrichum spp. Species, collection sitea Host (species code)b Common name Number of isolates Colletotrichum gloeosporioides NC1/96 Quercus spp. (sp12) Oak 2 Smilax rotundifolia (sp14) Smilax 1 S. rotundifolia, fruit (sp14B) Smilax berry 3 Vitis rotundifolia (sp16) Wild grape 8 NC1/97 Callicarpa americana, fruit (sp3) Beauty berry 1 Dioscorea bulbifera (sp4) Air potato 4 Ipomoea spp. (sp5) Morning glory 1 Liquidambar styraciflua (sp6) Sweet gum 2 Myrica cerifera L. (sp10) Wax myrtle 2 Parthenocissus quinquefolia (sp11) Virginia creeper 3 Quercus spp (sp12) Oak 1 Smilax rotundifolia fruit (sp14B) Smilax berry 1 Urena lobata (sp15) Caesar weed 1 NC2 Momordica charantia L. (sp9) Balsamapple 2 Richardia brasiliensi (sp13) Brazilian pusley 1 NC3 Magnolia virginiana L. (sp7) Magnolia, sweet bay 2 Myrica cerifera L. (sp10) Wax myrtle 1 Quercus spp. (sp12) Oak 1 S. rotundifolia (sp14) Smilax 2 S. rotundifolia fruit (sp14B) Smilax berry 1 V. rotundifolia (sp16) Wild grape 1 Unknown species (sp17) 2 NC4 V. rotundifolia (sp16) Wild grape 1 NC5 Asclepias spp. (sp1) Milkweed 2 aSites beginning with NC are areas with noncultivated plants ad jacent to strawberry fields and sites beginning with S are strawberry fields. Numbers followed by / represent year sa mple was collected at sites sampled more than once. bSpecies codes given in parentheses are combined with site numbers to identify the sample location and host for isolates displayed in Figure 3-1.

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58Table 3-1. Continued Species, collection sitea Host (species code)b Common name Number of isolates Bidens bipinnata (sp2) Bidens 1 C. americana -fruit (sp3) Beauty berry 1 Melia australis Sweet (sp8) Chinaberry 2 S. rotundifolia (sp14) Smilax 2 S1 Fragariae ananassa Strawberry crown 1 S2/95 Fragariae ananassa Strawberry crown 11 S2/97 Fragariae ananassa Strawberry crown 9 S3 Fragariae ananassa Strawberry crown 1 S4 Fragariae ananassa Strawberry crown 1 S5 Fragariae ananassa Strawberry crown 1 S6 Fragariae ananassa Strawberry crown 1 S7 Fragariae ananassa Strawberry crown 1 S8 Fragariae ananassa Strawberry crown 1 S9 Fragariae ananassa Strawberry crown 1 S10 Fragariae ananassa Strawberry crown 2 S11 Fragariae ananassa Strawberry crown 2 S12 Fragariae ananassa Strawberry crown 1 S13 Fragariae ananassa Strawberry crown 1 S14 Fragariae ananassa Strawberry crown 3 S15 Fragariae ananassa Strawberry crown 2 Lake Alfred, FL Citrus spp. Citrus 4 Homestead, FL Mangifera indica Mango 3 Colletotrichum acutatum NC5 Callicarpa americana fruit (sp3) Beauty berry 1 S9 Fragariae ananassa Strawberry crown 1 S16 Fragariae ananassa Strawberry crown 1 S17 Fragariae ananassa Strawberry crown 1

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59 Table 3-1. Continued Species, collection sitea Host (species code)b Common name Number of isolates Dover, FL Fragariae ananassa Strawberry Fruit 1 Lake Alfred, FL Citrus spp. Citrus 1

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60 Table 3-2. Isolates of Colletotrichum spp. collected from noncultivated hosts summarized according to site and pa thogenicity on strawberry plants Number of isolates Species, collection sitea Pathogenic Nonpathogenic Total Colletotrichum gloeosporioides NC1/96 8 6 14 NC1/97 8 8 16 NC2 1 2 3 NC3 1 9 10 NC4 0 1 1 NC5 0 8 8 All Sites 18 34 52 C. acutatum NC5 0 1 1 a Sites beginning with NC are areas with nonc ultivated plants in close proximity to strawberry fields in west-central Florid a. There was a significant association ( P = 0.02) between the pathogenicity of the C. gloeosporioides isolates and the site from which the isolates were collected, based on a Fishers exact test ( P = 0.05).

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61 Table 3-3. Isolates of Colletotrichum spp. collected from noncultivated hosts summarized according to host species and pathogenicity on strawberry plants Number of isolates Fungal species, host speciesa Pathogenic Nonpathogenic Total Colletotrichum gloeosporioides Asclepias spp (sp1) 0 2 2 Bidens bipinnata (sp2) 0 1 1 Callicarpa americana, fruit (sp3) 0 2 2 Dioscorea bulbifera (sp4) 2 2 4 Ipomoea spp. (sp5) 0 1 1 Liquidambar styraciflua (sp6) 0 2 2 Magnolia virginiana L. (sp7) 1 1 2 Melia australis Sweet (sp8) 0 2 2 Momordica charantia L. (sp9) 0 2 2 Myrica cerifera L. (sp10) 2 1 3 Parthenocissus quinquefolia (sp11) 2 1 3 Quercus spp (sp12) 2 2 4 Richardia brasiliensi (sp13) 1 0 1 Smilax rotundifolia (sp14) 0 5 5 S. rotundifolia fruit (sp14B) 1 4 5 Urena lobata (sp15) 1 0 1 Vitis rotundifolia (sp16) 6 4 10 Unknown species (sp17) 0 2 2 All host species combined 18 34 52 C. acutatum Callicarpa americana fruit (sp3) 0 1 1 aThere was no significant association ( P = 0.23) between the pathogenicity of the C. gloeosporioides isolates and the host species from which the isolates were collected, based on a Fishers exact test ( P = 0.05). Species codes given in parentheses are combined with site numbers to identify the sample location and host for isolates displayed in Figure 3-1.

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62 Table 3-4. Frequencie s of RAPD bands for Colletotrichum gloeosporioides isolates from strawberry and noncultivated hosts Host Noncultivated Primer, length Strawberry Nonhomothallic Homothallic (kb) ( n =39)a ( n =50) ( n =2) (ACTG)4 2.145 0.49 0.52 0.00 1.9 0.97 1.00 0.00 1.55 0.00 0.00 1.00 1.5 1.00 1.00 0.00 1.12 0.85 0.84 0.00 0.6 0.92 0.84 1.00 0.4 0.33 0.38 1.00 (GACA)4 1.5 0.05 0.02 0.00 1.35 0.95 0.96 0.00 1.3 0.00 0.00 1.00 1.2 0.97 0.96 0.00 1.15 0.03 0.04 0.00 0.95 0.69 0.74 0.00 0.9 0.46 0.38 1.00 0.8 0.13 0.30 0.00 0.75 0.00 0.02 0.00 0.5 1.00 0.96 0.00 (TCC)5 2 0.03 0.10 0.00 1.9 0.62 0.62 1.00 1.55 0.23 0.28 0.00 1.15 1.00 0.98 1.00 0.9 0.03 0.02 0.00 0.75 0.13 0.22 0.00 aExact test for population differentiation between strawberry and noncultivated (nonhomothallic) host isolates over all loci was not significant ( P =0.29). bExact test for population differentiation between strawberry and noncultivated (nonhomothallic) host isolates was significant at specified loci ( P < 0.05). cExact test for population differentiati on between pathogenic and nonpathogenic, noncultivated (nonhomothallic) host isolates was significant at specified loci ( P < 0.05).

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63 Table 3-4. Continued Host Noncultivated Primer, length Strawberry Nonhomothallic Homothallic (kb) ( n =39) ( n =50) ( n =2) OPC-2 2.2 0.08 0.06 0.00 1.9 0.62 0.64 0.00 1.7 0.03 0.00 0.00 1.2 0.08 0.02 0.00 1.1 1.00 1.00 1.00 0.5 0.03 0.00 0.00 OPC-5 2.7 0.18 0.34 0.00 2.6 0.00 0.00 1.00 2.5 0.21 0.44b 0.00 2.15 0.59 0.82b 1.00 2 0.21 0.22 0.00 1.8 0.18 0.16 1.00 1.75 0.08 0.08 0.00 1.65 0.10 0.30b 0.00 1.6 0.74 0.22bc 1.00 1.55 0.33 0.32 0.00 1.4 0.03 0.06 0.00 1.2 0.38 0.22 0.00

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64 Fig. 3-1. A phenogram using unweighted pair gr oup method with arithmetric averages showing similarity (Dice) between Colletotrichum gloeosporioides and C. acutatum isolates from noncultivated plants, strawber ry crowns, citrus, and mango. Numbers at nodes are the percentage of occurrence of the cluster to the right of the branch in 1,000 bootstrapped samples. All bootstrapped values ar e reported for clusters that are less than 0.50 similar to other isolates or clusters. For all other clusters, only bootstrapped values greater than 50 are reported. Nonstrawberry C. gloeosporioides isolates with asterisks (**) indicate they were pathogenic on stra wberry in greenhouse inoculation tests. Isolates are identified in Table 3-1.

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65 CHAPTER 4 SELECTION FOR PATHOGENICITY TO STRAWBERRY IN POPULATIONS OF Colletotrichum gloeosporioides FROM NATIVE PLANTS Introduction Colletotrichum crown rot of strawberry cau ses wilting and collapse of strawberry plants in production fields a nd nurseries in Florida. Currently most of the transplants used for the annual winter production season ar e propagated in nurseries located in the northern United States or provinc es of Canada. This has gr eatly decreased the incidence of crown rot in production fields, although mode rate plant losses still occur. At the present time, the vast majority of Colletotrichum species isolated from diseased strawberry crowns in Florida are from a nonhomothallic C. gloeosporioides population. This population is genetically diverse and reco mbination appears to occur at a relatively high frequency (92). As shown in chapter 3, C. gloeosporioides can be isolated from foliar lesions of a broad range of introduced and native noncultivated plant species growing adjacent to strawberry fields as well as from strawberry petioles where it forms latent infections (66). Isolates from these s ources vary in their abi lity to cause crown rot with some being aggressive pathogens on stra wberry and others lacking the ability to produce crown rot symptoms. Also from resu lts reported in chapter 3, a large proportion of the C. gloeosporioides isolate s on noncultivated hosts appear to be from the same population as those isolated from diseas ed strawberry crowns, indicating that noncultivated hosts may provide inoculum for crown rot epidemics. However, populations of C. gloeosporioides from noncultivated hosts at sites distant from strawberry production areas have not been st udied. Wild strawbe rry is not known to

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66 occur in the subtropical regions of Florid a where commercial stra wberries are produced (26). Thus, sampling of C. gloeosporioides at sites away from strawberry fields can provide information as to whether isolates on noncultivated hosts are recent migrants from strawberry or if there is an indigenous population of C. gloeosporioides on noncultivated hosts and stra wberry. In addition, the C. gloeosporioides /strawberry pathosystem provides a unique system for examination of local selection for pathogenicity. In Florida, strawberry produc tion is highly centralized. Mo re than 90% of the area dedicated to strawberry production is locat ed within Hillsborough County, with most plantings concentrated in the Dove r/Plant City area (5). If the C. gloeosporioides population on strawberry is derived from a wide ly dispersed population with a wide host range, the cultivation of strawberry in a particular area may influence the frequency of the pathogenic phenotype in the C. gloeosporioides population on noncultivated hosts. Studies examining mean host resistance a nd mean pathogen virulence have shown a positive correlation between these two traits in both natural and agricultural pathosystems (64,88), suggesting directional selection in the pathogen population for increased infectivity. Analogously, genes controlling path ogenicity on strawberry may be selected in C. gloeosporioides populations at sites where strawb erries are grown extensively. In this study we genetically characterized C. gloeosporioides isolates from two native hosts, oak ( Quercus spp.) and wild grape ( Vitis spp.), at four locations. Two locations were immediately adj acent to strawberry fields and two sites were distant from any commercial strawberry production. Sampli ng at all sites was conducted at least four months after strawberry plants had been rem oved from fields. The genetic data was used

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67 to identify a population to te st the hypothesis that ther e is local selection for pathogenicity to strawberry in areas wher e strawberries are grown extensively. Materials and Methods Sampling Strategy and Isolate Codes In Florida, strawberries are grown as an annual crop on raised, plastic mulched beds. Plants are set in late September and harvested from November to late March. At the end of the harvest season, plants are chem ically destroyed and the field tilled or a different crop planted on the mulched beds. Sampling was done in late August and early September 2002, 4 to 5 months after plants we re removed from fiel ds, to avoid sampling migrants from strawberry fiel ds that may have only transi ently established themselves on native hosts. At least 20 isolates identified as C. gloeosporioides based on morphology (43) were obtained from necrotic lesions on oak ( Quercus spp.) or wild grape ( Vitis spp.) leaves at each of four sites (Fig. 4-1). Samp les were taken from leaves of different trees and vines at each site to reduce the possibi lity of sampling clones. Two sites were adjacent to commercial strawberry fields in Dover, FL. These sites were 3.5 km apart within the strawberry production region in Hillsborough County. The native hosts sampled at these sites were located 9 to 50 m from the edges of fields. Two sites distant from strawberry production fields were sample d: University of Florida, Citrus Research and Education Center in Lake Alfred, FL and a residential area in Sarasota, FL. Both sites contained natural stands of vegetation along edges of ro ads or citrus groves and in preserved areas. The Lake Alfred and Saraso ta sites are located in Polk and Sarasota counties respectively. The combined acreage used for strawberry production in these counties is less than 2% of the acreage used in Hillsborough County. The nearest commercial strawberry farm was approximately 28 km from the Lake Alfred site and 15

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68 km from the Sarasota site. Native host isolates from the two sites in Dover were designated D1-oak, D1-grape, D2-oak, and D2-g rape. Those from Lake Alfred were designated LA-grape, LA-oak, and those from Sarasota labeled SS-grape and SS-oak. The site D2 was also sampled in the study pr esented in chapter 3, wh ere it was designated NS1. Site D1 had not been sampled previously. Twenty C. gloeosporioides isolates from diseased strawberry crowns seven citrus isolates, and one C. fragariae isolate from crown tissue were also included in the analys is. Strawberry isolates came from samples submitted by local growers to the diagnostic labora tory at the University of Florida, Gulf Coast Research and Education Center in D over, Florida from 1995 to 2000 and are coded strawberry. Citrus isolates came from sweet orange or tangelo fru it, twigs, or leaves from plantings near Lake Alfred, Avon Park, Vero Beach, or Frostproof, Florida. These isolates are coded citrus. C. gloeosporioides isolates from citrus have been shown to be distinct from those on strawberry (92). Fungal Isolation Isolates from strawberry were obtained by placing tissue from necrotic crowns of wilted strawberry plants directly onto CIM media (CIM 12 g potato dextrose broth, 17 g agar, 100 mg streptomycin, 250 mg ampicilli n, and 8 mg iprodione per L plus 0.02 % tergitol). For isolations from native hosts, por tions of oak and grape leaves with one or more circular necrotic lesions were surf ace sterilized with 0.525 % sodium hypochlorite for 1 min, rinsed in sterile water, and placed on CIM media. Plates were incubated under fluorescent light for 3 to 5 days and single-spore isolates ma de from growing colonies. Cultures were stored at C in 20% glycerol.

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69 DNA Extraction and PCR Amplifications Two to 3 days after seeding with an agar plug, mycelium was harvested from 50mL liquid shake cultures (Emerson media 4 g yeast extract, 15 g soluble starch, 1 g K2HPO4, and 0.5 g MgSO4H2O per L) and dried overnight in a centrifugal evaporator. DNA was isolated from 60 mg of the dried mycelia. Mycelia powder was suspended in 750 l of DNA extraction buffer consisting of 700 mM NaCl, 50 mM Tris(pH 8.0), 10 mM EDTA(pH 8.0), 1% cetyltrimet hylammonium bromide, and 1% -mercaptoethanol for 2 h with periodic shaking at 65C. Partic ulate material was pell eted by centrifugation at 12,000 g for 10 min, the supernatent re moved and extracted once with chloroform:isoamyl alcohol (24:1). Two vol umes of 100% ethanol were added to the aqueous extract and the mixture incubated at room temperature for 10 min. Nucleic acids were pelleted from the ethanol solution by centrifugation at 12,000 g for 10 min. The pellet was washed with 100% ethanol and suspended in 400 l 1 TE buffer containing 10 g/mL RNase for 1 hour at 37C. Ribonucle ase was removed from the nucleic acid solution by extraction with 400 l phenol/chloroform/isoamyl alcohol (25:24:1). To the aqueous extract, 1/10 volume 3 M sodium acet ate and 2.5 volumes of ethanol were added to precipitate the DNA. This solution wa s incubated at C for 1 h and the DNA was pelleted at 12,000 g for 10 min. The DNA pellet was washed once with 1 mL 80% ethanol, dried, suspended in 50 to 500 l 1 TE buffer, and stored at C. Isolates were identified to species using a C. gloeosporioides/C. fragariae specific ITS1 primer (5-GACCCTCCCGGCCTCCCGCC-3 ) and a conserved universal primer encoded within the 28S ribosomal subun it (5-TCCTCCGCTTATTGATATGC-3). This primer set amplifies the ITS regi on from subpopulations within the C. gloeosporioides

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70 species complex as well as that of C. fragariae (83,92). Amplifications from 5 ng of template DNA were carried out under mineral oil in 20 l containing 1 reaction buffer (10 mM Tris [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, and 0.001% gelatin), 200 M dNTP, 1 unit of Taq polymerase, and 10 mol of each primer/reaction. Cycling parameters consisted of a 5-min denaturing step at 94 C followed by 26 cycles at 94 C for 1 min, 60 C for 2 min, and 72 C for 2 min. RAPD -PCR using four primers (ACTG)4, (GACA)4, (TCC)5, and 5-GTGAGGCGTC-3 (OPC-2) (Operon Technologies, Alameda, CA) was used to differentiate C. fragariae from C. gloeosporioides and to identify subpopulations within isol ates identified as C. gloeosporioides Reactions were carried out using 5 ng of template DNA under mineral oil in 20 l containing 1 reaction buffer (50 mM Tris [pH 8.3], 0.25 mg/mL bovine serum albumin, 2 mM MgCl2, 0.5% Ficoll, and 1 mM Tartrazine; Idaho Technology), 200 M dNTP, 1 unit of Taq polymerase, and 20 mol primer/reaction (primers (ACTG)4, (GACA)4, and (TCC)5) or 8 mol OPC2/reaction. Cycling parameters consis ted of a 5-min denaturing step at 95 C, 30 cycles of 1 min at 95 C, 1 min at 48 C, and 2 min at 72 C for primers (ACTG)4 and (GACA)4; 34 cycles of 1 min at 95 C, 1 min at 46 C, and 1.5 min at 72 C for primer (TCC)5 or 38 cycles of 1 min at 95 C, 1 min at 35 C, and 2 min at 72 C for primer OPC-2. Amplified products were separated in 2% agarose gels made with 1 TAE. Samples were randomized before processing and two separate reactions performed for each primer/template combination. Bands were sc ored manually. Bands that were weak and those that failed to consistent ly amplify from the same template were excluded from the analysis.

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71 Pathogenicity Tests Conidia from 7 to 10-day old grape and oak C. gloeosporioides cultures grown on PDA were suspended in sterile deionized wa ter, passed through f our layers of cheese cloth, and adjusted to 106 conidia/mL. Approximately 0.1 mL of this solution was injected directly into the cr own of three Camarosa stra wberry plants in a greenhouse during the summer of 2003. Under the warm environmental conditions used for the assay, pathogenicity could be determined unambiguously since no is olate caused collapse of an intermediate number of plants. Is olates were categorized as pathogenic on strawberry if they caused collapse of 3 of 3 plants within 4 weeks and nonpathogenic if they failed to affect any of the 3 plants i noculated. The repeatability of the assay was determined from inoculations on subgroups of isolates done during different time periods between the winter of 2002 and the fall of 2004. Based on theses data only one of 89 isolates would have been classifi ed differently (data not shown). The validity of the assay was confir med by topically applying 1 mL of 106/mL conidia suspension to crowns in ten-plant plots of the cultivar Camarosa under field conditions. Twelve plots were treated with oak or grape isolates determined to be pathogenic in greenhouse tests and twelve plots treated with isolates determined to be nonpathogenic in greenhouse tests. As a positive c ontrol, four plots were inoculated with isolates from diseased strawberry crowns a nd four plots were inoc ulated with distilled water as a negative control. Transplant s were set on October 24, 2003 on plastic mulch covered beds and inoculati ons conducted on March 12, 2004. Captan 80WP (4.2 kg per ha) was applied to plants weekly, with applic ations suspended 2 weeks before plants were inoculated and subsequently resumed 2 week s following inoculations. The proportion of

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72 plants collapsed was recorded for each plot 45 days after in oculation, just before water and nutrient supplies to plants under cultiv ation were discontinued for the season. Statistical Analyses Cluster analysis was performed on Dice similarity coefficients using the unweighted pair group method with arithme tic averages (UPGMA) with NTSYS, PC version 2.0 (Exeter Software, Setauket, NY). All scored bands were used for this analysis. Statistical sup port for phenogram branches was based on 1,000 replications using the Winboot bootstrap algorith m (71). Values of theta () were estimated using the method of Weir and Cockerham (99), w ith confidence inte rvals generated by bootstrapping over loci using TFPGA (Utah Stat e University, Logan). Statistical support for population subdivision was concluded if the lower boundary of the 90% confidence interval generated by 10,000 bootstrap replica tions exceeded zero. Theta measures the correlation of alleles of different individua ls in the same population relative to all populations and is described by the equation (Q q)/(1 q) where Q is the probability that two randomly sampled genes within a population are the same allele and q is the probability that genes randomly selected from different populations are the same allele (22). Bands absent or fixed in those popul ations that were being compared were excluded from this analysis. The effect of proximity to strawbe rry production, specific sampling site, and native host species on the incidence of pathogenicity was determined using logistic analysis perf ormed with the GENMOD procedure in SAS (SAS Institute Inc., Cary, NC). For this analysis, isolates were categorized as being either close or distant from the strawberry production area. Effects of specific sampling sites were included in the model as nested effects with in the variable proxi mity to strawberry

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73 production and were compared to one another using specified contrasts. The incidence of plant collapse in field plots inoculated w ith isolates determined to be pathogenic or nonpathogenic in greenhouse experiments were compared using one-way analysis of variance or unpaired t tests. Proportions were transfor med using the arcsine-square root transformation prior to analysis. Results Species Identification and Population Structure Eighteen isolates from oak and grape le sions collected at Dover site 1 and 24 isolates collected at Dover site 2 produ ced a positive amplific ation product with the C. gloeosporioides / C. fragariae -specific ITS1 primer. From the two sites distant from strawberry production, Lake Alfred and Sarasota, 22 isolates and 25 isolates, respectively, were obtained that produced a positive amplification product with the species-specific ITS1 primer. Cluster analys is using data from 38 RAPD bands grouped isolates from grape and oak into 4 distinct cl usters with similarity among isolates within the cluster greater than 0.65 a nd similarity between clusters less than 0.35 (Fig. 4-2). The vast majority (82 of 89) of isolates from th ese native hosts formed a large cluster along with 20 C. gloeosporioides isolates from diseased stra wberry crowns. This cluster occurred in 89% of bootstrapped trees. Two homothallic isolates from Sarasota and one from Dover site 1 formed a distinct cluster, an isolate from an oak lesion in Lake Alfred clustered with a C. fragariae isolate, and three isolates fr om Lake Alfred clustered among six citrus isolates obtained from groves at various locations in Florida. Interestingly, one of the seven citrus isolates used as a cont rol clustered among isolat es from strawberry crown. The cluster containing only homothalli c isolates and the cluster with isolates

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74 related to citrus each occurred in 99% of boot strapped trees. The cluster that included the C. fragariae isolate occurred in 100% of bootstrapped trees. Of the 38 RAPD bands scored, nine were unique to the cluster defined by isolates of C. gloeosporioides from crown tissue, six were uni que to the cluster defined by isolates from citrus, four were unique to the homothallic isolate cluster and six were unique to the cluster containing C. fragariae (Table 4-1.). Within the cluster that included isolates from crown tissue, there were only two bands that were amplified from native hosts only and these occurred at an overall frequency within these populations of less than 0.04. When only isolates that clustered with t hose from crown rot were examined, there was no evidence for population differentiation be tween isolates from oak and grape hosts at any of the four sites (Table 4-2). In a hi erarchal analysis in wh ich native host isolates from all sites were used and sampling site was included in the analysis to delimit subpopulations, there was also no evidence for population subdivision based on host ( = 0.029, 90% C. I. = 0.054 0.000). When sampling sites were examined in pairwise tests for population differentiation, there was evidence for popul ation subdivision between isolates taken from the Sarasota sa mpling site and samples from Lake Alfred, Dover, and diseased crowns (Table 43). There was no ev idence for population subdivision for isolates from any of the ot her samples (Lake Alfred, Dover 1, Dover 2 and the population from dis eased crowns). Although ther e was evidence for population subdivision between the Sarasota sampling site and all others there were no bands unique to this population nor did it lack any bands present in a ll of the other populations examined.

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75 Pathogenicity In greenhouse tests, a higher proportion of isolates from the Dover sites located adjacent to strawberry fields were pathogenic to strawberry than isolates from the sites distant from strawberry fields ( P = 0.002; Tables 4-4 and 4-5). When the Sarasota population was excluded from the analysis, the proportion of isolates pathogenic on strawberry was still greater at the Dover sites ( P = 0.03). Isolates fr om oak did not differ in degree of pathogenicity from grape isolat es. There was also no significant difference in the proportion of pathogenic isolates at the two Dover site s located in close proximity to strawberry fields (Contrast P = 0.59), nor a difference in the proportion of pathogenic isolates at the Lake Alfred and Sarasota sites distant from strawberry production (Contrast P = 0.76). Homothallic C. gloeosporioides isolates from native hosts and C. gloeosporioides isolates from native hosts that clustere d with citrus isolates did not cause crown rot symptoms. The single isolate fr om Lake Alfred that clustered with a C. fragariae isolate also caused collapse of plants wh en injected into crown tissue, produced tapered conidia, and possessed setae that f unctioned as phialides These traits are characteristic of C. fragariae (43). In the field experiment using both nonpat hogenic and pathogenic isolates identified by direct injection of inoculum into crow n tissue, plots topica lly inoculated with pathogenic isolates had a higher inci dence of crown rot (38.3% vs. 3.3%, P < 0.001 unequal variances assumed, n = 12 per treatment). The relatively low incidence of crown rot in plots inoculated with isolates dete rmined to be nonpathogenic in greenhouse tests was not statistically significantly different from plots spraye d with water (3.3% vs. 2.5%, P = 0.783, n = 12 and 4), suggesting that inoculation of plots with nonpathogenic isolates

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76 did not increase disease inci dence above that caused by natu ral sources of inoculum. Incidence of plant collapse was not different in the field for pathogenic isolates from the four different sites ( F = 0.36, P = 0.781, n = 3 per site), nor was the percentage of plants collapsed for plots treated with native host isolates different from plots treated with isolates from diseased crown (38.3% vs. 60%, P = 0.174, n = 12 and 4). Discussion Colletotrichum isolates from native grape and oak leaves producing a positive PCR product with C. gloeosporioides/C. fragariaespecific ITS1 primers fell into four separate clusters based on RAPD marker data. The majo rity of the isolates from all sites sampled fell into the same cluster as C. gloeosporioides isolates from diseased strawberry crowns. However, one isolate clustered with C. fragariae another pathogen causing strawberry crown rot, and three is olates grouped with C. gloeosporioides from citrus. For the oak and grape isolates that groupe d with isolates from diseas ed crowns, band frequencies were not significantly different between the crown rot population and the population distant from strawberry produc tion in Lake Alfred. This supports the hypothe sis that the population of C. gloeosporioides on strawberry is derived from a population already present on hosts in Florida. Although band fr equencies in the Sarasota population were different from the crown rot population, no unique bands were found in the Sarasota population and the phenogram constructed fr om RAPD data provided no evidence that the population is monophyletic. This suggests that differences in band frequencies are due to restricted gene flow combined with ge netic drift rather than a speciation event. Fixation indices may also be estimated using the statistic GST, which differs from as a function of the number of individuals in each sample population and the number of

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77 sample populations used in the ca lculation (22). This statistic has been used in the past to examine genetic differentiation in pathoge n populations. For comparison to other studies, pairwise GST estimates between the Sarasota population and the other four sampled populations were estimated and range d from 0.11 to 0.18. These values are in the range of estimates for other fungal populations using RAPD, amplified fragment length polymorphism (AFLP), or RFLP ma rkers (42,67,74). However, in only one study where population structure of the chestnut blight fungus Cryphonectria parasitica was examined were distances between populations comp arable to those between Sarasota and the other sites (67). No popul ation subdivision was evident between oak and grape hosts at any location. In the study presented in chapter 3, band frequencies were not significantly different between a group of isolates from nu merous noncultivated hosts and strawberry crowns. However, due to limite d sampling, any host spec ificity within the noncultivated host population could not be examined. The observation of limited subdivision by host in areas where host specie s occur in close proximity further supports the hypothesis that the C. gloeosporioides population found on both native hosts and strawberry has a broad host range. Similar results were observed in a study examining host specificity of C. gloeosporioides isolates classified as endophytes collected from the foliage of trees in a tr opical forest (60). The three homothallic C. gloeosporioides isolates obtained from the Dover and Sarasota sites were compared to historical homothallic strawberry isolates (Florida isolates 311-1 and 329-1, C. M. Howard) (3 8) using RAPD markers and the two groups do not appear to be from the same population (data not shown). In chapter 3, homothallic isolates distinct from historical strawberry isolates were also collected from noncultivated

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78 hosts. Although many of the bands scored for isolates in the study presented here and in chapter 3 were the same, several were not. There also may have been differences in calculated band migration rates. For these reasons, the relatedness of isolates collected in the two separate studies cannot be determined by a simple analysis of marker frequencies reported in tables. However, a side by side comparison of bands amplified from isolates obtained in these two studies using the primer OPC-2 indicates that they are from the same population (data not shown). The single C. fragariae isolate was obtained in Lake Alfred, FL approximately 28 km from a commer cial strawberry farm. This species was previously shown to colonize Cassia obtusifolia growing in and around a strawberry nursery (50), but has never been reported away from a strawberry field. Its occurrence on oak at a considerable distance from a ny strawberry production suggests that C. fragariae may have a wider host range than previously believed and that it may be indigenous to Florida. In Louisiana, C. fragariae is responsible for most strawberry crown rot epidemics (79). Contaminated stock appears to be the major source of inoculum for these epidemics, as crown rot is not observed in production fields using di sease free transplants (65). However, this does not exclude the possi bility that a native host provided the initial inoculum for the population that persists on st rawberry. Also, native hosts may still play a role in disease caused by C. fragariae as runners taken from plants that were free of disease after one production s eason develop crown rot symptoms in subsequent years (65). Isolates of C. gloeosporioides from grape and oak that were closely related to citrus isolates were only observed in Lake Alfred. The grape vi nes and oak trees sampled at this site were next to a citr us grove and immigration of s pores from citrus hosts would

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79 likely be high in this area. The fact that only a small proportion of native host isolates from this site clustered with isolates from citrus undersco res the specific interaction between the population on citrus and its host. However, the finding of a few citrus type isolates at the Lake Alfred s ite also demonstrates that, in the presence of a sufficient amount of inoculum from an outside sour ce, alternate hosts can be colonized. In greenhouse tests, the proportion of isolat es from native hosts closely related to those on strawberry that were pathogenic on strawberry was greater at sampling sites close to strawberry fields. This provides support for the hypothesis that local selection for pathogenicity on strawberry occurs wh ere this host is grown in abundance. Experimental studies of pathogen evolution ha ve been conducted in pathosystems where the fungal pathogen displays a high degr ee of host specificity, such as the Hordeum vulgare-Rhynchosporium secalis pathosystem and the wild Linum marginaleMelampsora lini pathosystem (56,58,64,88). Within these pathosystems, pathogenic variation is governed by gene -for-gene interactions (15,56). Evidence that the biological relationship between C. gloeosporioides and strawberry is different from that found in these other pathosystems comes from research showing that isolat es indistinguishable from those on strawberry can be found on nume rous hosts. In addition, there is in no differential interaction between C. gloeosporioides isolates and strawberry cultivars as demonstrated in the following chapter. No microscopic studies investigating the infection of strawberry crown tissue by C. gloeosporioides have been conducted. The best available information on whether the interaction of C. gloeosporioides and strawberry is necrotrophic or biotrophic comes from a study examining C. fragariae on stolons. In this study, there was only a brief biotrophic phase before the pathogen entered

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80 an extended necrotrophic stage (25). There is also good evidence that C. gloeosporioides forms infections in strawberry petioles, citrus twigs, and citrus fruit that remain quiescent until infected tissue senesces (12,66). Ta ken together, these studies suggest that C. gloeosporioides does not form a biologically intimate relationship with its host and genefor-gene relationships do not play a major role. Because C. gloeosporioides has a broad host range and likely uses more than one pathogenic strategy to invade its host, it would be hard to identify an overridi ng factor shaping the evolution of this species in a natural environment. However, this may not be the ca se in agricultural areas where the presence of a large, genetically unif orm host population at a specif ic site would select for individuals that can grow on the overrepresented host. This would be consistent with research showing races immune to specific hos t resistance genes are overrepresented in samples from areas where resistance genes are deployed (98). It is not clear whether the isolates collected in this study were actually pathogens on oak and gr ape leaves. Isolates from these hosts came from typical anthracnos e type lesions, suggesting that they were pathogens. However, it is also possible th at isolates were growing as saprophytes on lesions caused by another pathogen, insect damage, or injuries. In greenhouse inoculations of oak leaves, is olates were recovered from in oculated leaves, but produced necrotic symptoms only if the tissue wa s first wounded (data not shown). One of the limitations of the crown inj ection assay may be that resistance to penetration by the pathogen may be circum vented (54). For this reason, topical inoculations of strawberry plot s in the field were examined. The field trial indicated that isolates classified as pathogens in gree nhouse tests caused plant collapse and those classified as nonpathogens did not The field experiment also provided quantitative data

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81 to differentiate pathogenic is olates. The aggressiveness of pathogenic isolates from native hosts was not statistica lly different between sampling sites, nor was aggressiveness of these native host isolates statistically di fferent from crown isolates. However, the comparison of isolates from different sites a nd hosts suffered from hi gh variability in the number of collapsed plants observed per plot as well as small sample sizes. The mean percentage of plants killed per plot for is olates from diseased crown tissue was 60%, whereas only 38% of plants in plots inoculated with pathogenic isolates from native hosts were killed. These numbers were not signi ficantly different from one another, but it would not be inconsistent with the data on th e incidence of pathogeni city that isolates from crown and pathogenic isolates from native hosts would differ from one another using a more quantitative measure for pathogenicity. In summary, there was no conclusive evidence that C. gloeosporioides isolates from diseased strawberry crowns in Fl orida are genetically distinct from the C. gloeosporioides population broadly distributed on oak and wild grape hosts both close to and distant from commercial strawberry fields Isolates pathogenic to strawberry were also broadly distribut ed on these native hosts, alt hough they occurred at a higher frequency at sites close to strawberry fields. The resu lts observed in this study are consistent with earlier work indicating that native plants can serve as a source of inoculum for crown rot epidemics. The high incidence of Collectotrichum crown rot that occurs in summer nurseries located in Florida is one of the reasons why transplants used for commercial fruit production in Florida are purchased from nurseries located at higher latitudes. The current study suggests that over the long term us ing transplants from nurseries not located in Flor ida will likely reduce the amount of initial inoculum in

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82 growers fields but will not prevent epid emics caused by introduced isolates, since C. gloeosporioides isolates pathogenic to strawberry are present away from strawberry fields and the frequency of pathogenic isolates app ears to respond to selective pressures.

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83Table 4-1. Frequencies of randomly amplified polymorphic DNA bands from Colletotrichum gloeosporioides (C.g.) and Colletotrichum fragariae (C.f.) isolates Clustera C.g. Strawberry C. g. Citrus C. g. Homothallic C. f. D1b D2 LA SS Strawberry LA Citr us SS & D1 LA Strawberry Primer, length (kb) ( n = 17) ( n = 24) ( n = 23) ( n = 20) ( n = 20) ( n = 3) ( n =6) ( n = 3) ( n = 1) ( n = 1) (ACTG)4 2.145 0.35 0.46 0.89 0.74 0.65 1.00 1.00 0.00 0.00 0.00 1.9 1.00 1.00 1.00 0.91 1.00 0.00 0.00 1.00 0.00 0.00 1.85 0.00 0.00 0.00 0.00 0.00 1.00 1.00 0.00 0.00 0.00 1.6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 1.5 1.00 1.00 1.00 1.00 1.00 0.33 1.00 0.00 0.00 0.00 1.45 0.00 0.00 0.00 0.00 0.00 0.67 0.00 0.00 0.00 0.00 1.3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 0.00 0.00 1.1 0.71 0.83 0.67 0.22 0.80 0.00 0.00 0.00 0.00 0.00 (GACA)4 1.6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 1.45 0.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.4 0.12 0.04 0.17 0.70 0.05 0.00 0.00 0.00 0.00 0.00 1.35 1.00 1.00 0.94 1.00 0.95 0.00 0.00 0.00 0.00 0.00 1.25 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 0.00 0.00 1.2 0.88 0.96 0.89 0.87 0.90 0.33 0.00 0.00 0.00 0.00 1.1 0.06 0.04 0.11 0.13 0.05 0.67 1.00 0.00 1.00 1.00 0.95 0.65 0.50 0.61 0.87 0.55 0.00 0.00 0.00 0.00 0.00 0.9 0.47 0.63 0.56 0.74 0.60 1.00 1.00 1.00 1.00 1.00 0.8 0.24 0.21 0.11 0.04 0.05 0.00 0.00 0.00 0.00 0.00 0.55 0.00 0.00 0.06 0.09 0.00 1.00 1.00 1.00 0.00 0.00 0.5 1.00 0.96 1.00 0.96 1.00 1.00 1.00 0.00 1.00 1.00 aClusters correspond to the four groups of isolates identified in fig. 4-2. bIsolates from native hosts oak and grape are identified by site c ode D1, D2, LA, or SS. Isolates from cultivated hosts used as controls are referred to by the name of the host.

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84 Table 4-1. Continued Clustera C.g. Strawberry C. g. Citrus C. g. Homothallic C. f. D1b D2 LA SS Strawberry LA Citr us SS & D1 LA Strawberry Primer, length (kb) ( n = 17) ( n = 24) ( n = 23) ( n = 20) ( n = 20) ( n = 3) ( n =6) ( n = 3) ( n = 1) ( n = 1) (TCC)5 2.0 0.00 0.08 0.28 0.00 0.05 0.00 0.00 0.00 0.00 0.00 1.95 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.67 1.00 1.00 1.9 0.59 0.42 0.44 0.61 0.60 0.33 0.83 0.33 0.00 0.00 1.55 0.00 0.00 0.00 0.00 0.00 1.00 1.00 0.00 0.00 0.00 1.1 0.59 0.63 0.50 0.87 0.40 0.00 0.00 1.00 0.00 0.00 1.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 0.75 0.24 0.17 0.39 0.83 0.25 0.00 0.00 0.33 0.00 0.00 0.7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 OPC-2 2.4 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 0.00 0.00 2.1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 1.9 0.65 0.58 0.67 0.96 0.50 0.00 0.00 0.00 0.00 0.00 1.85 0.00 0.00 0.00 0.00 0.00 0.67 0.50 0.00 0.00 0.00 1.7 0.00 0.00 0.00 0.00 0.00 1.00 1.00 0.00 0.00 0.00 1.3 0.00 0.00 0.00 0.00 0.00 1.00 1.00 0.00 1.00 1.00 1.1 1.00 1.00 1.00 1.00 1.00 0.00 0.00 0.00 0.00 0.00 1.08 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 0.00 0.00 1.05 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 0.00 0.7 0.00 0.00 0.00 0.00 0.00 1.00 0.33 0.00 0.00 0.00

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85 Table 4-2. Estimates of for pairwise comparisons of Colletotrichum gloeosporioides isolates from oak and grape hosts at four sites Site a 90% Confidence interval b Dover 1 -0.094 -0.114 -0.069 Dover 2 0.031 -0.032 0.091 Lake Alfred 0.056 -0.032 0.157 Sarasota -0.017 -0.057 0.040 aThirteen to 15 bands were used to estimate bThe 90% confidence interval was determin ed from 10,000 bootstrap replications.

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86 Table 4-3. Pairwise estimates of (above diagonal) for Colletotrichum gloeosporioides populations at four sites and the estimated 90% confidence interval for (below diagonal) Dover 1 Dover 2 Lake Alfred Sarasota Strawberry crown Dover 1 -0.021a -0.031 0.218* -0.005 Dover 2 -0.032 -0.010b 0.030 0.275* -0.007 Lake Alfred -0.039 0.129 -0.017 0.094 0.168* -0.004 Sarasota 0.116 0.310 0.141 0.396 0.087 0.239 0.281* Strawberry crown -0.032 0.028 -0.027 0.014 -0.025 0.023 0.131 0.373 aFifteen to 17 bands were used to estimate The lower boundary of the 90% confiden ce interval for estimates followed by an asterisk is greater than zero. bNinety percent confidence limits were determined from 10,000 bootstrap replications.

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87 Table 4-4. Percentage of isolates pathogenic on strawberry from oak ( Quercus spp.) and grape ( Vitis spp.) lesions at four sites Number of isolatesa Pathogenic Site Host Path Nonpath (%) Dover 1 Quercus spp. 4 4 50.0 Vitis spp. 3 6 33.3 Dover 2 Quercus spp. 6 7 46.2 Vitis spp. 6 5 54.6 Lake Alfred Quercus spp. 2 9 18.2 Vitis spp. 1 6 14.3 Sarasota Quercus spp. 2 10 16.7 Vitis spp. 1 10 9.1 aPath = pathogenic, Nonpath = nonpathogenic

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88 Table 4-5. Likelihood ratio st atistics examining the effect of local strawberry production, specific sampling site and nativ e host species on the proportion of native host isolates pathogenic to strawberry. Source of variance df 2 P > 2 Proximity to strawberry productiona 1 9.41 0.002 Site(proximity to strawbe rry production) 3 0.38 0.828 Host 1 0.17 0.677 aProximity to strawberry production was clas sified as either near or distant.

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89 Fig. 4-1. Map showing the three locations where Colletotrichum gloeosporioides isolates were sampled from lesions on oak and grape leaves. There were two separate sampling sites in Dover. Hillsborough County, the main strawberry-growing county in Florida, is shaded. Map template provided by th e Department of Geography, Geology, and Anthropology, Indiana State Univ ersity, Terre Haute 47809.

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90 Fig. 4-2. Unweighted pair group method with arithmetic averages phenogram showing genotypic similarity between Colletotrichum isolates. C. gloeosporioides isolates from oak and grape fell into three separate clus ters based on high similarity among isolates within each cluster (similarity 0.65), low similarity between each cluster and other isolates (similarity 0.35), and high bootstrap support for clusters ( 50%). The three clusters consisted of isolates that were homothallic in culture, isolates similar to strawberry crown isolates, and isolates similar to citrus isolates. An isolate and cluster of C. fragariae is identified by name. Isolates coded w ith the prefix D1 and D2 are from the two sites in Dover. Isolates coded LA ar e from Lake Alfred and SS from Sarasota. Numbers at branch points indicate the percent occurrence of the cluste r to the right of the branch in 1,000 bootstrapped phenograms. Only branches occurring in at least 50% or more of bootstrapped phenograms are labeled.

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91 CHAPTER 5 RESISTANCE OF STRAWBERRY CULTI VARS TO CROWN ROT CAUSED BY Colletotrichum gloeosporioides Introduction Previous studies examining variati on in strawberry resistance to C. fragariae found a broad range of susceptibility among cu ltivars and aggressiveness among isolates (28,48,81). One study found a significant cultivar isolate interaction (81), implying gene-for-gene interactions between the pathoge n and host. Host resistance studies using homothallic G. cingulata isolates were not as conclusi ve, as they included only a few isolates (31,81). One study found significant variation in cultivar susceptibility and isolate aggressiveness, and a significant cultiv ar isolate interacti on (81). In a later study using a different set of isolates, only cul tivar susceptibility varied (31). Variation in cultivar susceptibility to self-sterile C. gloeosporioides isolates from Florida has not been investigated. Although closely related to C. fragariae (83), genotypes of self-sterile C. gloeosporioides isolates are diverse, whereas little diversity is observed among C. fragariae isolates (92). In addi tion, as the name implies, C. fragariae is thought to have a much narrower host range (25). In this chapter, the levels of resistan ce to crown rot caused by self-sterile C. gloeosporioides isolates in cultivars commonly grown in Florida as well as the patterns of virulence and aggressiveness among C. gloeosporioides isolates under field conditions are investigated. Resistance da ta for progeny from crosses usi ng a highly resistant parent and a susceptible parent are also presen ted. The study was designed to generate

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92 information that can be useful to growers fo r cultivar selection as well as selection of nursery locations from which to purchase pl ants. It was also designed to generate information useful to breeders in terms of the mechanism and sources of resistance to crown rot caused by C. gloeosporioides Materials and Methods Plant Materials and Cultivation Cultivars for experiments examining cultivar isolate interactions were selected from those commercially available as l eaf-on, bare-root transplants imported from Canada at the beginning of the 2001-2002, 2002-2003, and 2003-2004 seasons. The cultivars used included the standard cultivar s in the industry, severa l new cultivars, and an advanced selection from the Florida Ag ricultural Experime nt Station breeding program. The cultivars Aromas, Camarosa, Ea rlibrite, and Sweet Charlie were evaluated during the 2001-2002 season. During the 2002-2 003 season, in addition to the above four cultivars, the cultivars Gaviot a, Strawberry Festival, and Tr easure were also tested. Strawberry Festival and Treasure were evaluated again du ring the 2003-2004 season, but Gaviota was not available. Additional material used over the 2003-2004 season included the cultivars Camino Real, Carmin e, Camarosa, and selection FL 99-164. Aromas, Camarosa, Gavio ta, and Camino Real are University of California cultivars; Carmine, Earlib rite, Strawberry Festival, and Sweet Charlie are University of Florida cultivars. The cultivar Treasure is from J & P Research in Naples, FL. For the experiment examining inheritan ce of resistance to crown rot, progeny from crosses using Treasure and Camarosa as parents were s upplied by the Florida Agricultural Experiment Station breeding program and propagated from runners during

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93 the summer of 2004 in Florida. The parent cultivars were impor ted from Canada and selected to match crown diameters cons istent with those observed among progeny propagated in Florida. The number of progeny for four crosses were: 24 Camarosa Treasure, 17 Treasure Camarosa, eleven Trea sure self, and ten Camarosa self. The number of plants propagated from individual progeny was variable as some produced few runners or transplants died. For the cro sses between Camarosa and Treasure, the number of plants propagated of each genotype ranged from nine to twelve plants and eight to twelve plants were obtained for pr ogeny from the Camarosa self. For Treasure self between nine and twelve plants were propagated for seven genotypes and between two and five plants were propagated for f our genotypes. The low numbers of plants propagated for three of these genotypes was due to death of transplants and one genotype only produced three runners. Plants were grown on rais ed, plastic mulch covered be ds 71 cm wide, 15 cm high at the edge, and 18 cm high in the center. Each bed contained two rows of strawberry plants with 30.5 cm separating rows and plants within the rows se parated by 38 cm. The distance between bed centers wa s 1.22 m. Before planting, th e beds were fumigated with methyl bromide/chloropicrin (98:2) at 350 kg/ ha. Leaf-on, bare-root transplants were set on Oct 18 for the 2001-2002 season, Oct 16 fo r the 2002-2003 season, and Oct 29 for the 2003-2004 season for experiments evaluating cultivar isolate interactions. For the experiment examining inheritance of resist ance, plants were set on Oct 21 of the 20042005 season. Plants were overhead irrigate d for ten to twelve days to facilitate establishment. After establishment, water a nd fertilizer was provide d through drip tape. To prevent secondary spread of crown rot, w eekly applications of Captan 80WP (Micro

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94 Flo, Memphis, TN; captan) at 4.2 kg/ha were be gun 2 weeks after plants were inoculated. Freeze and frost protection was provided by overhead sprinklers when necessary. Fungal Isolates and Inoculation Procedure C. gloeosporioides isolates were selected from thos e characterized in chapters 2, 3 and 4. Most were from strawberry crow n tissue, although during the 2002-2003 season an isolate from a lesion on wild grape ( Vitis rotundifolia ) and during the 2003-2004 season an ascospore isolate from a perithecium emerging from a strawberry petiole were included in the study (Table 5-1). All isol ates were determined to be pathogenic on strawberry by injection of inoculum into cr own tissue, also reporte d in chapters 2, 3 and 4. The isolates had distinct RAPD bandi ng patterns and were recovered from seven different strawberry cultivars. Two isolates of C. fragariae one from a strawberry crown and the other from an oak ( Quercus sp.) leaf lesion in Lake Alfred, FL, were included in the cultivar isolate interaction study dur ing the 2003-2004 season. Isolate 96-83R was used to inoculate plots in the inheritance of resistance study. This isolate was chosen because it generates numerous spores and ha s the ability to produce crown rot symptoms on both parents examined. Inoculum was prepared from 6to 8day old cultures gr own under continuous fluorescent light at 24C on potato dextrose agar. Conidial su spensions used for inoculations were prepared in sterile dei onized water, filtered through four layers of cheese cloth, and diluted to 5 105 conidia/mL. For cultiv ar isolate interaction experiments, inoculations were performed by spraying 2 mL of conidial suspension with a hand mister directly into the crown of plants 15 to 17 days after the plants were set in the field. The inoculation date was 2 Nov 2001 for the 2001-2002 season, 31 Oct 2002

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95 for the 2002-2003 season, and 15 Nov 2003 for the 2003-2004 season. For the inheritance of resistance experiment inoculations were delayed until 14 Feb 2005. Experimental Design and Statistical Analysis The cultivar isolate interaction experime nt was designed as a two-factor complete block. Blocks consisted of a single 73-m bed for the 2001-2002 season or two parallel 73to 76-m beds for the 2002-2003 and 2003-2004 s easons. There were four blocks in each experiment. All cultivar-isolate combinations were randomly assigned to ten-plant plots within each block along with one uninoc ulated plot of each cultivar. After inoculation, the number of collapsed plants within each plot was recorded weekly until the experiment was terminated at the end of the growing season. Termination dates were 15 Mar 2002, 28 Mar 2003, and 26 Mar 2004. The pr oportion of plants that collapsed in each plot on a specific date was transformed to the arc-sine-square root and used for statistical analysis. The formula for the transformation was arcsin(sqrt((y + )/(n + ))), where y = the number of collapsed plants per plot and n = the total number of plants per plot (4). Data for each season were an alyzed separately usi ng PROC MIXED of SAS (SAS institute, Cary, NC). In the analysis using C. gloeosporioides and C. fragariae data alone, block was considered a random ef fect and cultivar and isolate were considered fixed effects. In the analysis where C. gloeosporioides and C. fragariae data were combined, species, isolate, and cul tivar were all considered fixed effects with isolate nested within species. Block wa s considered a random effect. Uninoculated plots were not included in any reported an alysis. The risk of type I error ( ) was 0.05 for tests comparing means and was not ad justed for multiple comparisons.

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96 For the inheritance of resistance experi ment, progeny and parent clones were randomly assigned to plots on three beds. E ach plot contained plants of the same progeny genotype or ten to twelve plants of the cultivars Camarosa or Treasure. There were four plots of each parent genotype a nd one plot for each progeny genotype. The number of collapsed plants was recorded fo r plots on Monday and Friday each week after inoculation. The experiment was terminat ed on 25 May 2005. Proportions of collapsed plants were not statistically analyzed in this experiment due to vari ation in the number of plants of each genotype and the effect that truncating distributions at 0% and 100% might have on likelihood ratio tests for mixed normal di stributions. As an alternative, the sum of average daily temperatures preceding the point where 50% of the plants of each genotype collapsed was estimated from the da ta by regressing the proportion of plants collapsed over time points immediately before and after this threshold was exceeded. Because temperature affects the rate of collaps e (70), an adjustment for this variable was necessary. Assuming that resistance to cr own rot is a quantitatively inherited trait determined by genes at multiple loci, progeny of a cross should have resistance clustered around a value close to the midpoint of both pa rents. If a major gene contributes to resistance in one parent and not the other, then resistance among progeny will be bimodal with equal proportions of progeny in each mode. This assumes that the parent only possesses one copy of the resistan ce gene. A likelihood ratio test statistic was used to test for bimodality of resistance among progeny from the cross between the susceptible cultivar Camarosa and the resist ant cultivar Treasure. Before the statistical analysis, the sum of average daily temperatures between in oculation and the time point at which 50% of plants collapsed was transformed to elimin ate any residual skewness in data that could

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97 confound likelihood ratio analysis (61). The formula for the transformation was ( average daily temperature)1/4. Transformed data was described using models fit with the program NOCOM (Ott, J., NOCOM and COMP MIX programs. New York, Rockefeller University. 1992). The test statistic G2 was calculated from maximum likelihood estimates under the hypothesis of a single nor mal distribution or a mixture of two normal distributions with equal va riance. The formula for G2 is 2[ln(L1) ln(L0)] where L0 is the maximum likelihood under the hypothesi s of a single normal distribution and L1 is the maximum likelihood under the hypothesis of two normal distributions. When two normal components in a mixture have equal variance, the distribution of G2 can be approximated by the chi-square distribution with 2 degrees of freedom. However, statistical tests based on this distribution have been shown to be liberal and therefore estimated P -values were obtained from simulated data sets (90). In addition to the analysis of progeny from crosses using T reasure and Camarosa as parents, a likelihood ratio analysis on progeny from self pollinated Treasure plants was conducted using the same transformation described a bove, also under the assumption of equal variances for multiple distributions. Assu ming that a dominant gene contributes to resistance in Treasure, a bimodal distributi on with a 3:1 ratio of progeny in the resistant versus susceptible distribution should be observed. Results Disease Progression in Cult ivar Isolate Experiments The rate at which plants colla psed after inoculation with C. gloeosporioides differed during the three seasons (Fig. 5-1A B and C). During the 2001-2002 season, the majority of plants that developed crown rot symptoms did so in the first 55 days after inoculation. For the 2002-2003 season, rapid pl ant collapse within this time period only

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98 occurred for Gaviota and Camarosa. Du ring this season, the ra te at which plants developed symptoms slowed between day 50 and day 130 after inoculation. However, unlike the 2001-2002 season in which the pr ogression of symptom development also slowed, there was a spike in plant death late in the season. For the 2003-2004 season, symptom development appeared to continue at about the same rate throughout the season for all of the cultivars tested. At each time point, the rankings of plants with respect to susceptibility were approximately the same. Ho wever, some cultivars such as Earlibrite in the 2002-2003 season, Strawberry Festiv al in the 2003-2004 season, and FL 99-164 in the 2003-2004 season initially had relatively low disease inci dence that increased at a faster rate relative to other cultivars toward the end of the season. Only Camarosa was examined for susceptibility to C. gloeosporioides in all three seasons. At the end of each season, the incidence of plant collapse for this cultivar was consistently high ranging from 62% to 84%. Plants were challenged with C. fragariae only during the 2003-2004 season (Fig. 5-1D). During this season th e progression of symptom development showed a similar pattern to that observed for C. gloeosporioides Secondary spread of pathogens did not appear to affect results in any of the experiments since di sease incidence at the end of the season in the c ontrol plots ranged from 0% in 2002-2003 to 0.12% in 20032004. Cultivar and Isolate Evaluation Transformed disease incidence data used for statistical analysis was obtained at different time points after inoculation for each season. Data for analysis were taken on 4 Jan 2002, 28 Mar 2003, and 19 Mar 2004. These dates corresponded to 63, 148, and 125 days after inoculation, respectively. On these dates the absolute value of the difference

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99 between the number of plot s with 0% plant mortality and 100% plant mortality was minimized. This reduced compression bias toward one extreme value or the other (0% or 100% mortality). In addition, on these date s the number of plot s with 0% or 100% mortality was low and variance among trea tment combinations was relatively high. Analysis of data for C. gloeosporioides revealed strong isolate a nd cultivar effects, but no significant cultivar isolate interaction in each of the three seasons studied (Table 5-2). The variance component block was not estimate d to be greater than zero in any of the experiments ( P > 0.05). Graphs displaying disease in cidence for each isolate-cultivar combination in all three seasons showed that cultivar rankings were usually consistent across isolates (Fig. 5-2A, B and C). Wher e rankings did change, the cultivars with different ranks had similar levels of re sistance suggesting that random error could account for the changes. There was also a cultivar and isolate effect on disease incidences caused by C. fragariae during the 2003-2004 seas on, but no significant cultivar isolate interaction (Table 5-3). The gr aph showing cultivar sensitivity to each C. fragariae isolate also shows that cultivar rankings were consistent across isolates (Fig. 5-2D). When the C. gloeosporioides and C. fragariae data from the 2003-2004 season were combined, in addition to a species, cultivar, and isolate effect on disease incidence, there was also a small but significant species cultivar interaction (Table 5-3). This interaction resulted from a ch ange in the rankings of Str awberry Festival and Camino Real across fungal species (F ig. 5-3). With the exception of this one rank change, disease reactions to isolates of the different Colletotrichum species were very similar. The cultivars and isolates included in experiments during the different seasons were altered to increase the chance of observi ng any cultivar isolat e interactions. In

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100 addition, cultivars and isolates were carried over to the next year to assess the repeatability of the assay. During th e 2001-2002 season, Camarosa was the most susceptible cultivar followed by Aromas. Earlibrite and Sweet Charlie were more resistant and had essentially the same level of susceptibility (Table 5-4). The ranking of these cultivars for the 2002-2003 seas on was the same as for 2001-2002, although statistically Aromas could not be separate d from Earlibrite and Sweet Charlie. During the 2002-2003 season Treasure, Strawbe rry Festival, and G aviota also were evaluated. Treasure was more resistant and Gaviota more susceptible than any of the other cultivars examined. Strawberry Festival had an intermediate level of resistance, similar to that of Aromas. During the 2003-2004 season, rankings among cultivars carried over from the previous season remain ed the same. Treasure was once again the most resistant cultivar and Camarosa was more susceptible than Strawberry Festival. Although Camarosa and Strawberry Festival were not statistically significant from one another for the 2003-2004 season as they were for the 2002-2003 season. During the 2003-2004 season, disease symptoms on Strawbe rry Festival likely had more time to attain the ratings observed on Camarosa. The three genotypes grown only during the 2003-2004 season, FL 99-164, Carmine, and Cam ino Real were intermediate between the resistant Treasure and the relativel y susceptible Strawberry Festival and Camarosa. During the 2001-2002 season, isolates fe ll into four groups based on average aggressiveness to the four cultivars tested (Table 5-5). Isolate 97-15A was the most aggressive and isolates 95-63A, 97-45A, and 97-47C had relatively high, comparable levels of aggressiveness. Isolates 98-285 and 97-63 were not as aggressive and 97-63

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101 was even less aggressive than 98-285. Isolates 97-45A, 98-285, and 97-63 were reevaluated in the 2002-2003 season. These thr ee isolates along with 00-59, 96-83R, and 96-83H also produced four isol ate clusters based on aggressiveness. The three isolates from the 2001-2002 season had intermediate le vels of aggressive ness similar to the previous year. The rankings of these isolates with respect to one another also remained the same, although in the second season, isolat es 98-285 and 97-63 were not significantly different from each other. Isolates 00-59 and 96-83R were highly aggressive and isolate 96-83H, a nonstrawberry isolate, was the leas t aggressive isolate examined. For the 2003-2004 season only isolate 00-59 was re-eva luated. Once again it was a highly aggressive isolate. Other isolates examin ed fell into two groups with isolate 02-172 being more aggressive than isolate 96-15A and ascospore isolate 00-117. In separate analyses comparing inoculated pl ots to uninoculated controls, all Colletotrichum inoculated plots had signifi cantly more crown rot than c ontrols (data not shown). Cultivar rankings for resistance to C. fragariae were very similar to those for C. gloeosporioides during the season that this species was included in the study (Table 5-4). Treasure was highly resistant to C. fragariae ; FL 99-164 and Carmine displayed moderate levels of resistance and Strawberry Festival and Camaros a were relatively susceptible. The rankings only differed in that Camino Real appeared to be more resistant and Strawberry Festival more susceptible to C. gloeosporioides than to C. fragariae although not dramatically. Only two C. fragariae isolates were included in the study. These isolates displayed different levels of aggressive ness: isolate 02-135, a C. fragariae isolate from a nonstrawberry host, was mo re aggressive than C-16, the isolate from strawberry (Table 5-5).

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102 Inheritance of Crown Rot Resistance Progeny inoculations were not done until Febr uary due to differences in the size of transplants. It was also n ecessary to determine whether a ny plants were infected with C. gloeosporioides during propagation over the summer. During the course of the experiment, average daily temperatures ra nged from 9.1C to 25.1C with a mean daily temperature of 18.8C. The average temp erature dropped below 15C on only eleven days, with all but three of these days occurr ing before any significant plant death. The experiment extended well beyond the normal grow ing season until at least 50% of plants had collapsed in all the plots. Once 50% mortality in all plots was achieved, for each progeny genotype the cumulative average temper ature was calculated for days up to and including the date when 50% mortality was re ached. The mean and standard deviation of transformed cumulative temperature for pr ogeny of crosses Camarosa Treasure and Treasure Camarosa was 5.25 0.35 C1/4. For crosses Camarosa self and Treasure self they were 4.82 0.24C1/4 and 5.24 0.45C1/4, respectively. The means and standard deviations for pare nt genotypes were 5.08 0.08C1/4 for Camarosa and 5.76 0.18C1/4 for Treasure. In the cross between T reasure and Camarosa, female parent did not affect resistance. (Mean sta ndard error, Camarosa Treasure = 5.23 0.08C1/4 and Treasure Camarosa = 5.27 0.08C1/4, P = 0.67). Both visual and statistical analysis of th e distribution of transforme d cumulative temperature for Camarosa Treasure and Treasure Camaro sa progeny suggests bimodal inheritance (Fig. 5-4, G2 = 7.39, P = 0.042). Means for the mixed normal distribution model that maximized likelihood were 4.91 and 5.51 with standard deviation of 0.177. Estimated proportions of progeny belonging to each distribution were 0 .45 and 0.55. Analysis of

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103 the distribution of progeny from self pollinated Treasure plants suggests that there were eight relatively resistant pr ogeny and three highly suscepti ble progeny (Fig. 5-4). The log likelihood ratio supports bim odality for this distribution ( G2 = 8.83, P = 0.035). Means for the mixed normal distribution m odel that maximized likelihood were 4.60 and 5.483 with standard deviation of 0.170. Es timated proportions of progeny belonging to each distribution were 0.27 and 0.73. Progeny from self pollinated Camarosa were mostly susceptible to crown rot with one outlier having a rela tively high level of resistance. Visual analysis of the proportion of co llapsed plants for progeny on 28 March 2005 supports conclusions based on the analysis us ing cumulative average temperature (Fig 55). On this date the number of progeny w ith either 0% or 100% plant collapse was minimal for progeny from crosses between Cam arosa and Treasure (three with 100% mortality and six with 0% mortality of 41 pr ogeny). Plant collapse in most plots of progeny from Camarosa Treasure and Treas ure Camarosa were close to extreme values of 0% or 100% with few progeny at mi dpoint values. Approximately half of the progeny had mortality greater than 50% and half had mortality less than 50%. Eight of eleven progeny from self pollinated Treasure plants had relatively low or intermediate mortality whereas three proge ny had high mortality. Discussion Pathogenicity trials conducted over three years using twelve distinct C. gloeosporioides isolates and ten strawberry cultiv ars identified differences in disease resistance among cultivars and differences in ag gressiveness among isolates, but failed to identify any cultivar is olate interactions. A more limited study using two C. fragariae isolates inoculated on six st rawberry cultivars conducted dur ing the third ye ar identified

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104 differences in disease resistance among cult ivars and differences in aggressiveness among the isolates, but failed to identify a cul tivar isolate interaction. When cultivar resistance rankings were compared between the two pathogen species, there was a small but significant cultivar species interaction. Horizontal resistance is effective agai nst all isolates of a pathogen, whereas vertical resistance is effective against a s ubset of isolates (96). These terms are synonymous with race-nonspecific and race-spe cific resistance, respectively (7). In pathosystems where race-specific resistance occurs, an incompatible interaction between the host and pathogen often requi res a dominant host resist ance gene and a dominant pathogen avirulence gene. Such interacti ons are described by the gene-for-gene hypothesis (41). Race-nonspecific resistance is less understood, but is believed to be governed by many host genes that incrementally contribute to the ove rall resistance of the plant (96). Although both resist ance mechanisms can be empl oyed within a pathosystem, race-specific resistance is often associated with biotrophic plant-microbe interactions and race-nonspecific resistance w ith necrotrophic interactions (41). VanderPlank proposed using analysis of variance to determine the contribution of race-nons pecific and/or racespecific resistance within a hos t population against a group of pathogen isolates (96). Absence of a cultivar isolate interaction is evidence for race-nonspecific resistance using this method. A cultivar isolate interaction sugge sts that race-specific mechanisms contribute to the resistance observed within the host population, although deviations from additivity could be responsib le for the statistical interaction (73). Deviations from additivity result from the scale used to measure resistance, whereas rank changes are consistent with physiological inte rdependency between the host and isolates.

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105 No cultivar isolate interactions were de tected in this study suggesting that racenonspecific resistance mechanisms are res ponsible for the differences in resistance observed between cultivars. Thus, screening with many isolates might not be necessary to examine resistance to C. gloeosporioides Using transformed percentages taken at a time point in which disease incidence was at an intermediate level effectively reduced interactions attributable to deviations from additivity. This finding may be useful in examining resistance to other wilt diseases where use of arbitrary rating scales or measurements of area under the disease pr ogress curve may produce results in which there are no transformations avai lable to eliminate undesirable scale effects. A drawback of measuring resistance using the proportion of collapsed plan ts is that the rankings of cultivars could change at diffe rent time points after inoculati on as resistance may occur at different levels of the infection process. Te mporally distinct resistance mechanisms have been demonstrated for Phytophthora palmivora on cacao (54). The first level of resistance, referred to as pene tration resistance, was attri buted to morphological factors and the second level of resist ance, referred to as post pene tration resistance, impeded tissue invasion by the pathogen after coloniza tion. In the current study, rank changes between cultivars over the course of the season were infrequent and occurred only between cultivars displaying similar levels of resistance, suggesting that sequential deployment of resistance mechanisms had little effect on the evaluation of cultivars. However, the relatively high levels of inocul um used could have saturated structural mechanisms that limit ingress into the host. Similarly, individual isolates within the pathogen population might differ in their ability to overcome plant defenses at different times during and after invasion of tissues. Ho wever, like the cultivar resistance rankings,

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106 pathogen aggressiveness rankings did not change over the course of the experiment (data not shown). During the 2001-2002 season, the rate of pl ant collapse was greater than the 20022003 or 2003-2004 seasons. Disease probably pr ogressed more rapidly in the 2001-2002 season because the mean daily temperature in December was 18.3 C, 3.7 C higher than it was in the other seasons. Th e mean daily temperature of 11.6 C in January of the 20022003 season was 2.5 C lower than the mean temperatur e during January for the other two seasons and may have slowed symptom development. In a previous study examining resistance to G. cingulata and C. fragariae in strawberry, cultivar isolat e interactions were observ ed (81). The population of G. cingulata used in that study was homothallic and th erefore it was probably distinct from the one we used. Also, in that study, it is conceivable that scale effects produced by the severity rating system used to evaluate di sease accounted for th e interaction, but the interaction may also have been due to use of isolates or cultivars not included in the current study. We attempted to use isolates from different cultivars and isolates that were genetically distinct. Commercial cultivars shipped from Canada were used to obtain the relatively large number of plants free of crown rot required for the experiment. A disadvantage of using these cultivars was th at some of them we re closely related, restricting the dive rsity of the germplasm evaluated. For example, Earlibrite (17) and Strawberry Festival (18) each share Rosa Li nda as a parent and Strawberry Festival (18) and Treasure (Plant Patent 12,414) shar e Oso Grande as a parent. Nevertheless, a great deal of variance in resist ance was observed among them.

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107 Race-specific resistance is found most frequently in biotrophic plant-microbe interactions where there is prolonged contact between the pathogen and the living host (41). Colletotrichum species use nutritional strategi es ranging from necrotrophy to hemibiotrophy (57). Co lletotrichum species such as C. orbiculare C. graminicola C. sublineolum C. destructivum C. truncatum and C. linicola are all considered hemibiotrophs, as there is an asymptomatic biotrophic interaction between these species and host cells before the reaction becomes necrotrophic (57). The occurrence of a number of dominant race-specific resistance genes in bean to C. lindemuthianum a member of the C. orbiculare species aggregate, suggest that gene-for-gene interactions play an important role in host resistance to these hemibiotrophic pa thogens (104). No microscopic studies of strawberry crown invasion by C. gloeospioides have been conducted, although invasion of subtropical fru its (6,11), northern jointvetch (87), and Stylosanthes scabra (91) by C. gloeosporioides has been investigated. On citrus and avocado fruit, C. gloeosporioides is a quiescent epiphyte that resorts to necrotrophy upon ripening of the fruit (6,11). On foliage of S. scabra and northern jointvetch, C. gloeosporioides has a brief biotrophic phase before entering an extended necrotrophic stage (87,91). The evidence for this biotrophic interaction is not near ly as clear as it is for interactions between Colletotrichum spp. commonly referred to as hemibiotrophs and is limited to the occurrence of a spherical vesi cle inside an epidermal cell just beneath the appressorium from which infection hyphae eman ate. A differential interaction between S. scabra cultivars and biotype A C. gloeosporioides isolates has also been demonstrated (91), although a great d eal of the variation in resistance among cultivars is due to racenonspecific mechanisms (16). The hi stopathology of the related pathogen C. fragariae

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108 on strawberry stolons showed that a brief bi otrophic phase possibly occurred before the pathogen entered an extended necrotrophic pha se (25). The biotrophic phase was less than 12 h and it was considered a modification of necrotrophy rather than an example of hemibiotrophy. The lack of race-specific resistance observed in the current study suggests that biotrophic interaction between C. gloeosporioides or C. fragariae and strawberry is brief and limited. This findi ng is also consistent with evidence from chapters 3 and 4 that both of these species infect hosts unrelated to strawberry. An isolate of C. gloeosporioides from grape, a C. gloeosporioides ascospore isolate from a strawberry petiole, and an isolate of C. fragariae from oak were included in this study. Evidence that these isolates came from populations responsible for crown rot on strawberry was presented in chapters 3 and 4. The ascospore isolate was one of the least aggressive isolates dur ing 2003-2004, although it was as aggressive as one of the crown rot isolates. During the 2002-2003 season the grape isolate was less aggressive than the isolates from strawberry crowns. Th is may result from more aggressive isolates being selected in the C. gloeosporioides population on strawberry. The C. fragariae isolate from oak was more aggressive and ha d essentially the same virulence pattern as the isolate from strawberry. This is further evidence that C. fragariae from strawberry is derived from a population with a very broad host range, since the oak isolate was found 28 km from the closest strawberry production area. The only interaction detected was between the species of the isol ate and cultivar. This interaction was small and only one rank change occurred between cultiv ars, suggesting that resistance to C. gloeosporioides is correlated with resistance to C. fragariae and that large scale screening of plants for resistance to both pathogens may not be necessary.

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109 Only one cultivar, Treasure, was highly resistant to C. gloeosporioides It was also highly resistant to C. fragariae Symptoms eventually developed in response to inoculation with both pathogens and therefore Treasure wa s not immune. Resistance among progeny from crosses to the suscepti ble cultivar Camarosa was distributed bimodally with progeny distributed evenly am ong the two distributio ns, suggesting that a major gene contributes to resi stance. However, the method used to determine resistance had several drawbacks which might cast so me doubt on this conclusion. Ideally the experiment would have been conducted at a c onstant temperature, but due to the large number of mature plants incl uded in the study, temp erature could not be controlled. It is well documented that th e growth rate of C. fragriae and G. cingulata is affected by temperature and that low temper atures inhibit progression of crown rot (70,81). For this reason cumulative temperature until 50% of plants collapsed was used to evaluate resistance. The relationship between grow th rate and temperature on agar for both C. fragariae and G. cingulata is approximated by a linear function between 8C and 30C (81), suggesting that the temp erature adjustment is appropriate. However, the growth rate may not be linear in plant tissue or the fungus may fail to grow in crown tissue below a specific temperature. As a result, it is conceivable that the bimodal response was observed when no major gene had an effect on resistance. An example would be if a sudden drop in temperature during the expe riment suspended plant death completely, splitting the distribution which otherwise woul d have been normal. An analysis of temperatures during the time period when fe w plots reached the 50% threshold suggests that this did not happen, as temperatures were relatively high (data not shown). Data examining proportions of plants collapsed at a time point where plant mortality for most

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110 progeny were at values intermediate betw een 0% and 100% also supports a bimodal distribution of resistance among progeny, although the dist ribution was truncated at extreme values for close to 25% of the plot s. Progeny from self pollinated Treasure plants supports that a single dominant gene eff ects resistance in Tr easure, as statistical analysis indicates a bimodal distribution with th e ratio of isolates from the more resistant distribution to the more suscep tible distribution very close to 3:1. However, these data must be viewed with caution as several pl ots had only a few plants and limited progeny were examined. Numerous plants of three of the progeny genotype s collapsed early in the season before inoculation. The cause of death was Colletotrichum crown rot. These three genotypes were also determined to be highly susceptible to crown rot in the experiment. It is conceivable that the plants were already infected with crown rot, but given that they did not develop symptoms dur ing the early part of the season and that plant death occurred relativel y synchronously after inoculati on it appears that they were infected by applied inoculum. The high sus ceptibility of these plants also suggests resistance apart from that conferred by a ma jor gene is very low within Treasure. Currently a much larger population of progeny fr om self pollinated T reasure plants are being propagated for evalua tion during the 2005-2006 season. Sibling families were used in a previous study examining resistance to C. fragariae and a bimodal distribution was obser ved for disease severity among the population (44). That study did not examine bi modality in single crosses and the disease rating scale consisted only of 6 classes. Plants were rated for their ability to resist lesion formation on stems as well as appearance of wilt and collapse. That study also used three C. fragariae isolates applied separately to plants and it is conceivable that the bimodality

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111 resulted from differences in aggressiveness of isolates as opposed to resistance. Although the field method used to evaluate pr ogeny in the current study has drawbacks, it provides an objective variable to evaluate genotypes. From a practical perspective, it was able to show that a large proportion of progeny of a cross involving Treasure and a susceptible parent had resistance approaching or surpassing that of Treasure. Three of the 41 progeny were rated as being more resist ant to crown rot than Treasure. Given the good fruit quality of this cultivar, crosse s to Treasure could produce new genotypes with crown rot resistance and improved hor ticultural characteristics in just one generation.

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112Table 5-1. Description of Colletotrichum isolates used in inoculation experime nts conducted over three seasons in Dover, Florida Species Isolate Host cultivar Tissue Collection site and year Season used C. gloeosporioides 95-63A Strawberry Oso Grande Crown Dover, FL 1995 1-02 96-15A Strawberry Oso Grande Crown Dover, FL 1996 3-04 96-83H Vitis rotundifolia Leaf lesion Dover, FL 1996 2-03 96-83R Strawberry Selva Crown Dover, FL 1996 2-03 97-15A Strawberry Sweet Charlie Crown Dover, FL 1997 1-02 97-45A Strawberry Camarosa Crown Dover, FL 1997 1-02, 02-3 97-47C Strawberry Camarosa Crown Dover, FL 1997 1-02 97-63 Strawberry Oso Grande Crown Dover, FL 1997 1-02, 02-3 98-285 Strawberry Sweet Charlie Crown Dover, FL 1998 1-02, 02-3 00-59 Strawberry Strawberry Festival Crown Dover, FL 2000 2-03, 03-4 00-117 Strawberry Rosa Linda Ascospore petiole Dover, FL 2000 3-04 02-172 Strawberry Gaviota Crown Dover, FL 2002 3-04 C. fragariae C-16 Strawberry Camarosa Crown Dover, FL 2002 3-04 02-135 Quercus species Leaf lesion Lake Alfred, FL 2002 3-04

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113 Table 5-2. Analysis of variance for th ree experiments evaluating incidence of crown rot in relation to strawberry cultivar and isolate of Colletotrichum gloeosporioides Season Source of variation df F P > F 2001-2002a Cultivar 3 39.16 <0.001 Isolate 5 45.04 <0.001 Cultivar isolate 15 1.52 0.123 2002-2003b Cultivar 6 35.50 <0.001 Isolate 5 31.63 <0.001 Cultivar isolate 30 1.34 0.134 2003-2004c Cultivar 5 36.66 <0.001 Isolate 3 21.08 <0.001 Cultivar isolate 15 0.65 0.823 aF values for 2001-2002 season were calcula ted using a residual variance estimate equal to 0.024 having 69 degrees of freedom. bF values for 2002-2003 season were calcula ted using a residual variance estimate equal to 0.038 having 123 degrees of freedom. cF values for 2003-2004 season were calcula ted using a residual variance estimate equal to 0.035 having 69 degrees of freedom.

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114 Table 5-3. Analysis of variance for 20032004 experiment evaluating incidence of crown rot in relation to strawberry cultivar and isolate of Colletotrichum fragariae alone or in comparison to C. gloeosporioides Species included Source of variation df F P > F C. fragariaea Cultivar 5 24.66 <0.001 Isolate 1 9.01 0.005 Cultivar isolate 5 0.91 0.485 C. fragariae and C. gloeosporioidesb Species 1 27.10 <0.001 Cultivar 5 56.91 <0.001 Isolate(species) 4 19.00 <0.001 Species cultivar 5 2.72 0.023 aF values for the analysis examining C. fragariae only were calculated using a residual variance estimate equal to 0.034 having 33 degrees of freedom. bF values for the analysis examining both C. fragariae and C. gloeosporioides were calculated using a residual variance es timate equal to 0.033 having 125 degrees of freedom.

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115 Table 5-4. Mean percent plant colla pse of cultivars inoculated with Colletotrichum gloeosporioides or C. fragariae during three seasons in Dover, Florida Disease incidence (%)a C. gloeosporioides C. fragariae Cultivar 2001-2002b 2002-2003 2003-2004 2003-2004 Treasure 10.0 A 11.3 A 0.0 A Sweet Charlie 30.3 A 29.9 B Earlibrite 30.7 A 33.9 B FL 99-164 33.7 B 12.0 B Carmine 41.5 BC 17.1 B Camino Real 50.6 C 56.4 C Aromas 59.6 B 43.1 BC Strawberry Festival 45.9 C 80.6 D 51.1 C Camarosa 71.8 C 64.7 D 83.8 D 76.3 D Gaviota 82.2 E aStatistical tests were conduc ted on mean transformed disease incidences. Reported values were calculated by back transformi ng the mean arc-sine-square root disease incidences for all isolates inoculated on a cultivar. bStatistical tests only compare means for pl ants inoculated with the same species within the same season. Means in each co lumn followed by the same letter are not significantly different, leas t significant difference ( P = 0.05).

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116 Table 5-5. Mean percent plant collapse for Colletotrichum gloeosporioides or Colletotrichum fragariae isolates used to inoculate strawberry cultivars during three seasons in Dover, Florida Disease incidence (%)a Inoculated species Isolate 2001-2002b 2002-2003 2003-2004 C. gloeosporioides 97-15A 86.8 A 00-59 69.4 A 71.8 A 96-83R 66.9 A 02-172 59.1 B 95-63A 53.8 B 97-45A 53.8 B 48.6 B 97-47C 50.7 B 96-15A 36.3 C 98-285 36.0 C 34.3 C 00-117 33.4 C 97-63 9.5 D 31.4 C 96-83H 15.3 D C. fragariae 02-135 40.9 A C-16 24.7 B aStatistical tests were conduc ted on mean transformed disease incidences. Reported values were calculated by back transformi ng the mean arc-sine-square root disease incidences for all cultivars inoculated with an isolate. bStatistical tests only compare means for is olates of the same species within the same season. Means in each column followed by the same letter are not significantly different, leas t significant difference ( P = 0.05).

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117 Fig. 5-1. Mean percent mortality for strawberry cultivars calculated at weekly intervals over the course of the growing season using data from all isolates. (A) Colletotrichum gloeosporioides inoculated plants duri ng the 2001-2002 season. (B) C. gloeosporioides inoculated plants during the 2002-2003 season. (C) C. gloeosporioides inoculated plants during the 2003-2004 season. (D) C. fragariae inoculated plants during the 2003-2004 season.

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118 Fig. 5-2. Cultivar isolate means for arc-sine-square root transformed disease incidences calculated on a speci fic date during or at the e nd of the season. Ticks on the y-axis show position of back transformed incidences rang ing from 0% to 100% at intervals of 10%. (A) Disease incidences calculated for pl ots 63 days after inoculation with Colletotrichum gloeosporioides during the 2001-2002 growi ng season, (B) 148 days after inoculation with C. gloeosporioides during the 2002-2003 gr owing season, (C) 125 days after inoculation with C. gloeosporioides during the 2003-2004 gr owing season, and (D) 125 days after inoculation with C. fragariae during the 2003-2004 growing season.

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119 Fig. 5-3. Cultivar species means for arc-sine-square root transformed disease incidences calculated 125 da ys after inoculation with Colletotrichum fragariae or C. gloeosporioides during the 2003-2004 season. The y-axis shows the position of backtransformed incidences ranging fr om 0% to 100% at intervals of 10%.

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120 Fig. 5-4. Distribution of transformed cumulative temperature until 50% plant death for progeny inoculated with Colletotrichum gloeosporioides from (A) Camarosa Treasure and Treasure Camarosa; (B) Treasure self; and (C) Camarosa self. Means for the four plots of the parents Tr eaure and Camarosa are depi cted by arrows in graphs B and C, respectively.

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121 Fig. 5-5. Distribution of percent plant mortality 42 days after inoculation for progeny inoculated with Colletotrichum gloeosporioides from (A) Camarosa Treasure and Treasure Camarosa; (B) Treasur e self; and (C) Camarosa self. Means for the four plots of the parents Treaure and Camarosa are depicted by arrows in graphs B and C, respectively.

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122 CHAPTER 6 CONCLUSION It was clear that C. gloeosporioides not C. fragariae was the species of Colletotrichum responsible for most of the crown rot on strawberry observed in Florida (92) prior to the initi ation of my research. In add ition, genetic data indicated that C. gloeosporioides isolates collected from collapsed crowns were genetically diverse and that they were part of a recombining population (92). C. gloeosporioides was also known to colonize petiole tissue and some isolates from petioles had been found to be incapable of producing crown rot symptoms (66). On several occasions, perithecia with morphology consistent with that of G. cingulata had been found on petioles, but the relationship of isolates from these perithecia to those res ponsible for crown rot had never been determined (66). Based on the research presented in chapter 2, a clearer picture of the population on strawberry has now emerge d. Perithecia found on petioles resulted from recombination and single-ascospore isolat es from these perithecia were genetically indistinguishable from self-ste rile crown isolates, providing direct evidence that sexual recombination had occured among indi viduals within th e population of C. gleosporioides responsible for crown rot. It was also apparent that C. gloeosporioides isolates collected from crown tissue were part of the broade r population found on strawberry and that some individuals within th is population were not capable of causing crown rot. This conclusion was based on the apparent lack of a reproductive barri er between isolates pathogenic and those nonpathogenic to stra wberry in laborato ry crosses and the

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123 occurrence of progeny with both phenotypes from at least one perithecium on plant material collected from the field. An important epidemiological question concerning the C. gloeosporioides /strawberry pathosystem that had remained unanswered until now was whether primary inoculum could come from sources outside of strawberry fields. Indirect evidence such as disease clusters at the edges of fields, the inability to find infections on transplants (66), and lack of evidence that the fungus could survive on debris between seasons (93) suggested th at the primary inoculum was coming from outside of fields. In chapters 3 and 4, the relationship of the C. gloeosporioides population from crown tissue to populations of C. gloeosporioides from hosts other than strawberry in Florida was examined. Among C. gloeosporioides isolates from these hosts, genetically distinct subpopulations were detected. Isolates from the C. gloeosporioides population on citrus app eared to be host specif ic and a homothallic population, distinct from both the self-sterile and self-f ertile crown population, was identified from noncultivated hosts. A lthough these subpopulations existed, the vast majority of isolates from noncultivated hosts were indistinguishable from the self-sterile population on strawberry and, like the population on strawberry petioles, both pathogenic and nonpathogenic isolates were obtained fr om the noncultivated hosts. This same alternative host population also occurred at sites distant from strawberry production, although the frequency of isolat es pathogenic to strawberry was lower. Based on the finding that pathogenic isolates genetically indistinguishable from the population on crowns were present at sites close to and di stant from strawberry fields, it appears that primary inoculum for crown rot epidemics can come from alternative hosts. In an

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124 unpublished study conducted in a Florida strawberry field, C. gloeosporioides spread from infected to healthy strawberry plants during the season. However, a high proportion of plants had to be infected to observe spread in the field and spread from plants infected early in the season was not detected until the very end of the season. Isolate genotypes from newly infected plants were mostly the sa me as the genotype of the strain used for the initial inoculation, indicating that disease had spread by conidia. In fields using transplants from Canada, C. gloeosporioides isolates from crowns were found to be genetically diverse (92). Since C. gloeosporioides has not been recovered from imported transplants and there was a long time lag between infection and spread in field trials, not only is it possible that primary infections come from noncultivated hosts, but it also appears likely that the infections currently observed in production fiel ds come from this source. However, in summer nurseries in Florida, it is probable that spread among strawberry plants perpetuates the disease, since higher temperatures would decrease the length of the disease cycle and rainfa ll would be more plentiful. Genetic and pathogenicity data generated in the current study also provided insight into the evolution of C. gloeosporioides pathogenicity on strawberry. Whether C. gloeosporioides is a pathogen on the native or nonc ultivated species from which it was isolated is not apparent, since C. gloeosporioides was isolated at a high frequency on asymptomatic tissue when epiphytic populations on tropical forest foliage were examined (60). Given that both epiphy tic and pathogenic strains of C. gloeosporioides were observed on strawberry in the current study and in a previous study (66), both saprophytic and pathogenic nutri tional strategies are prob ably employed. The genetic requirements for pathogenicity on strawberry occurred at a detectable frequency within

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125 the C. gloeosporioides population present on native hosts even though strawberry is not native to Florida. An increase in the pathogenic phenotype was also observed where strawberries were abundant. These observat ions were consistent with the idea that C. gloeosporioides occurs as a broadly distributed ep iphytic population on numerous hosts with a portion of individuals possessing pathogenicity genes for some but not all hosts. In the presence of a host that is suitable for colonization, selec tion for pathogenicity genes might then occur. Experiments examin ing the inheritance of pathogenicity did not give clear results. The ratio of pathogenic to nonpathogenic progeny in a cross between parents with different pathoge nicity phenotypes deviated fr om the expected 1:1 ratio under the assumption that a single segregati ng locus determines pathogenicity. This suggests that multiple genes contribute to pathogenicity on strawberry. However, nonmendelian segregation of whole chromoso mes and genetic markers was also observed indicating that nonmendelian inhe ritance of one or a cluster of pathogenicity genes could also explain the skewed inhe ritance. It is conceivabl e that these genes lie on a dispensable chromosome as has been observe d for other fungal species (24,45). No cultivar isolate interactions were obser ved in field trials reported in chapter 5. This finding is consistent with the hypothesis that C. gloeosporioides employs a necrotrophic nutritional strategy, with little biological inte raction between the pathogen and its host. The occurrence of C. gloeosporioides on numerous hosts other than strawberry is also consistent wi th this strategy. Resistance to C. gloeosporioides and C. fragariae among cultivars was positively correlated, indicating that resistance mechanisms to these fungi overlap. In prev ious studies examining resistance to crown rot, C. fragariae was used to screen for resistan ce (28,44,48,81). The positive correlation

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126 between resistance to C. fragariae and C. gloeosporioides among cultivars suggested that cultivars identified in previous studies as being resistant to C. fragariae would also be useful in breeding programs for C. gloeosporioides resistance. Although a cultivar immune to crown rot was not id entified, resistance in the cultivar Treasure appeared superior to others examined. When crossed with a susceptible cult ivar, segregation of resistance among progeny indicated that a ma jor gene contributes to resistance in Treasure. Given that resist ance in Treasure is confe rred by a major gene product, breeding and selection of crown rot resistant offspring from Treasure is possible in a short time.

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135 102. Whiting, E. C., and Roncadori, R. W. 1997. Occurrence of Colletotrichum gloeosporioides on pokeweed and sicklepod stems in Georgia and pathogenicity on black locust. Can. J. Plant Path. 19:256-259. 103. Wingfield, B. D., Harrington, T. C., and Steimel, J. 1996. A simple method for detection of mitochondrial DNA polymorphi sms. Fungal Genet. Newsl. 43:56-60. 104. Young, R. A., and Kelly, J. D. 1996. Charact erization of the genetic resistance to Colletotrichum lindemuthianum in common bean differential cultivars. Plant Dis. 80:650-654. 105. Zhan, J., Kema, G. H. J., and McDonald, B. A. 2004. Evidence for natural selection in the m itochondrial genome of Mycosphaerella graminicola Phytopathology 94:261-267.

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136 BIOGRAPHICAL SKETCH Steven was born in Mount Clemens, Mi chigan, and grew up in Grand Haven, Michigan, on the shore of Lake Michigan. He received a Bachelor of Science degree in biology from the University of Michigan, Ann Arbor in 1988. From 1989 to 2000, he was employed at the Detroit Medical Center, where he was a research assistant in the Department of Endocrinology. He also rece ived a Master of Business Administration degree from Wayne State University in 1997, while living in Detroit. His mother, Marjory MacKenzie, was a psychiatric nurse before retirement and his father, Donald MacKenzie, was a general surgeon until his de ath in 2000. He has one brother and three sisters. He married Dr. Enas Sallam in 2000. Their daughter, Leila, was born in 2004.


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POPULATION STRUCTURE AND PATHOGENICITY OF Colletotrichum
gloeosporioides FROM STRAWBERRY AND NONCULTIVATED HOSTS IN
FLORIDA
















By

STEVEN JOHN MACKENZIE


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

UNIVERSITY OF FLORIDA


2005

































Copyright 2005

by

Steven John MacKenzie















ACKNOWLEDGMENTS

Most of all, I would like to thank my wife, Enas, for her support during the years I

spent in graduate school. I would also like to thank the staff and faculty at the Gulf Coast

Research and Education Center for their help. In particular, I appreciate the assistance of

Teresa Seijo for collecting oak and grape leaves, conducting C. gloeosporioides

isolations, PCR reactions and pathogenicity assays for me when I was in Gainesville,

Florida; of Jim Mertely for providing single-ascospore isolates from perithecia he found

on strawberry, helping with isolate identifications, and assisting with pathogenicity

assays; of Chang Xiao for doing the initial collection of C. gloeosporioides from

noncultivated hosts; of Natalia Peres for use of her laboratory and for providing

assistance and helpful discussions; and of Jim Sumler for his help in identifying wild

plants and doing crosses. I would also like to thank the plant pathology faculty at the

University of Florida in Gainesville. Thanks to all of the members of my graduate

committee for their efforts and use of their equipment, especially Pete Timmer, who

accepted me as a graduate student when I needed a major professor, and Dan Legard for

his continuing interest in the studies designed under his guidance.
















TABLE OF CONTENTS



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

LIST OF TABLES ......... ........ ................................... .......... .... ............ vi

LIST OF FIGURES .............................................. ........ ...... ............. viii

A B ST R A C T .......... ..... ...................................................................................... x

CHAPTER

1 IN T R O D U C T IO N ............................................................................. .............. ...

2 SEXUAL RECOMBINATION AND PATHOGENIC VARIATION AMONG
ISOLATES OF Colletotrichum gloeosporioides ON STRAWBERRY .....................8

In tro d u ctio n .................................................................................. 8
M materials and M methods ..............................................................................................10
Perithecium Production on Strawberry Petioles................................................. 10
Im aging and M orphology ......... .... ................................ ............ ............... 11
Fungal Isolates and Analyses ...................... .................11
L laboratory C roses .. .... .. ..... ..... .. ...... .... ....... ...... .. .................. ... 12
D N A Iso latio n ............................................................................................... 13
M arker A analysis ............... ................. .. .............................. .. ... ............... 14
Contour-clamped Homogeneous Electric Field Gel Electrophoresis..................17
P ath o g en city T ests............ .......................................................... .. .... .. .. .. .. 17
Statistical A analysis .......................... ............ ............... ........... 18
Results ......... .... ............................... ...............19
Perithecial M orphology ......................................................... .............. 19
A T -rich D N A A analysis ......................................................................... ...... 19
Characterization of Naturally Occurring Isolates..............................................20
Laboratory Crosses .................. ............................. .... .............. ... 21
D iscu ssio n ...................................... ................................................. 2 2

3 GENETIC AND PATHOGENIC ANALYSIS OF Colletotrichum gloeosporioides
ISOLATES FROM STRAWBERRY AND NONCULTIVATED HOSTS .............41

Intro du action ...................................... ................................................ 4 1
M materials and M methods ....................................................................... ..................43









Fungal Isolate Collections ............................................................................ 43
Extraction of Fungal D N A ........................................................ ............... 45
Species Identification ........................ .. ................................ .... ...... ...... 46
Pathogenicity Tests....................................................... ........ ..... ...... .. 47
Randomly Amplified Polymorphic DNA Markers ..........................................47
S statistical A n aly ses.................................................................... ................ .. 4 8
R e su lts ...............................................................................................4 9
Identification of Isolates ......................................................... .............. 49
Pathogenicity Tests............................... ............................... .. .. ...... .. 49
Randomly Amplified Polymorphic DNA Analyses................. ............. .....50
D discussion ............. .......................... .................... ........... ...... 52

4 SELECTION FOR PATHOGENICITY TO STRAWBERRY IN POPULATIONS
OF Colletotrichum gloeosporioides FROM NATIVE PLANTS ............................65

In tro d u ctio n ...................................... ................................................ 6 5
M materials and M methods .................................................................... ................... 67
Sampling Strategy and Isolate Codes ............ .............................................67
Fungal Isolation ............................................................. ... ....... ............... 68
DNA Extraction and PCR Amplifications .................................. ...............69
P ath og en city T ests............. ... ....................................................... .... .... .. ....7 1
Statistical A n aly ses........... ................................................ .......... ..... .... .. 72
R e su lts ..................... ...... ......... ..... .......... ....................... ................ 7 3
Species Identification and Population Structure...............................................73
P ath o g en city .................................................... ................ 7 5
D iscu ssion ............... .................................... ............................76

5 RESISTANCE OF STRAWBERRY CULTIVARS TO CROWN ROT CAUSED
BY Colletotrichum gloeosporioides ...................................................................91

In tro d u ctio n ...................................... ................................................ 9 1
M materials and M methods ....................................................................... ..................92
Plant M materials and Cultivation .................................... .................................... 92
Fungal Isolates and Inoculation Procedure............................... ...............94
Experimental Design and Statistical Analysis.........................................95
R results ..................................................................... ...... ................. 97
Disease Progression in Cultivar x Isolate Experiments ....................................97
Cultivar and Isolate Evaluation ........................................ ........ ............... 98
Inheritance of Crown Rot Resistance............... ......................................102
D iscu ssion ................................................................................................ ..... 103

6 C O N C L U SIO N .......... .................................................................. ......... ....... .. 122

L IST O F R E FE R E N C E S ........................................................................ ................... 127

BIOGRAPHICAL SKETCH ............................................................. ...............136




v















LIST OF TABLES


Table p

2-1 Glomerella cingulata/Colletotrichum gloeosporioides isolates from Dover, FL
used to compare AT-rich DNA banding patterns and for recombination studies.....32

2-2 Crown rot pathogenicity phenotype of single-ascospore isolates from eight
perithecia from naturally infected strawberry petioles ..........................................33

2-3 Segregation of mitochondrial DNA, fungicide sensitivity, RAPD bands, and
(CAT)5 bands from a cross between pathogenic crown isolate 97-15A and
nonpathogenic ascospore isolate P3-8 ........................................... ............... 34

3-1 Collection sites and host species for Colletotrichum spp. ........................................57

3-2 Isolates of Colletotrichum spp. collected from noncultivated hosts summarized
according to site and pathogenicity on strawberry plants............... ...................60

3-3 Isolates of Colletotrichum spp. collected from noncultivated hosts summarized
according to host species and pathogenicity on strawberry plants .........................61

3-4 Frequencies of RAPD bands for Colletotrichum gloeosporioides isolates from
strawberry and noncultivated hosts................................... .......................... 62

4-1 Frequencies of randomly amplified polymorphic DNA bands from
Colletotrichum gloeosporioides (C.g.) and Colletotrichum fragariae (C.f)
iso la te s ........................................................................ 8 3

4-2 Estimates of 0 for pairwise comparisons of Colletotrichum gloeosporioides
isolates from oak and grape hosts at four sites. ............................. ...................85

4-3 Pairwise estimates of 0 (above diagonal) for Colletotrichum gloeosporioides
populations at four sites and the estimated 90% confidence interval for 0 (below
d ia g o n a l) .......................................................................... 8 6

4-4 Percentage of isolates pathogenic on strawberry from oak (Quercus spp.) and
grape (Vitis spp.) lesions at four sites ............................................ ............... 87

4-5 Likelihood ratio statistics examining the effect of local strawberry production,
specific sampling site and native host species on the proportion of native host
isolates pathogenic to straw berry......................................... ......................... 88









5-1 Description of Colletotrichum isolates used in inoculation experiments
conducted over three seasons in Dover, Florida.....................................................112

5-2 Analysis of variance for three experiments evaluating incidence of crown rot in
relation to strawberry cultivar and isolate of Colletotrichum gloeosporioides .......113

5-3 Analysis of variance for 2003-2004 experiment evaluating incidence of crown
rot in relation to strawberry cultivar and isolate of Colletotrichumfragariae
alone or in com prison to C. gloeosporioides ........................................................ 114

5-4 Mean percent plant collapse of cultivars inoculated with Colletotrichum
gloeosporioides or C. fragariae during three seasons in Dover, Florida...............115

5-5 Mean percent plant collapse for Colletotrichum gloeosporioides or
Colletotrichumfragariae isolates used to inoculate strawberry cultivars during
three seasons in D over, Florida..................................... ............................ ........ 116
















LIST OF FIGURES


Figure p

2-1 Glomerella cingulata from strawberry petiole............ ................................ 35

2-2 AT-rich DNA banding pattern produced by digestion of total DNA with the
restriction enzym e H aeIII......... ................. ................... ................. ............... 36

2-3 Randomly amplified polymorphic DNA banding patterns of single-ascospore
isolates from eight Glomerella cingulata perithecia removed from strawberry
p e tio le s ........................................................................... 3 7

2-4 Randomly amplified polymorphic DNA banding patterns of two pathogenic and
two nonpathogenic isolates to strawberry and RAPD banding patterns of
progeny from crosses using these four isolates as parents................ ............ 38

2-5 Molecular markers for 97-15A, an isolate pathogenic on strawberry, and isolate
P3-8, an ascospore isolate nonpathogenic to strawberry ............... ..................... 39

2-6 Chromosomes ranging in size from 300 kb to 1100 kb for pathogenic crown
isolate 97-15A, nonpathogenic ascospore isolate P3-8, and progeny from a cross
betw een these isolates ...................................... ............... ..........40

3-1 A phenogram using unweighted pair group method with arithmetric averages
showing similarity (Dice) between Colletotrichum gloeosporioides and C.
acutatum isolates from noncultivated plants, strawberry crowns, citrus, and
m ango ..................................... .................. ................. ......... 64

4-1 Map showing the three locations where Colletotrichum gloeosporioides isolates
were sampled from lesions on oak and grape leaves.................................... 89

4-2 Unweighted pair group method with arithmetic averages phenogram showing
genotypic similarity between Colletotrichum isolates .......................................... 90

5-1 Mean percent mortality for strawberry cultivars calculated at weekly intervals
over the course of the growing season ............................................................. 117

5-2 Cultivar x isolate means for arc-sine-square root transformed disease incidences
calculated on a specific date during or at the end of the season............................ 118









5-3 Cultivar x species means for arc-sine-square root transformed disease incidences
calculated 125 days after inoculation with Colletotrichumfragariae or C.
gloeosporioides during the 2003-2004 season......................................................119

5-4 Distribution of transformed cumulative temperature until 50% plant death for
progeny inoculated with Colletotrichum gloeosporioides ...................................120

5-5 Distribution of percent plant mortality 42 days after inoculation for progeny
inoculated with Colletotrichum gloeosporioides ............................................. 121















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

POPULATION STRUCTURE AND PATHOGENICITY OF Colletotrichum
gloeosporioides FROM STRAWBERRY AND NONCULTIVATED HOSTS IN
FLORIDA

By

Steven John MacKenzie

December 2005

Chair: Lavern W. Timmer
Major Department: Plant Pathology

Colletotrichum crown rot, caused by Colletotrichum gloeosporioides, limits

strawberry transplant production in Florida summer nurseries and causes moderate plant

losses during the winter season. Marker data have shown that C. gloeosporioides crown

isolates are diverse and recombination contributes to this diversity. Glomerella

cingulata, the teleomorph of C. gloeosporioides, has been observed on petioles, but the

role of the meiotic cycle in crown rot disease is unknown. There is little evidence that the

primary inoculum for infections comes from imported transplants or from debris from

past seasons. Colletotrichum gloeosporioides has a broad host range and hosts other than

strawberry could contribute inoculum. In the current study, isolates from strawberry

crowns, noncultivated hosts, and perithecia were characterized using randomly amplified

polymorphic DNA markers, AT-rich DNA banding patterns, pathogenicity assays, and

laboratory crosses. Genetic data indicated the population from crowns produced









recombinant ascospores from perithecia and was found on noncultivated hosts close to

fields. Pathogenicity to strawberry was variable among perithecia and noncultivated host

isolates, but laboratory crosses indicated that sexual recombination occurred between

isolates with different pathogenicity phenotypes. The same C. gloeosporioides

population on strawberry was also found on two native hosts at sites distant from

strawberry fields. A low frequency of isolates pathogenic to strawberry at these sites

relative to sites close to strawberry fields suggests that there was selection for

pathogenicity.

Although strawberry transplants for winter production are rarely propagated in

Florida, crown rot resistant cultivars could make this desirable. To identify resistant

cultivars and to determine if resistance is isolate specific, plant mortality was evaluated

for cultivars following inoculation with an isolate differential. Repeatable differences in

resistance were observed among cultivars, but no cultivar x isolate interactions. The lack

of a cultivar x isolate interaction suggested limited biotrophic interaction between

pathogen and host. Crosses using a cultivar with superior resistance and a susceptible

cultivar as parents indicated that major genes contributing to resistance could be useful

for breeding. In conclusion, it is unlikely that crown rot introductions into nurseries or

fields can be prevented in Florida, but resistant cultivars could help make propagation of

transplants between seasons possible.














CHAPTER 1
INTRODUCTION

At least three distinct species of Colletotrichum are responsible for anthracnose

diseases of strawberry (Fragaria x aanassa Duchesne): Colletotrichum acutatum J. H.

Simmonds (80), C. gloeosporiodes (Penz.) Penz. & Sacc. (teleomorph Glomerella

cingulata (Stoneman) Spauld. & H. Schrenck) (52) and C. fragariae A. N. Brooks (9).

All three of these Colletotrichum species have been reported to cause fruit rot (49,52,80),

although in Florida and strawberry-producing regions around the world, C. acutatum

appears to be responsible for most fruit rot epidemics (30,92). Colletotrichum crown rot,

another economically important anthracnose disease, is caused by C. gloeosporioides and

C. fragariae (10,52). Both species produce a reddish-brown necrosis in crown tissue that

eventually causes plants to collapse (49). Infection of roots by C. acutatum may also

cause plants to collapse, but this pathogen does not appear to colonize the crown tissue

(37). Colletotrichumfragariae or C. gloeosporioides infection can also cause black leaf

spot (51), lesions on petioles, and lesions on runners (9,27).

The first report of C. fragariae causing crown rot was nearly 50 years ago in 1935

(10). In 1984 isolates described as G. cingulata, the teleomorph of C. gloeosporioides,

were reported to cause crown rot (52). These isolates were referred to by their

teleomorphic name based on their ability to form perithecia when grown alone in culture

and were distinguished from C. fragariae isolates by the color of their conidia in addition

to the ability to form perithecia (52). Although G. cingulata was reported from

strawberry plants in Florida, the plants appeared to have been infected in Arkansas,









Tennessee or North Carolina nurseries (53). Glomerella cingulata has also been isolated

from strawberry plants in Europe and Japan (30). In 1992, it was noted that, among

isolates classified as C. fragariae, a subset lacked pointed conidia and failed to produce

phialidic setae and these isolates were reclassified as C. gloeosporioides (43). Molecular

studies have confirmed that these isolates are distinct from those initially described as C.

fragariae (38). The reason that these populations were not distinguished earlier may

have been due to the belief that C. fragariae was simply C. gloeosporioides from

strawberry (51), since they have been considered synonymous (97). Although never

explicitly stated in the literature, molecular marker data from self-fertile G. cingulata

isolates and self-sterile C. gloeosporioides isolates indicate that they are from genetically

distinct populations (38,43). Currently, the self-sterile population from strawberry

appears to be prevalent in Florida, although genotypes of these isolates have not been

compared to reference homothallic isolates. Unfortunately, isolates often used in

molecular studies to represent the G. cingulatalC. gloeosporioides population from

strawberry were self-fertile and were not representative of the population in Florida,

where crown rot caused by C. gloeosporioides is a problem (13,30).

A population study examining variation of randomly amplified polymorphic DNA

(RAPD) marker data among C. gloeosporioides isolates on strawberry in Florida found

that the C. gloeosporioides population was diverse and recombining, as most markers

were in linkage equilibrium (92). Colletotrichum gloeosporioides also forms quiescent

infections on petioles that can be detected after petiole tissue senesces and the fungus

sporulates (66). Some of the isolates derived from these infections are pathogenic to

strawberry in crown inoculation tests, whereas others are not. Occasionally perithecia









with morphology consistent with that of G. cingulata are found among C.

gloeosporioides acervuli. Single-ascospore isolates from these perithecia are self-sterile

in culture, suggesting that they may be from the same population responsible for crown

rot. However, the genetic relationship between ascospore isolates and isolates from

diseased crown tissue has not been examined. In addition, the ability of these isolates to

cause crown rot has not been confirmed and recombination among progeny from

perithecia has not been validated.

In southeastern states such as Florida and Louisiana, strawberries are grown as an

annual crop. Plants are established from late September to early November onto raised,

methyl-bromide-fumigated beds covered with polyethylene film. Harvest usually begins

during the month of December in Florida. In this region, Colletotrichum crown rot is a

serious disease and is especially devastating if growers produce their own transplants in

summer nurseries (53), because both C. fragariae and C. gloeosporioides grow and

reproduce best under moist conditions at temperatures exceeding 25C (81). Along with

providing chilling to induce floral bud initiation, the high incidence of crown rot in

summer nurseries in Florida is one of the primary reasons why transplant production for

the winter season has moved to northern states and provinces of Canada (53). Although

the movement of summer nurseries out of Florida has dramatically reduced the incidence

of crown rot in production fields in this state, a portion of plants still become infected.

Analysis of the Colletotrichum species infecting crowns revealed that they were primarily

C. gloeosporioides (92). In contrast, C. fragariae accounted for most of the mortality due

to crown rot in Louisiana production fields (65).









In Florida, C. gloeosporioides does not appear to have the capability of surviving

over the summer between seasons on plant debris (93). Colletotrichum gloeoporioides

cannot be isolated from petioles of transplants shipped from northern latitudes into

Florida, although it can be obtained from petiole tissue after plants are set in the field

(66). Depending on the nursery source, C. acutatum can be isolated from petiole tissue of

transplants. Because C. gloeosporioides does not appear to be introduced into fields each

season on transplants or plant debris from the previous season, alternative host species

may play a role in providing primary inoculum for crown rot epidemics. Although there

is no genetic or pathogenicity data to confirm this hypothesis, several characteristics of

the C. gleoesporioides species assemblage indicate that this may be the case.

Colletotrichum gloeosporioides has a broad host range (69). Cross-inoculations indicate

that isolates can produce disease symptoms on hosts other than those from which they

were isolated (1). Although use of rapidly evolving genetic markers has identified host-

specific subpopulations within C. gloeosporioides (47,92), some isolates appear to lack

any host specificity (1). Colletotrichum gloeosporioides is also a common endophyte on

numerous tropical forest plants and there does not appear to be any discernible host

specificity among isolates from these plants (60).

The best way to control crown rot in Florida production fields is to eliminate it

from nurseries where plants are propagated (53). Movement of transplant production

from Florida to temperate regions has effectively done this. If Colletotrichum crown rot

is observed during the production season, it is usually at either the beginning or end of

the season when temperatures are high and generally no more than a few percent of

plants in a field die. Because the conditions for growth and spread of Colletotrichum









gloeosporioides are not ideal during the relatively cool weather that coincides with the

production season, it is not likely to be the focus of chemical control programs. Weekly

fungicide applications are generally designed to control gray mold, anthracnose fruit rot,

and powdery mildew. However, both preventive and systemic fungicides used in these

programs have activity against C. gloeosporioides. Prior to the migration of nurseries to

cooler climates, management of soil fertility, fungicide applications, host resistance, and

sanitary measures were all employed to control the disease. Measures included reduction

of fertilizer to maintenance levels during July and August, two fungicide applications per

week and after rain events, careful selection of runners to be used in nurseries, and use of

resistant cultivars such as Dover (53). Fungicides with activity against C.

gloeosporioides include captain, benzimidazoles, and Qol fungicides (J. Mertely, personal

communication). Resistant cultivars can be successfully propagated in Florida and local

production of transplants is one means of lowering costs to growers, but cultivars such as

Dover that are relatively resistant to crown rot are not grown on a large scale because

they lack desired yield and fruit characteristics. Levels of resistance to crown rot in

cultivars currently used for strawberry production in Florida have not been documented

and it is conceivable that one or more of these cultivars could be sufficiently resistant to

crown rot to justify attempts at local propagation. Identification of cultivars with

resistance in a genetic background with desirable fruit and yield properties might also

help future breeding efforts. The resistance of cultivars to multiple C. fragariae isolates

has been investigated (28,48,81). The resistance of cultivars to self-sterile C.

gloeosporioides isolates that are responsible for most of the crown rot observed in Florida









has not been. This species is more variable than C. fragariae and isolate specific

resistance could be more prevalent.

This dissertation was designed to address some of the fundamental questions that

remain regarding crown rot caused by C. gloeosporioides in Florida. Original research is

presented in the following four chapters. Chapter 2, the first chapter following this

introduction, examines the genetic relationship of single-ascospore isolates from

perithecia collected from senescent petioles to isolates known to produce crown rot on

strawberry. Both pathogenicity and mating compatibility of isolates are examined to

determine if the perithecia observed on strawberry petioles represent the teleomorph of

the self-sterile population that causes crown rot. Chapter 3 examines the genetic

relationship between isolates from strawberry crown and isolates from noncultivated

hosts growing close to strawberry fields. In this chapter, marker frequencies, as opposed

to the ability of isolates to mate, are examined along with the occurrence of the

pathogenic phenotype to determine if noncultivated hosts can provide inoculum for

crown rot epidemics. In chapter 4, C. gloeosporioides from two native host species are

examined at sites distant from strawberry production and compared to those close to

strawberry production. Because the strawberry industry in Florida is highly concentrated

(5) and strawberry plants are not native to Florida (26), sampling host populations away

from strawberry fields may provide information regarding the origin of the pathogen and

whether selection for pathogenicity on strawberry might occur in areas where it is grown

in abundance. In chapter 5, resistance of cultivars to a group of C. gloeosporioides

isolates is examined to determine the mechanism of resistance to C. gloeoporioides and

to identify cultivars with genes that could be used in future breeding programs. In






7


chapters 2 and 4, data regarding the inheritance of genes controlling pathogenicity for a

cross between C. gloeosporioides isolates and resistance to crown rot for a cross between

strawberry cultivars are presented.














CHAPTER 2
SEXUAL RECOMBINATION AND PATHOGENIC VARIATION AMONG
ISOLATES OF Colletotrichum gloeosporioides ON STRAWBERRY

Introduction

Crown rot of strawberry caused by self-fertile Glomerella cingulata strains was

first reported in Florida in 1984 (52). In addition to the ability of these strains to produce

fertile perithecia when grown alone in culture, they were distinguished from

Colletotrichumfragariae by the production of white rather than salmon-colored conidia.

A more recent evaluation of Colletotrichum species responsible for strawberry diseases

indicated that isolates previously characterized as C. fragariae consisted of two

morphologically distinct groups of isolates (43). One group of isolates conformed to the

initial description of C. fragariae and the other group was reclassified as C.

gloeosporioides. Isolates in the second group produced more oblong conidia that were

rounded at both ends and no conidia were formed on the setae. Unlike isolates previously

classified as G. cingulata, fertile perithecia were not formed when these isolates were

grown singly in culture. However, these isolates formed fertile perithecia

morphologically similar to G. cingulata when paired in culture. No teleomorph has been

described for isolates classified as C. fragariae. Molecular analysis using AT-rich DNA

band patterns, arbitrarily primed polymerase chain reaction (PCR), and sequence data

from the internally transcribed spacer 1 region of the rDNA repeat revealed that isolates

classified as C. fragariae were similar to each other, whereas G. cingulatalC.

gloeosporioides isolates fell into two groups with a high level of similarity among









isolates within each group (38,39,83). These two G. cingulatalC. gloeosporioides groups

were designated Cgl-1 and Cgl-2 (38,39). Isolates found to form the G. cingulata state

when grown singly fell in the Cgl-1 group whereas those isolates that reproduce only

clonally or outcross were in group Cgl-2 (39,43).

In Florida, C. gloeosporioides is the Colletotrichum species most frequently

isolated from the crowns of wilted strawberry plants (92). Self-fertility within this C.

gloeosporioides population is rare. Analysis of RAPD marker data revealed a high level

of diversity among isolates and a low level of linkage disequilibrium among markers

(92). Both findings are consistent with genetic recombination within this population,

although there is no direct evidence. Colletotrichum gloeosporioides also infects

strawberry petioles producing asymptomatic quiescent infections in Florida (66).

Acervuli are produced after the petiole tissue has senesced. Some isolates from petiole

tissue are capable of causing crown rot and plant collapse and others are not. In addition

to acervuli of C. gloeosporioides, perithecia morphologically similar to G. cingulata are

occasionally observed on senescent petioles (66). The relationship of isolates produced

from ascospores from these perithecia to isolates from crown tissue has not been

determined. It is also not certain whether the perithecia on petioles are from a self-fertile

strain or the result of recombination between two heterothallic strains nor whether

ascospore isolates vary in pathogenicity as do isolates from acervuli.

This study examines the relationship between isolates of G. cingulata from

perithecia on strawberry petioles to C. gloeosporioides isolates known to cause disease

on strawberry. AT-rich DNA banding patterns of single-ascospore isolates from

perithecia were compared to those from the two genotypically distinct G. cingulata/C.









gloeosporioides groups known to cause disease on strawberry. Recombination among

progeny was evaluated by comparing RAPD markers of isolates from the same

perithecium. The ability of crown rot isolates, pathogenic isolates from single

ascospores, and nonpathogenic isolates from single ascospores to recombine was

evaluated in laboratory crosses. In addition to studies conducted to define biological

species boundaries, segregation analysis of marker data and pathogenicity phenotype was

conducted for a cross between a nonpathogenic ascospore isolate and a crown rot isolate.

Materials and Methods

Perithecium Production on Strawberry Petioles

Perithecia were collected from senescent petioles during a study to evaluate the

effectiveness of freezing tissue to detect latent infections of Colletotrichum spp. on

strawberry (66). In the study, healthy petioles with no visible lesions were removed from

field-grown plants in plots untreated with any fungicides, cut to lengths of 5 to 7 cm and

frozen for 1 to 2 h at -150C. After thawing, petioles were surface sterilized for 1 min in

0.5% NaOCl plus 20 tl/L Tween 20, rinsed with sterile water, and placed on moistened

filter paper inside petri dishes. Petri dishes were placed in clear plastic boxes and

incubated on a laboratory bench at 23-250C under continuous fluorescent light. Petioles

were monitored for perithecia production for 21 days. When detected, a single

perithecium was transferred with a scalpel to a microscope slide and gently crushed in a

drop of sterile water between the slide and a cover slip. If ascospores were present in a

perithecium and Colletotrichum conidia were not observed, as much vegetative tissue as

possible was removed and the suspension containing a cluster of asci and ascospores was

transferred to 0.75 mL sterile water in a test tube. Ascospore release from asci was









stimulated by repeatedly pipetting the suspension. The suspension was then spread on

semi-selective media (16 g potato dextrose broth, 14 g agar, 250 mg ampicillin, 150 mg

streptomycin sulfate, 5 mg iprodione, 100 [l tergitol, and deionized water to 1 L) and

incubated overnight. Germinating ascospores were subsequently transferred from the

semi-selective media with a sterile scalpel and transferred onto potato dextrose agar

(PDA).

Imaging and Morphology

Perithecia from field samples were examined using a Zeiss Stemi SV-6 stereo

dissecting scope (Carl-Zeiss-Stiftung, Oberkochen, Germany) and asci with a Zeiss

Axiolab microscope using brightfield optics at 400x magnification. Microscopic images

were captured using a Spot digital camera system (Diagnostic Instruments, Inc., Sterling

Heights, MI). Dimensions and morphology of conidia were based on measurements

taken on 75 conidia from three isolates.

Fungal Isolates and Analyses

Eight perithecia (P1-P8) were recovered from strawberry cultivars 'Camarosa',

'Strawberry Festival', and 'Rosa Linda' during the 1998-1999 and 2000-2001 growing

seasons at the University of Florida Gulf Coast Research and Education Center (GCREC)

in Dover, Florida. Seven to ten ascospore isolates were obtained from each perithecium.

Pathogenicity to strawberry was determined for all ascospore isolates. The AT-rich DNA

banding pattern was determined for one isolate from each perithecium and compared to

banding patterns from Cgl-1 and Cgl-2 genotype isolates known to be pathogenic on

strawberry. To determine if ascospores from perithecia on petioles were produced by

recombination between two or more fungal strains, RAPD DNA bands were amplified









from a subset of four to six isolates from each perithecium. In addition to progeny from

perithecia found on petiole tissue, RAPD bands from progeny of laboratory crosses

between two pathogenic ascospore isolates, two nonpathogenic ascospore isolates and a

cross using pathogenic and nonpathogenic ascospore isolates as parents were analyzed

for recombination. Pathogenic ascospore isolates P1-9 and P8-1 and nonpathogenic

ascospore isolates P2-6 and P3-3 were used as parents in these crosses. For quick

reference regarding the perithecium and pathogenicity phenotype of these isolates, they

are also referred to as PI-path, P8-path, P2-nonpath and P3-nonpath respectively.

Isolates 97-15A and 99-51, representative of C. gloeosporioides genotype Cgl-2 came

from crown rot samples submitted to the GCREC diagnostic clinic by local growers in

1997 and 1999. Cgl-1 genotype isolates 311 and 329 were collected by C. M. Howard in

Florida. These isolates were characterized previously with respect to morphological traits

and AT-rich banding pattern (38,43). Crown isolate 97-15A was also used as a parent in

a cross with nonpathogenic ascospore isolate P3-8. A description of all isolates used in

this study and specific analysis in which they were employed is given in table 2-1.

Laboratory Crosses

Laboratory crosses were performed in 9 cm diameter plastic petri plates on sucrose-

free Czapek-Dox medium (2 g/L NaNO3, 1 g/L K2HPO4, 0.5 g/L MgSO4-7H20, 0.5 g/L

KC1, and 0.01 g/L FeSO4-7H20) containing 1.5% agar overlaying a piece of Whatman

No. 3 filter paper (Whatman International, Maidstone, UK) (21). Isolates were

inoculated on opposite sides of a plate approximately 7 cm apart and the plates incubated

under fluorescent light at 240C. Perithecia formed a line at the point of contact between

the two isolates. Few, if any, acervuli were present along the line of intersection between









isolates, greatly reducing the likelihood of conidial contamination. Single-ascospore

isolates were obtained from fertile perithecia as described above for isolations from

petiole tissue.

DNA Isolation

Mycelia was collected from 2- to 4-day-old cultures grown in 100 mL of Emerson

media (4g/L yeast extract, 15 g/L Soluble Starch, lg/L K2HPO4, and 0.5 g/L MgSO4) by

vacuum filtration through Whatman No. 3 filter paper. After transfer to 15 mL tubes,

mycelia was dried overnight in a centrifugal evaporator. Sixty mg of the dried mycelia

were then suspended in 750 [tl of DNA extraction buffer consisting of 700 mM NaC1, 50

mM Tris(pH 8.0), 10 mM EDTA(pH 8.0), 1% CTAB, and 1% P-mercaptoethanol and

heated to 650C for 2 h with periodic shaking. Particulate material was pelleted by

centrifugation at 12,000x g for 10 min, the supernatant removed, and extracted once with

chloroform:isoamyl alcohol (24:1). Two volumes of 100% ethanol were added to the

aqueous extract and the mixture incubated at room temperature for 10 min. Nucleic acids

were pelleted from the ethanol solution by centrifugation at 12,000x g for 10 min.

Subsequently the pellet was washed with 100% ethanol and resuspended in 400 [tl Ix TE

buffer containing 10 [tg/mL RNase for 1 h at 37C. Ribonuclease was removed from the

nucleic acid solution by extraction with 400 dtl phenol/chloroform/isoamyl alcohol

(25:24:1). To the aqueous extract, 1/10 volume 3 M sodium acetate and 2.5 volumes of

ethanol were added to precipitate DNA. This solution was incubated at -200C for 1 h and

the DNA pelleted at 12,000x g for 10 min. The DNA pellet was washed once with 1 mL

80% ethanol, dried, suspended in 100 [tl lx TE buffer, and stored at -200C.









Marker Analysis

AT-rich DNA bands were identified by digesting 5 to 10 tg genomic DNA with

the restriction enzymes HaeIII or MspI (103). Digested DNA was separated on a 1%

agarose gel in 1 xTBE buffer for 24 h at 50 V and subsequently stained with ethidium

bromide. Successful differentiation of Colletotrichum species and subpopulations using

this technique has previously been demonstrated (38).

Randomly amplified DNA fragments were obtained using the tetranucleotide repeat

primers (ACTG)4 and (GACA)4, the trinucleotide repeat primer (TCC)5, and two short

oligonucleotides 5'-GTGAGGCGTC-3' and 5'-GATGACCGCC-3' referred to as OPC-2

and OPC-5 (Operon Technologies, Alameda, CA). Amplifications were carried out

under mineral oil in a 20-tl volume containing Ix reaction buffer (50 mM Tris(pH 8.3),

0.25 mg/mL BSA, 2 mM MgCl2, 0.5% Ficoll, and 1 mM Tartrazine), 200 [tM dNTP, 1

unit Taq polymerase and 20 pmol primer/reaction [primers (ACTG)4, (GACA)4, and

(TCC)5] or 8 pmol primer/reaction [primers OPC-2 and OPC-5]. Cycling parameters for

the PCR reactions consisted of a 5-min denaturing step at 950C followed by 30 cycles of

1 min at 950C, 1 min at 480C, and 2 min at 720C for primers (ACTG)4 and (GACA)4; 34

cycles of 1 min at 950C, 1 min at 460C, and 1.5 min at 720C for primer (TCC)5 or 38

cycles of 1 min at 950C, 1 min at 350C, and 2 min at 720C for primers OPC-2 and OPC-

5. The amplified products were separated by electrophoresis through a 1.5% high

resolution blend agarose (3:1) gel or 2% analytical grade agarose gel in Ix TAE buffer.

Gels were photographed on a UV transilluminator after staining with ethidium bromide.

DNA fingerprints were obtained for progeny from the cross between crown rot

isolate 97-15A and nonpathogenic perithecium isolate P3-8 using a digoxigenin-labeled









(CAT)5 oligonucleotide fingerprinting probe. For this analysis, 5 to 10 pg ofPstI

digested DNA were separated on a 1% gel in x TAE for 16 to 24 h at 25 to 35 V. Gels

were incubated subsequently for 30 min in 1.5 M NaCl/0.5 N NaOH buffer followed by

30-min and 15-min incubations in 1 M Tris (pH 7.4)/1.5 M NaC1. Capillary transfer of

DNA to nylon membrane was conducted with 10x SSC buffer overnight. DNA was UV

cross-linked to the membrane prior to prehybridization in 5x SSC buffer, 0.1% N-lauryl-

sarcosine, 0.02% SDS, and 1% blocking reagent (Roche Applied Science, Mannheim,

Germany) at 300C for 4 h. Hybridization was carried out in the same buffer with 10

pmol/mL oligonucleotide probe at 300C overnight. Following hybridization, blots were

washed twice with 2x SSC/0.1 % SDS for 5 min at room temperature and twice with

0.5x SSC/0.1 % SDS at 30C for 5 min. The probe was detected with an anti-

digoxigenin Fab fragment conjugated to alkaline phosphatase and NBT/BCIP color

substrate according to instructions supplied with a DIG nucleic acid detection kit (Roche

Applied Science, Mannheim, Germany).

Resistance to benomyl among progeny of the cross between isolates 97-15A and

P3-8 was determined by growing fungi on PDA amended with 5 [g/mL benomyl.

Susceptible isolates did not grow at this concentration, whereas growth of resistant

isolates was not affected. Benomyl resistance was found to segregate at a 1:1 ratio in

crosses with Glomerella graminicola, and a point mutation in a P-tubulin gene was

shown to confer benomyl resistance in C. gloeosporioides f. sp. aeschynomene (14,95).

A polymorphism observed among AT-rich DNA bands was identified with the

restriction enzyme MspI and used to determine mitochondrial inheritance among progeny

of the cross between 97-15A and P3-8. That the polymorphic MspI band was comprised









of mitochondrial DNA was determined by comparison to Mspl-digested DNA from

purified mitochondria. Purified mitochondrial DNA was isolated from approximately 6 g

wet weight mycelia collected onto a filter disk using a modification of the method

described for isolation ofEpichloe typhina mitochondrial DNA (77). Mycelia was

ground in 30 mL buffer containing 15% sucrose, 10 mM Tris-HCl(pH 7.5), and 0.2 mM

EDTA(pH 7.5) at 4C. Nuclei and cellular debris was removed by centrifugation at

1,000x g for 10 min and the supernatant was saved. The pellet was re-extracted in 20 mL

buffer, ground, and debris removed by centrifugation at 1,000x g for 10 min. The

supernatant from both centrifugations was pooled and centrifuged at 15,000x g for 15

min. The pellet was suspended in 10 mL 20% sucrose, 10 mM Tris-HCl(pH 7.5), and

0.01 mM EDTA(pH 7.5) and centrifuged at 15,000x g for 15 min. Following

centrifugation, the pellet was resuspended in 5 mL buffer containing 1.75 M sucrose, 10

mM Tris-HCl(pH 7.5), 5 mM EDTA(pH 7.5), 12 mM MgC12, 100 [g/mL DNase, and 50

[g/mL RNase and incubated for 1.5 h at 40C. Mitochondria were pelleted from this

solution at 20,000x g for 10 min. The pellet was suspended in 5 mL of the same buffer

without MgC12 or nuclease and pelleted a second time at 20,000x g. The pellet was then

suspended in 0.5 mL lysis buffer (0.44 M sucrose, 1% SDS) and incubated at 370C for 30

min. Lysis buffer was extracted once with phenol :chloroform:isoamyl alcohol (25:24:1)

followed by an extraction with chloroform:isoamyl alcohol. DNA was precipitated by

addition of 1/10 volume 3 M sodium acetate and 2.5 volumes ethanol. DNA was

subsequently washed with 70% ethanol, dried, and resuspended in x TE.









Contour-clamped Homogeneous Electric Field Gel Electrophoresis

High molecular weight DNA was prepared from agarose embedded conidia using a

modification of the method described for C. lindemuthianum (72). Conidia from 5- to 7-

day-old cultures were harvested in sterile water and the concentration of conidia

determined using a haematocytometer. Conidia were pelleted and suspended in SCE (1

M sorbitol, 50 mM sodium citrate, 50 mM EDTA, pH 5.7, and 20 tM DTT) containing

75 mg/mL glucanex at a concentration of 8 x 108 conidia/mL. An equal volume of 1.2%

low melting point agarose in SCE was then added to the conidial suspension at 420C and

the mixture cast into 10 mm x 5 mm x 0.5 mm wells. Once solidified, plugs (1 to 4 each)

were incubated in 0.5 mL SCE buffer at 370C overnight. The plugs were then rinsed with

1 mL of EST (0.5 M EDTA, 0.1 M Tris-HC1, and 1% lauryl sarcosyl, pH 9.5) and

incubated in 1 mL EST containing 2 mg/mL proteinase K at 500C for 48 h with one

change of solution. After the proteinase digestion was complete, the buffer was removed

and plugs incubated 0.5 h in 1 mL 50 mM EDTA (pH 8.0) at room temperature. This

incubation was repeated twice with fresh buffer. Plugs were then stored at 40C in 50 mM

EDTA (pH 8.0) until used. Chromosomes were separated on a CHEF-DR II pulsed field

electrophoresis system (Bio-Rad, Hercules, CA) at 200 V in 0.5x TBE buffer using a run

time of 24 h and 60 to 120 s ramped switch time. This run condition resolves

chromosomes less than 1200 kb in length.

Pathogenicity Tests

To determine if isolates were pathogenic on strawberry, approximately 0.1 mL of 1

x 106 conidia/mL inoculation solution was injected directly into crown tissue of

greenhouse grown strawberry plants (cv. Camarosa) as described previously (66).









Conidial suspensions were prepared in sterile water from 7-day-old cultures grown on

PDA. Plants injected with sterile water only served as controls. Plants were monitored

weekly over 28 days for symptoms of crown rot (wilting and collapse of plants). For

single-ascospore isolates from petiole tissue, three plants were inoculated with each

isolate on two dates. Isolates that caused the collapse of two or more plants for both

inoculation dates were classified as pathogenic and those which failed to cause collapse

of at least two plants for both inoculations were classified as nonpathogenic. If an isolate

caused collapse of two or more plants after one inoculation date and not the other, the

assay was repeated a third time and data from this experiment used to classify the isolate.

For progeny from a cross between the crown rot isolate 97-15A and nonpathogenic

perithecium isolate P3-8, pathogenicity was determined from inoculation of three plants

on a single date.

Statistical Analysis

A hierarchal analysis using theta (0) was employed to determine if there was

population subdivision between perithecia that yielded pathogenic isolates and perithecia

that had only nonpathogenic isolates. The perithecium that an isolate was obtained from

defined the subpopulation. Values of 0 were calculated using the method of Weir and

Cockerham (99) with confidence intervals generated by bootstrapping over loci. Theta

measures the correlation of alleles from individuals in the same population relative to all

populations and is described by the equation (Q q)/(1 q), where Q is the probability

that two randomly sampled genes within a population are the same allele and q is the

probability that genes randomly selected from different populations are the same allele

(22). A hierarchal analysis was required due to repeated sampling within perithecia. All









values of were calculated using the software program TFPGA (Utah State University,

Logan, UT). Chi-square analyses and a Wilcoxon-Mann-Whitney test were done using

the SPSS 8.0 statistics package (SPSS Inc., Chicago, IL).

Results

Perithecial Morphology

The proportion of petioles with at least one perithecium ranged from 0% to 10% of

those examined, depending on the sample date. Perithecia were typically globose,

approximately 100 to 400 |tm in diameter, dark, covered with gray mycelia, and found

among acervuli of C. gloeosporioides (Fig. 2-1A). Each perithecium contained tens of

asci holding three to eight ascospores each (Fig 2-1B). Asci were cylindrical to clavate

and contained ascospores in both linear and alternately biserrate arrangements. Ostioles

were not discernible. Ascospore shape was variable both within and among perithecia.

Allantoid, ellipsoid, and oblong ascospores were observed in squashes. No fertile

perithecia were observed in cultures of single-ascospore progeny, indicating that isolates

were not homothallic when cultured on artificial substrate. The size and shape of conidia

from isolates grown in culture were consistent with C. gloeosporioides from both

strawberry and nonstrawberry hosts. Average conidial length was 16.8 0.32 |tm and

width 5.07 0.037 |tm. Ninety-five percent were oblong with obtuse ends.

AT-rich DNA Analysis

AT-rich DNA banding patterns for the two Cgl-1 genotype isolates from strawberry

(isolates 311 and 329) were identical to one another and the two isolates with a Cgl-2

genotype (isolates 97-15A and 99-51) were identical to one another (Fig. 2-2). Dice

similarity between the two genotypes was 0.33. Isolates from six of the eight naturally









occurring perithecia (P1, P3, P4, P5, P6, P8) had AT-rich DNA banding patterns identical

to the pattern of the Cgl-2 genotype isolates (Similarity = 1.0). The banding patterns of

the two unique perithecial isolates (P2 and P7) were more similar to the Cgl-2 genotype

than to the Cgl-1 genotype (0.83 vs 0.33 and 0.5) with the gain or loss of a single

restriction site able to account for the difference between the pattern for these isolates and

those for Cgl-2 isolates from strawberry.

Characterization of Naturally Occurring Isolates

RAPD markers were used to determine if segregation was occurring among

isolates from the same perithecium. Using five RAPD primers, eleven polymorphic

bands consistently amplified from the template DNA of single-ascospore isolates

collected from perithecia on petioles. Bands at 2.15 kb and 1.15 kb for the primer

(ACTG)4; 0.9 kb for primer (GACA)4; 2.0 kb and 1.9 kb for primer (TCC)5; 1.9 kb for

primer OPC-2; and bands at 2.15 kb, 2.0 kb, 1.8 kb, 1.6 kb, and 1.2 kb for primer OPC-5

segregated among progeny from at least one perithecium (Fig 2-3). For each of the eight

perithecia, between one and five of the bands were different among progeny. Four of

eight perithecia (P2, P3, P4, P6) contained only nonpathogenic isolates as determined by

the greenhouse bioassay and two perithecia contained only pathogenic isolates (P5 and

P8) (Table 2-2). Of the two perithecia with progeny that segregated for pathogenicity,

only perithecium 1 had multiple representatives of each phenotype. Of the 68 isolates

tested, the pathogenicity phenotype was the same in repeated experiments for 62 isolates.

Of the six isolates in which there was a conflict in scoring, two were classified as

pathogens and four as nonpathogens based on a third assay. Three of the four isolates

classified as nonpathogens came from perithecia with progeny that were both pathogenic









and nonpathogenic. Based on RAPD markers, there was no evidence for population

subdivision between perithecia with progeny pathogenic on strawberry and those with

only nonpathogenic progeny (0 = 0.037, 90% C. I. -0.078 to 0.149), although there was

strong support for correlation of RAPD bands among progeny from the same perithecium

(0 = 0.515, 90% C. I. 0.393 to 0.634).

Laboratory Crosses

Thirty-six of 80 crosses attempted between single-ascospore isolates or between

single-ascospore isolates and isolates from diseased crown tissue yielded perithecia with

mature ascospores. Some isolates successfully mated with both parents that were

compatible with one another in a third cross, indicating that mating compatibility was not

determined by a single mating type locus with two alleles. Viable ascospore progeny

were obtained from crosses in which both parents were nonpathogenic on strawberry,

both parents were pathogenic on strawberry, as well as with parents that had different

pathogenicity phenotypes. Recombination among progeny from one cross for each of

these parent combinations was examined using RAPD markers. Of four progeny

examined from each cross, at least three were recombinants (Fig 2-4), indicating that

recombination had occurred in these crosses.

Forty-six progeny from the cross between isolate 97-15A, from a crown-rot

affected plant and isolate P3-8, a nonpathogenic ascospore isolate, were evaluated for

pathogenicity using the greenhouse bioassay. Of these 46 isolates, two were

nonpathogenic and 44 were pathogenic on strawberry. A 2.5-kb MspI band observed

only in mitochondrial DNA of isolate P3-8 was inherited by all progeny (Fig 2-5A, Table

2-3). Putative genomic DNA markers included benomyl sensitivity, three RAPD bands,









and three (CAT)5 repeat bands (Fig 2-5B, 2-5C and 2-5D). Segregation of the 1.6-kb

OPC-5 RAPD band, the 0.9-kb (GACA)4 band, and two of the three bands identified by

the (CAT)5 repeat probe diverged from the expected 1:1 ratio assumed under the

hypothesis that these markers randomly assort and identify a single site within the

genome (P < 0.05) (Table 2-3). Segregation of the 1.8-kb OPC-5 RAPD band, benomyl

resistance, and one of the (CAT)5 repeat bands did not differ significantly from a 1:1

ratio. Of 25 progeny scored for all seven markers, three isolates inherited all of the

dominant markers examined. It is unlikely that progeny were derived from

contaminating parental material since only one of the progeny had a phenotype that was

identical to either parent. The genotypes of the two isolates that were not pathogenic on

strawberry were distinct from either parent.

Chromosomes in the size range of 220 to 1100 kb were very different between the

parental isolates (Fig 2-6). Isolate 97-15A had four chromosomes ranging from 730 to

940 kb, whereas isolate P3-8 had five chromosomes ranging from 350 kb to 640 kb. The

sum of sizes in this range was greater for isolate 97-15A than it was for isolate P3-8

(3310 kb vs. 2240 kb). Chromosome numbers for progeny ranged from three to six.

Most progeny had either a different number of chromosomes or different sized

chromosomes from either parent. The total length of chromosome DNA for the two

isolates that failed to cause crown rot on strawberry was less than it was for ten isolates

pathogenic on strawberry (1875 kb vs 3411 kb, P = 0.03, Mann-Whitney-Wilcoxon test).

Discussion

Perithecia with morphology consistent with G. cingulata were observed on

strawberry petioles in a previous study, however the genetic relationship between single-

ascospore isolates from these perithecia and isolates known to infect crown tissue was not









investigated (66). Self-sterile C. gloeosporioides isolates are most commonly isolated

from crown tissue in Florida, although homothallic G. cingulata strains have also been

isolated from strawberry in this state. It was conceivable that the population that

produced the perithecia observed on petiole tissue came from either one of these

populations or a population unrelated to those responsible for crown rot. In addition to

differences in the ability of isolates to self-fertilize, the G. cingulata C. gloeosporioides

populations on strawberry can be distinguished from one another based on AT-rich DNA

banding patterns. Self-fertile isolates have a Cgl-1 genotype banding pattern and self-

sterile isolates have a Cgl-2 genotype banding pattern. The AT-rich DNA banding

patterns observed for perithecial isolates indicate that they are from the same population

or are closely related to isolates with the Cgl-2 genotype, which are most frequently

isolated from crowns in Florida. Sexual reproduction has been suggested as playing a

role in the reproduction of this population, since it is comprised of genetically diverse

isolates and linkage disequilibrium among RAPD bands is not observed (92). The

occurrence of ascospore isolates with identical or similar AT-rich DNA banding patterns

to isolates from crown tissue supports the hypothesis that isolates responsible for crown

rot reproduce sexually on strawberry. Further evidence for the occurrence of sexual

recombination in this population comes from the analysis of RAPD banding patterns.

From each perithecium examined, at least two unique banding patterns were observed

among progeny indicating that ascsopores were produced by recombination of parental

strains.

Because there is no information on parental genotypes for perithecia collected from

field material, it is more difficult to be certain that unique banding patterns do not result









from poor reproducibility of RAPD bands. Evidence that RAPD genotype differences

were representative of true genetic differences comes from the ability to reproducibly

amplify scored bands from the same isolate and the observation that bands polymorphic

among progeny from laboratory crosses were also different between parents. Further

evidence that band differences were real and not artifacts of the PCR reaction comes from

the correlation of marker data among isolates from the same perithecium. As measured

by 0, the correlation of marker data among isolates from the same perithecium was 0.515.

Assuming that only one male parent gives rise to ascogenous hyphae, RAPD bands

segregate among progeny in a 1:1 ratio, and that there is random mating among

individuals within a population, the expected value of 0 for a heterothallic fungus would

be 0.5. This value is well within the 90% confidence interval calculated for 0. The

expected value of 0 would be 1 for completely self-fertile isolates, and 0, if ascospores

from the same perithecium are unrelated. Assuming that a fungal population is

heterothallic, processes such as nonmendelian inheritance of markers would tend to bias

measurements of 0 towards 1 and multiple male parents toward 0. Although no attempt

was made to determine the effect these processes might have on estimates of 0 in this

study, segregation of markers from crosses conducted in the laboratory indicate that

nonmendelian inheritance might bias the estimate of 0 upwards, whereas analysis of

ascospore isolates collected from the field for other fungi indicate that multiple male

parents could bias 0 downward (34).

Both pathogenic and nonpathogenic single-ascospore isolates were obtained from

perithecia. Most of the perithecia yielded ascospores with only one phenotype or the

other. Although three of four perithecia yielding pathogenic ascospore isolates had AT-









rich DNA genotypes identical to three of the perithecia yielding only nonpathogenic

ascospore isolates, it is conceivable that isolates with different pathogenicity phenotypes

derived from different populations. This would occur if the mutation rate in

mitochondrial DNA, which accounts for the bulk of AT-rich bands, was not fast enough

to produce detectable polymorphisms to distinguish the two groups. Several additional

lines of evidence, however, support the hypothesis that pathogenic and nonpathogenic

isolates derive from the same population. These include the ability of pathogenic and

nonpathogenic isolates to produce recombinant offspring when crossed on agar, the

occurrence of both phenotypes among isolates from perithecia 1 and 7, and there was no

evidence for population subdivision based on RAPD band frequencies, although this

analysis probably suffered from reduced statistical power as progeny from the four

perithecia in each group only represented eight parental genotypes.

The most frequently occurring AT-rich DNA banding pattern from perithecia

isolates matched that for isolates from strawberry crown, indicating that they are both

derived from the same population. However, as noted above, mitochondrial DNA may

fail to evolve fast enough to identify a recent divergence between the two groups.

Additional evidence that crown and perithecia isolates are from the same population

comes from the ability of strawberry crown isolates to successfully cross with perithecial

isolates on agar. RAPD markers were not amplified from a population of crown isolates

in this study. However, in the next chapter band frequencies from a population of

strawberry crown isolates are reported. A comparison of these frequencies to those

observed among perithecia provided no evidence for population subdivision (data not

reported).









The cross between a crown isolate and a nonpathogenic perithecium isolate yielded

a highly skewed distribution of progeny pathogenic on strawberry, suggesting that

pathogenicity on strawberry is determined by genes at more than one locus or

nonmendelian segregation of pathogenicity determinants encoded at a single locus.

Plasmids and double-stranded RNA viruses localized within mitochondria have been

shown to induce hypovirulence in several species of fungi (29,68). Mitochondrial

restriction fragment length polymorphisms (RFLP) also correlate with host preference in

Mycosphaerella graminicola populations (105). These studies suggest that mitochondrial

inheritance of a pathogenicity or hypovirulence factor could account for the skewed

distribution of pathogenicity among progeny. However, all progeny examined inherited

the 1.6-kb MspI mitochondrial DNA fragment found in nonpathogenic isolate P3-8.

Given that inheritance was skewed toward the pathogenic phenotype, it does not appear

that factors associated with the mitochondria affect pathogenicity. Inheritance of

assumed genomic markers and chromosomes indicate nonmendelian mechanisms govern

inheritance in portions of the genome. In most studies examining segregation of markers

in fungal crosses, a subset of markers typically deviate from expected ratios (36,76).

However, in the cross between 97-15A and P3-8, the number of markers that deviated

from a 1:1 segregation ratio was greater than that typically observed. Use of markers that

require hybridization to simple sequence repeats might account for the skewed ratios, as a

high proportion of bands identified with the (CAT)5 probe fail to segregate at expected

ratios in other fungi (32). However, simple sequence repeat loci do not segregate in a

nonmendelian fashion at a higher frequency than restriction fragment length

polymorphisms in plants (82). The occurrence of dominant markers at more than one









locus, inheritance of more than one homologous chromosome within progeny, or simply

preferential inheritance of one or more chromosomes with limited recombination could

also account for the skewed ratios. All of the skewed ratios result from a larger

proportion of progeny inheriting the dominant marker, suggesting inheritance of more

than one copy of the marker or more than one copy of homologous chromosomes. If a

significant number of progeny were heterokaryons, one would expect skewed inheritance

of dominant markers among progeny, but heterokaryon formation could be excluded for

all but three of 25 isolates examined. The meiotic events producing the skewed ratios

could also have arisen from genetic incompatibility of the isolates used in the cross as has

been observed with geographically isolated strains of Uromyces appendiculatus (62) or

alternatively they may be a normal occurrence in crosses of C. gloeosporioides.

Segregation of markers in crosses between perithecial isolates was detected at a high rate

by sampling only four progeny, indicating that segregation of markers in these crosses

probably did not deviate substantially from mendelian ratios. This suggests that the

diseased crown isolate and the ascospore isolate might have lacked genetic compatibility.

In crosses of C. gloeosporioides from jointvetch and pecan most genomic markers

displayed normal mendelian segregation ratios on agar, although use ofjointvetch as

substrate tended to skew inheritance of markers (21). Ultimately, segregation of co-

dominant markers using more than one combination of parents will be required to

determine normal mechanisms of segregation among isolates of this species from

strawberry.

Distinct electrophoretic karyotypes have been observed for C. gloeosporioides

isolates from .,,nllyh,,ih/, sp., with all of the variation within biotypes due to differences









in the size and number of small chromosomes that range from 0.27 Mb to 1.2 Mb in

length and comprise approximately 15% or less of the total genome (63). In this size

range, there were also differences in the size and number of chromosomes between

isolates 97-15A and P3-8. Most progeny from the cross of these isolates had

chromosome banding patterns distinct from each other and both parents. Although

recombination appears to have produced some chromosomes that migrate at very

different rates from those of the parental isolates, assortment of chromosomes produced

most of the differences in banding patterns. This conclusion is based on the observation

that most chromosomes inherited by progeny were identical or close to the size of

chromosomes from parent isolates. No specific probes were generated for chromosomes,

so it is not possible to determine with any certainty which ones might be homologous or

if one isolate has coding sequence absent in the other. However, for isolate 97-15A, the

sum of chromosome lengths was substantially greater than it was for isolate P3-8,

indicating that this isolate may possess genes not found in isolate P3-8. Also, based on

the size of progeny chromosomes it appears that chromosomes from parent 97-15A were

inherited preferentially. Thus, inheritance of pathogenicity determinants on these

chromosomes might account for the high proportion of offspring pathogenic to

strawberry. There are a number of examples of genes encoded on chromosomes less than

2.0 Mb in length that are required for virulence on specific hosts, but dispensable for

saprophytic growth (24,45). Often the small, dispensable chromosomes that encode these

genes display nonmendelian inheritance in crosses (24).

The occurrence of progeny pathogenic to northern j ointvetch seedlings has been

reported from a cross between a pathogenic isolate from northern j ointvetch and a









nonpathogenic isolate from pecan (19). None of the progeny from this cross consistently

killed northern jointvetch seedlings. Because there was little evidence from marker data

to suggest that nonmendelian mechanisms contributed to the skewed ratio of pathogenic

to nonpathogenic progeny, it was suggested that multiple avirulence genes or multiple

pathogenicity genes differentiate isolates pathogenic to jointvetch from those that are not

and that the genetic requirements for successful infection ofjointvetch are complex. In

the cross between pathogenic crown isolate 97-15A and nonpathogenic ascospore isolate

P3-8 results opposite to those observed for northern jointvetch were obtained, as most of

the progeny were pathogenic to strawberry. Discounting the role that nonmendelian

inheritance might play in the inheritance of pathogenicity to strawberry, it appears that

there may be more plasticity with respect to genetic requirements for causing crown rot

on strawberry. However, the nonpathogenic isolate in the current experiment was

obtained from a latent infection on strawberry, not a distantly related host, and could

already have met most of the physiological requirements for pathogenicity. Also of note,

laboratory crosses done in the current study did not support that a single locus with two

alleles regulated mating compatibility, a finding consistent with studies examining the

mating system of C. gloeosporioides and other Colletotrichum species (20,94).

The bioassay used to determine pathogenicity phenotype was highly reproducible

for most isolates, although several isolates from perithecium 1 were difficult to

categorize. This perithecium yielded both pathogenic and nonpathogenic isolates. In

field experiments examining plant mortality in response to inoculation with multiple C.

gloeosporioides isolates presented in chapter 5, the isolates display quantitative

differences in aggressiveness. This suggests that multiple genes affect pathogenicity on









strawberry and that the crown injection assay may identify isolates as pathogens only if

they possess physiological capabilities that exceed a threshold. Since perithecium 1

yielded both pathogens and nonpathogens, the disease-causing ability of isolates from

this source would lie close to the threshold. Also, given that many of the isolates from

this perithecium are weak pathogens, one would expect environmental variation between

experiments to have a greater effect on the outcome of pathogenicity tests using these

isolates. Differences in temperature across experiments is one variable which may

account for the conflicting results, as temperature has been shown to alter final mortality

of plants inoculated with G. cingulata (70).

Colletotrichum gloeosporioides has been reported from a wide range of host

species (69). Although reproductively isolated subgroups with narrow host ranges likely

exist within the C. gloeosporioides species aggregate, there are populations of C.

gloeosporioides in which isolates from different hosts are not genetically distinct from

one another and isolates from different hosts can successfully recombine in culture

(19,60). The acquisition of single-ascospore isolates that are able to interbreed and

display variation in pathogenic ability on strawberry will benefit future studies examining

the specific genetic requirements for pathogenicity and how they are distributed within

the population on strawberry and other plant species that the population from strawberry

might colonize.

In summary, it appears that C. gloeosporioides from strawberry is part of a

recombining population that consists of strains both pathogenic and nonpathogenic to

strawberry. The mechanism by which the pathogenicity phenotype is inherited in this

population remains unclear. It is apparent that a gene or cluster of genes at a single






31


mendelian-segregating locus does not determine pathogenicity. However, based on the

data derived from this study no conclusions could be made regarding wether

pathogenicity is determined at a nonmendelian-segregating locus or that it is affected by

multiple unlinked genes each having a quantitative effect on pathogenicity.












Table 2-1. Glomerella cingulata/Colletotrichum gloeosporioides isolates from Dover, FL used to compare AT-rich DNA
banding patterns and for recombination studies
Analysisa


Isolates
Pl-1 to Pl-10b
P2-1 to P2-10
P3-1 to P3-10
P4-1 to P4-10
P5-1 to P5-7
P6-1 to P6-7
P7-1 to P7-7
P8-1 to P8-7
311
329
97-15A
99-51


Description
Perithecium 1 ascospore
Perithecium 2 ascospore
Perithecium 3 ascospore
Perithecium 4 ascospore
Perithecium 5 ascospore
Perithecium 6 ascospore
Perithecium 7 ascospore
Perithecium 8 ascospore
Cgl-1 self-fertile
Cgl-1 self-fertile
Cgl-2 conidial
Cgl-2 conidial


Tissue
Petiole
Petiole
Petiole
Petiole
Petiole
Petiole
Petiole
Petiole
?
?
Crown
Crown


AT-rich DNA
P1-9
P2-6
P3-3
P4-7
P5-3
P6-3
P7-3
P8-1
Yes
Yes
Yes
Yes


Recombination on petiole
P1-3 to P1-4, P1-7 to P1-10
P2-7 to P2-10
P3-7 to P3-10
P4-5 to P4-7, P1-9 to P1-10
P5-1 to P5-4
P6-1 to P6-4
P7-1 to P7-4
P8-1 to P8-4


Parent agar crosses
P1-9 (P1-Path)'
P2-6 (P2-Nonpath)
P3-3 (P3-Nonpath), P3-8





P8-1 (P8-Path)


aSubsets of isolates used to compare AT-rich DNA banding patterns, to compare RAPD bands among progeny from perithecia on
petioles, and as parents for crosses on agar.
bPrefixes P followed by numbers 1 through 8 represent the individual perithecium sampled. Numbers following '-' represent
specific ascospore isolates sampled from each perithecium, with only the range of these designations given in column 1. All
isolates in column 1 were tested for pathogenicity to strawberry.
cIsolate codes in parentheses represent alternate names for isolates to the left. Alternate names identify the perithecium and
pathogenicity phenotype on strawberry of the isolate.












Table 2-2. Crown rot pathogenicity phenotype of single-ascospore isolates from
eight perithecia from naturally infected strawberry petioles
Phenotype No. of ascospore
Perithecium Pathogenic Nonpathogenic isolates evaluated
1 6 4 10
2 0 10 10
3 0 10 10
4 0 10 10
5 7 0 7
6 0 7 7
7 6 1 7
8 7 0 7






34


Table 2-3. Segregation of mitochondrial DNA, fungicide sensitivity, RAPD bands,
and (CAT)5 bands from a cross between pathogenic crown isolate 97-15A and
nonpathogenic ascospore isolate P3-8


97-15A genotype P3-8 genotype


P > k


2.5-kb Msp I mitochondrial band 0 39
Benomyl sensitivity 29 22 0.33
1.8-kb OPC-5 band 11 14 0.55
1.6-kb OPC-5 band 22 3 0.001
0.9-kb (GACA)4 band 7 18 0.04
9.9-kb (CAT)5 band 1 27 0.001
7.7- kb (CAT)5 band 17 11 0.26
3.5-kb (CAT)5 band 20 8 0.02












A ::



































Fig. 2-1. Glomerella cingulata from strawberry petiole. (A) Perithecia of G. cingulata
growing on a field-infected strawberry petiole after the petiole was freeze-killed and
incubated at room temperature 2-3 wk. Bar = 1000 atm. (B) Squash showing asci and
ascospores of a perithecium collected from a strawberry petiole. Bar = 50 atm.










inn f
N- d6I T-C-4M q^
CO M 00> QMaQ.


kb








11.5 -






5.1-
4.7 -
4.5-






2.8 -

2.4 -

2.1 -
2.0 -

1.7 -

Fig 2-2. AT-rich DNA banding pattern produced by digestion of total DNA with the
restriction enzyme HaeIII. Isolates 311 and 329 are Cgl-1 genotype isolates from
strawberry. Isolates 97-15A and 99-51 are Cgl-2 genotype isolates from strawberry.
Isolates P1 P8 are perithecium isolates from strawberry petioles.










kb P1 P2 P3 P4
(ACTG)4 2.:
2.0- .

(GACA)4 20
1.0- AD
0.5-
(TCC) 2:8:



PC-2 2.0-
i i:
'-iiii ; ,.;. 1: il


kb P5 P6 P7 P8
(ACTG)4 2!:


(GACA)42- 0
oi. U 44C
0.5-- I '
(TCC)5 28 ?r 2:IT

o-E


OPC-2 2.0-

1.0-


'1-

i '. ...s


i i


OPC-5 2.8- OPC-5 2.8- 1
20- 20-

1.0- LA 1.0-


Fig 2-3. Randomly amplified polymorphic DNA banding patterns of single-ascospore
isolates from eight Glomerella cingulata perithecia removed from strawberry petioles.
Frames within columns labeled P1-P8 contain banding patterns of isolates from the same
perithecium amplified using primers (ACTG)4, (GACA)4, (TCC)5, OPC-2, or OPC-5.
Arrows to the left of the frame show the position of bands that were polymorphic among
isolates from the perithecium identified by the label at the top of the column.







kb
2.8-
2.0 -
1.0 -
1.5-


1.0-
0.5 -
(TCC)5 2.8-
2.0-
1.0-


OPC-2

OPC-5


& a =
cR
00 0. 0.0.
. .L R RRR R P8R R R P3R R


I


2.0-
1.0-
2.8 -
2.0 -
1.0-


I


Ii


Il


ME


I


I

U


Fig 2-4. Randomly amplified polymorphic DNA banding patterns of two pathogenic and
two nonpathogenic isolates to strawberry and RAPD banding patterns of progeny from
crosses using these four isolates as parents. The first column of five frames contains
bands amplified from the four parents using primers (ACTG)4, (GACA)4, (TCC)5, OPC-
2, and OPC-5. The arrows to the left of these frames show the positions of bands
polymorphic among parents. The next two columns show bands amplified using the
same primers from four progeny of crosses using two nonpathogenic and two pathogenic
isolates as parents. The final column shows bands from progeny of a cross using isolates
of each phenotype as parents. Arrows to the left of frames show the position of bands
polymorphic among progeny. Progeny with banding patterns distinct from both parents
are identified by the letter R, those that had banding patterns identical to a parent are
identified by the parent perithecium.


(ACTG),


(GACA)4














kb
4.5-


SH 5 I
.ti
i& "; "T


2.0 -


1.5-



1.0 -


2.0-


e-1

C.
in S


kb
1.5 -


1.0-





0.5 -


11.5 -




5.1 -
4.5-


2.8 -


1.7 a ...



Fig 2-5. Molecular markers for 97-15A, an isolate pathogenic on strawberry, and isolate
P3-8, an ascospore isolate nonpathogenic to strawberry that were used to examine
mitochondrial and genomic inheritance among progeny from a cross between these
isolates. (A) The frame on the left is 700-rig mitochondrial DNA digested with Mspl.
The frame at the right is 10 ag of total DNA digested with Mspl, which was used to
identify mitochondrial inheritance among progeny. (B) Bands amplified with the primer
OPC-5. (C) Bands amplified with the primer (GACA)4. (D) Pstl-digested DNA probed
with (CAT)5. Arrows indicate bands used to examine inheritance of mitochondria and
recombination among progeny.











.-

Co


A Z
L Z


kb
1100 -

950 -

820 -
750 -

600 -

440-


220 -


kb

1100-

950 -

820 -
750 -

600 -

440-


220-


Fig 2-6. Chromosomes ranging in size from 300 kb to 1100 kb for pathogenic crown
isolate 97-15A, nonpathogenic ascospore isolate P3-8, and progeny from a cross between
these isolates. The only two progeny that were nonpathogenic on strawberry are in lanes
3 and 4 of frame 1. Ten representative isolates of those pathogenic on strawberry are in
lanes 5 through 10 of frame 1 and lanes 3 through 6 of frame 2.















CHAPTER 3
GENETIC AND PATHOGENIC ANALYSIS OF Colletotrichum gloeosporioides
ISOLATES FROM STRAWBERRY AND NONCULTIVATED HOSTS

Introduction

Under west central Florida climatic conditions, C. gloeosporioides does not appear

to have the capacity to survive on strawberry plant debris in soil during the summer

(53,93). In addition, soil is fumigated with a mixture of methyl bromide and chloropicrin

before bare-root transplants are set in the fall (3), making transfer of inoculum between

plants from different production seasons even less likely. Due to the low probability that

infections are transferred directly between plants grown in different seasons, inoculum

for the initiation of epidemics in Florida must come from sources outside of production

fields. Infected transplants are one potential source and there are some apparent

correlations between crown rot epidemics in Florida production fields with the nursery

providing transplants (59). Another potential source of inoculum is alternate host species

growing in the vicinity of strawberry fields. In Florida, strawberry fields are generally

surrounded by noncultivated trees, shrubs, and herbaceous plants and in some fields it




Most of the material in this chapter is reprinted with permission from Xiao CL, MacKenzie SJ, and Legard
DE. 2004. Genetic and pathogenic analyses of Colletotrichum gloeosporioides from strawberry and
noncultivated hosts. Phytopathology 94: 446-453. Additional permission to reproduce this material came
from The American Phytopathological Society. Chang L. Xiao and Daniel E. Legard originally conceived
of the experiments presented here. Chang L. Xiao and Steven J. MacKenzie performed pathogenicity
assays. Steven J. MacKenzie collected the genetic data, analyzed all data, and wrote the manuscript
submitted to Phytopathology under the guidance of Daniel E. Legard. Both Daniel E. Legard and
Chang L Xiao agree that it is appropriate for this material to be included in Steven J. MacKenzie's
dissertation.









appears as though strawberry plants with Colletotrichum crown rot symptoms are

aggregated near the edge of the field adjacent to these noncultivated plants.

Colletotrichum gloeosporioides has been isolated from numerous host species

suggesting that it is a physiologically adaptable fungus (69). This is supported by cross

infection experiments using strains of C. gloeosporioides from different cultivated plants,

the occurrence of crop pathogens on noncultivated plants, and genetic analysis of C.

gloeosporioides populations from different hosts. Colletotrichum gloeosporioides from

noncultivated host species have been shown to be pathogenic on black locust (102).

Isolates of C. gloeosporioides recovered from plant material of seven tropical fruits

tended to be more pathogenic on leaves of the plant species from which they are isolated,

but they still produced symptoms on alternative host leaves and molecular data did not

indicate that isolates from different hosts were derived from genetically distinct

populations (1). Among C. gloeosporioides living as endophytes on tropical forest trees

no host specificity could be detected using molecular fingerprints (60). Colletotrichum

fragariae, closely related to C. gloeosporioides, has been reported on a noncultivated

host (50) and was shown to produce disease symptoms on an alternative host (100). In

addition to being physiologically adaptive, the C. gloeosporioides taxonomic group

probably includes more than one biological species as well as clonally reproducing

lineages with specific host requirements. Based upon ribosomal DNA sequence analysis,

C. gloeosporioides isolates cluster into at least two distinct groups (55,78). In addition,

RAPD, RFLP, and isozyme markers have been used to identify genetically distinct

subgroups on hosts such as citrus and coffee (84,93).









The goal of the study presented in this chapter was to determine whether

noncultivated hosts adjacent to strawberry fields serve as potential sources of C.

gloeosporioides inoculum for infection of strawberry crown. The specific objectives

were to collect and identify isolates of Colletotrichum spp. from noncultivated plant

species adjacent to strawberry fields, evaluate C. gloeosporioides isolates from

noncultivated hosts for their pathogenicity on strawberry, and determine the genetic

relatedness of C. gloeosporioides isolates from noncultivated plant species to isolates

recovered from diseased strawberry plants.

Materials and Methods

Fungal Isolate Collections

From 1995 to 1998, isolates of Colletotrichum spp. with morphology consistent

with descriptions for C. gloeosporioides (43,81) were collected by staff at the University

of Florida, Gulf Coast Research and Education Center in Dover, FL from various

noncultivated hosts growing adjacent to strawberry production fields and from diseased

strawberry plants in west-central Florida (Table 3-1). Isolations were made from

diseased tissues, such as foliar and fruit lesions of noncultivated host plants and diseased

crowns of strawberry plants. Diseased plant tissues were surface disinfested for 5 min

with 0.525% sodium hypochlorite and plated onto either potato dextrose agar (PDA)

amended with neomycin sulfate (20 mg/L), chloramphenicol (6.5 mg/L), tetracycline

hydrochloride (25 mg/L), and erythromycin (7.5 mg/L) or a semi-selective medium for

Collectotrichum (16 g of Difco potato dextrose broth, 14 g ofDifco agar, 250 mg of

ampicillin, 150 mg of streptomycin sulfate, 5 mg of iprodione, 100 [tl of Tergitol, and 1 L

deionized water). Isolation plates were incubated under continuous fluorescent light at









room temperature (-220C) or in an incubator at 240C for 3 to 7 days. Cultures of

Colletotrichum spp. were single-spored and stored in 20% glycerol at -850C.

A total of 53 Colletotrichum spp. isolates were obtained between 1996 and 1997

from noncultivated plants growing on land adjacent to strawberry fields at five locations

(Table 3-1). Among these sites, there were 16 known and two unknown noncultivated

hosts from which Colletotrichum spp. were isolated. A Colletotrichum species was

found growing on both foliar tissue and fruit of one host species, Smilax rotundifoli.

Isolates coming from strawberry or noncultivated hosts can be identified by the letter "S"

for strawberry or "NC" for noncultivated at the beginning of the isolate code (Table 3-1).

Approximately half of the isolates included in the noncultivated population were

collected from site NC1. This site was sampled once in 1996 and again in 1997. The

population of Colletotrichum spp. obtained from infected strawberry crowns consisted of

42 isolates collected from 17 sites between 1995 and 1998 (Table 3-1). Site S2 was

sampled in both 1995 and 1997. Approximately half of the strawberry crown rot isolates

evaluated in the study were from this site. If isolates were collected from a site more than

once, the site number is followed by a slash and the year of collection. For isolates from

noncultivated hosts, the host species are indicated by the letters "sp" followed by a

number referring to the particular host species (Table 3-1 lists species corresponding to

the code number). Three C. gloeosporioides isolates from mango, four C.

gloeosporioides isolates from citrus, and one C. acutatum isolate from citrus were used as

representative outgroup populations for genetic comparisons. These isolates are believed

to be from or have previously been shown to be distinct from populations on strawberry

(47,92). One C. acutatum isolate from strawberry fruit was also included as an outgroup









in the genetic analysis and used to confirm the identity of any C. acutatum isolates that

may have been isolated from crown tissue.

Extraction of Fungal DNA

Total fungal DNA was extracted from mycelia obtained from cultures grown in 100

mL of Emerson Media (4 g/L yeast extract, 15 g/L soluble starch, Ig/L K2HPO4 and 0.5

g/L MgSO4) for 2 to 4 days at room temperature (-22 C). Mycelium was harvested

from the liquid cultures by vacuum filtration through Whatman no. 3 filter paper and

transferred into a 15 mL tube. Mycelia were then dried overnight in a centrifugal

evaporator and subsequently ground into a fine powder using a sterile glass rod. Sixty

milligrams of the dried powder for each isolate was suspended in 750 tl of DNA

extraction buffer consisting of 700 mM NaC1, 50 mM Tris(pH 8.0), 10 mM EDTA(pH

8.0), 1% cetyltrimethylammonium bromide, and 1% P-mercaptoethanol for 2 h with

periodic shaking at 650C. Particulate material was pelleted by centrifugation at 12,000x

g for 10 min, the supernatent removed and extracted once with chloroform:isoamyl

alcohol (24:1). Two volumes of 100% ethanol were added to the aqueous extract and the

mixture incubated at room temperature for 10 min. Nucleic acids were pelleted from the

ethanol solution by centrifugation at 12,000x g for 10 min. The pellet was washed with

100% ethanol and suspended in 400 tl 1 x TE buffer containing 10 [tg/mL RNase for 1 h

at 37C. Ribonuclease was removed from the nucleic acid solution by extraction with

400 tl phenol/chloroform/isoamyl alcohol (25:24:1). One-tenth volume 3 M sodium

acetate and 2.5 volumes of ethanol were added to the aqueous extract to precipitate the

DNA. This solution was incubated at -200C for 1 h and the DNA was pelleted at









12,000x g for 10 min. The DNA pellet was washed once with 1 mL 80% ethanol, dried,

suspended in 50 to 500 [pl x TE buffer, and stored at -200C.

Species Identification

A species-specific internal transcribed spacer region 1 (ITS1) primer and the

conserved universal primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3') encoded in the

28S ribosomal subunit were used in pairs to identify isolates to species (101). The ITS1

primers used were either the C. gloeosporioides specific ITS primer

5'-GACCCTCCCGGCCTCCCGCC-3' or the C. acutatum specific ITS primer

5'-GGGGAAGCCTCTCGCGG-3' (83,85). Isolates where assigned to the species group

for which a positive amplification with a specific ITS1 primer was obtained.

Amplifications were carried out under mineral oil in a 20-rl volume containing 1 x

reaction buffer (10 mM Tris(pH 8.3), 50 mM KC1, 1.5 mM MgC12, and 0.001% Gelatin),

200 pM dNTP, 1 unit Taq polymerase and 10 pmol of each primer/reaction. The reaction

buffer for the C. acutatum specific primer also contained 5% glycerol. Temperature

cycling parameters for the C. gloeosporioides specific/ITS4 pair consisted of a denaturing

step for 5 min at 940C, followed by 26 cycles at 940C for 1 min, 60C for 2 min, and

720C for 2 min. Temperature cycling parameters for the C. acutatum specific/ITS4 pair

consisted of a denaturing step for 5 min at 940C followed by 32 cycles at 940C for 1 min,

600C for 2 min, and 720C for 2 min. The amplified products were separated by

electrophoresis through a 2% agarose gel containing 1 x TAE buffer. Gels were

photographed on a UV transilluminator after ethidium bromide staining.









Pathogenicity Tests

Pathogenicity of the 53 Colletotrichum isolates recovered from noncultivated plants

was evaluated on the susceptible strawberry cv. Camarosa in a greenhouse. Conidial

suspensions were made from 7- to 10-day-old PDA cultures grown at 240C under

continuous fluorescent light and adjusted to 1 x 106 conidia per mL in sterile deionized

water. Infections were begun by injecting approximately 100 [tl of a conidial suspension

into the crown of mature transplants with a 25G1 syringe needle. Strawberry plants were

evaluated weekly for the development of Colletotrichum crown rot symptoms (i.e.,

wilting and collapse of plant). An isolate was considered to be pathogenic to strawberry

if at least 2 of 3 inoculated plants collapsed within the 4 weeks after inoculation. Each

isolate was tested at least twice in separate inoculation experiments. In addition, ten,

three, and four isolates of C. gloeosporioides recovered from diseased strawberry plants,

mango, and citrus, respectively, were also tested for pathogenicity on strawberry in the

same manner as described above.

Randomly Amplified Polymorphic DNA Markers

Five primers, including two tetranucleotide repeat primers (ACTG)4 and (GACA)4,

the trinucleotide repeat primer (TCC)5, and two short oligonucleotides

5'-GTGAGGCGTC-3' (OPC-2) and 5'-GATGACCGCC-3' (OPC-5) (Operon

Technologies, Alameda, CA), were selected for the population studies based on their

ability to consistently amplify bands that demonstrated a high level of fluorescence under

UV light. DNA amplifications were carried out under mineral oil in a 20 [tl volume

containing lx reaction buffer [50 mM Tris (pH 8.3), 0.25 mg/mL BSA, 2 mM MgC12,

0.5% Ficoll, and 1 mM Tartrazine], 200 tM dNTP, 1 unit Taq polymerase, and 20 pmol









primer/reaction [primers (ACTG)4, (GACA)4, and (TCC)5] or 8 pmol primer/reaction

(primers OPC-2 and OPC-5). Cycling parameters for the PCR reactions consisted of a 5

min denaturing step at 950C followed by 30 cycles of 1 min at 950C, 1 min at 480C, and 2

min at 720C for primers (ACTG)4 and (GACA)4; 34 cycles of 1 min at 950C, 1 min at

460C, and 1.5 min at 720C for primer (TCC)5 or 38 cycles of 1 min at 950C, 1 min at

350C, and 2 min at 720C for primers OPC-2 and OPC-5. The amplified products were

separated by electrophoresis through a 1.5% high resolution blend agarose (3:1) gel

containing 1 x TAE buffer. Gels were photographed on a UV transilluminator after

ethidium bromide staining.

Statistical Analyses

The SPSS 8.0 statistics package (SPSS Inc., Chicago, IL) was used to perform

Fisher's exact tests to determine the association between the pathogenicity of isolates and

the site or noncultivated host species from which the isolates were recovered. The

genetic relationship of isolates to one another was summarized in a phenogram

constructed from dice similarity coefficients using the unweighted pair group method

with arithmetic averages (UPGMA) clustering algorithm (NTSYS, PC version 2.0, Exeter

software, Setauket, NY). Statistical support for branches was based on 1,000

bootstrapped samples using Winboot (35,71). The probability of obtaining identical

genotypes among strains in the sample population assuming a random distribution of

alleles was determined using a custom written QBASIC program which shuffles alleles

among strains at each locus to mimic recombination and subsequently determines the

frequency of the most common genotype observed in the shuffled data set (86,89).

Probabilities of obtaining clone frequencies were based on analysis of 10,000 randomized









data sets. Population differentiation was examined using an exact test for population

differentiation at each locus and Fisher's combined probability test to obtain a probability

estimate over all loci (75). Tests for population differentiation were calculated using the

software program TFPGA (Utah State University, Logan).

Results

Identification of Isolates

Of the 53 isolates recovered from noncultivated hosts, 52 produced a

characteristic PCR product when the C. gloeosporioides specific primer was used and

they were therefore identified as C. gloeosporioides. One isolate produced an

amplification product when the C. acutatum species specific primer was used (Table 3-1).

The C. acutatum isolate obtained from the noncultivated host came from the fruit of

Callicarpa americana. It was genetically distinct from any of the C. acutatum isolates

from strawberry (Fig. 3-1) and did not produce either crown rot (Table 3-2) or fruit

lesions when inoculated on strawberry (data not shown). Of the 42 isolates recovered

from diseased strawberry crowns, 39 were identified as C. gloeosporioides and three as

C. acutatum. The species identities of Colletotrichum isolates from mango and citrus

(Table 3-1) were also confirmed with C. gloeosporioides or C. acutatum specific primers.

Pathogenicity Tests

Of the 52 C. gloeosporioides isolates from noncultivated hosts, 18 produced typical

symptoms of Colletotrichum crown rot (i.e. wilting and collapse of plants) on inoculated

plants. Therefore, these isolates were considered to be pathogenic to strawberry crowns.

The 18 pathogenic isolates were recovered from nine different noncultivated host species

from three separate sites (Tables 3-2 and 3-3). There was a significant association

between the pathogenicity of the isolates and the site from which the isolates were









collected (Table 3-2, Fisher's exact test P = 0.02). Of the 18 pathogenic isolates, 16

came from a single site (NC1). Half of the pathogenic isolates from this site were

collected in 1996 and the other half in 1997. There was no significant association

between the pathogenicity of isolates and the noncultivated host species from which the

isolates were recovered (Table 3-3, Fisher's exact test P = 0.23).

Often isolates that were recovered from diseased strawberry plants, nine produced

typical crown rot symptoms on inoculated strawberry plants. Two of the three isolates

from mango and none of the four citrus isolates were pathogenic to strawberry.

Randomly Amplified Polymorphic DNA Analyses

RAPD amplifications using five primers yielded 60 scorable bands from all isolates

examined. Forty-one scorable bands were amplified from C. gloeosporioides isolates

recovered from strawberry and noncultivated hosts. When isolates from citrus and

mango were included, 44 scorable bands were obtained. Thirty scorable bands were

amplified from C. acutatum isolates. Isolates identified as C. gloeosporioides grouped

into three clusters with between cluster similarities less than 0.50 (Fig. 3-1). All of the C.

gloeosporioides isolates from citrus had a level of similarity to one another greater than

0.75 and formed a cluster in 93% of bootstrapped phenograms. A second cluster

consisted of two clonal isolates from different noncultivated host species at site NC3 and

occurred in 100% of bootstrapped trees. The two isolates in this cluster were homothallic

(data not shown). Self-fertility was not observed for any other isolates, although it is

possible that low numbers of fertile perithecia may have been overlooked.

A third cluster contained C. gloeosporioides from noncultivated hosts, strawberry

and mango. This cluster occurred in only 41% of bootstrapped trees, giving weak









support for a monophyletic origin of this cluster. A large amount of the genetic variation

within this cluster can be attributed to the mango isolates. The three mango isolates were

distinct from most strawberry and noncultivated host isolates based on Dice similarity

coefficients, but isolates from this host did not compromise a group of closely related

organisms. When mango isolates are excluded from the analysis the cluster containing

strawberry and nonhomothallic, noncultivated host isolates occurred in 59% of

bootstrapped samples.

Bootstrapping did not provide support for strawberry isolates forming an

evolutionary lineage distinct from noncultivated isolates, as strawberry isolates were

interspersed among these isolates in the phenogram. However, clustering did occur

among subsets of strawberry and noncultivated host isolates. Several of these clusters

were supported by relatively high bootstrap values (Fig. 3-1), although the bootstrap

values may not be reliable as there are few informative sites separating the clusters from

other isolates (46). Seven clusters of nonhomothallic isolates contained two or more

clonal individuals based on RAPD profiles. Of these seven clonal lineages, two consisted

of isolates from strawberry and five contained isolates from noncultivated hosts. Two of

the clonal groups contained three or more isolates. One clonal genotype on strawberry

was found at two sites (four isolates at site S2/97; two isolates at S14). Of the five clonal

genotypes found on noncultivated hosts, four contained only isolates from the same field

and in only one of these fields were all of the isolates from the same host species.

Pathogenicity phenotypes were the same among all individuals possessing identical

genotypes. Based on probabilities obtained by repeated shuffling of the data set, two or

more individuals having the same genotype occurred in 4.39% of the randomized data









sets and three or more individuals with the same genotype did not occur in any of the

10,000 randomized data sets examined.

Allele frequencies were significantly (P < 0.05) different between strawberry

isolates and nonhomothallic isolates from noncultivated hosts at only four of 36 (11 %)

polymorphic loci based on exact tests with ca, the level of type I error, equal to 0.05

(Table 3-4). A test for population differentiation combining all polymorphic loci was not

significant (P = 0.29). The 1.6 kb band amplified with primer OPC-5, one of four loci

displaying allele frequency differences between these isolates, also displayed frequency

differences between pathogenic and nonpathogenic isolates when only isolates from

noncultivated hosts were compared (frequency = 0.50 among pathogens and 0.06 among

nonpathogens). There were no significant differences in allele frequencies between

pathogenic and nonpathogenic isolates from noncultivated hosts at the three other loci.

Discussion

In this study we found that approximately one-third of the C. gloeosporioides isolates

recovered from noncultivated hosts grown in the areas adjacent to strawberry fields were

pathogenic to strawberry in greenhouse tests. Phylogenetic analysis of RAPD data and

tests for genetic differentiation between C. gloeosporioides from noncultivated hosts and

those from diseased strawberry crowns suggest that they were from a single population.

These results indicate that noncultivated hosts growing adjacent to strawberry fields may

serve as a source of inoculum for epidemics of strawberry crown rot caused by C.

gloeosporioides.

Isolates of C. gloeosporioides from a wide range of temperate, subtropical, and

tropical fruits have shown cross infection potential (1,40). However, pathogenicity tests









in artificial inoculation experiments are not conclusive evidence to support that cross

infection occurs under natural conditions. For this reason, in addition to pathogenicity

tests, the genetic relationship between isolates from strawberry and different

noncultivated hosts was determined in the present study. Based upon bootstrap analysis

of a phenogram constructed using RAPD markers, isolates of C. gloeosporioides from

noncultivated hosts fell into two genetically distinct populations. The largest population

consisted of 50 isolates and the smaller population consisted of two genotypically

identical isolates. The populations from noncultivated hosts also could be distinguished

from one another based upon the presence of homothallism in isolates from the smaller

population. Homothallic isolates of C. gloeosporioides from strawberry appear to be

genetically distinct from interbreeding heterothallic isolates (38,43). However, the

homothallic isolates from strawberry do not appear to be related to those isolated from

noncultivated hosts (data not shown).

Isolates of C. gloeosporioides from strawberry crowns had a high level of diversity

and were not genetically distinct from isolates from noncultivated hosts that were not

homothallic in culture when all polymorphic loci were included in the analysis. Tests for

differences in allele frequencies at single loci revealed a 1.6-kb OPC-5 amplification

product that occurred at a higher frequency in the strawberry population relative to the

noncultivated host population and also occurred at a higher frequency among pathogenic

isolates from the noncultivated host population relative to nonpathogenic isolates. Allele

frequencies from this locus do not provide strong evidence for population subdivision,

given that the frequency of the vast majority of allelic markers, assumed to be neutral, are

not different between populations. The positive correlation between the 1.6-kb OPC-5









product and pathogenicity on strawberry does, however, suggest that it may be linked to a

genetic factor conferring pathogenicity on strawberry. In the previous chapter, this

marker also displayed highly skewed nonmendelian segregation in a cross between a

pathogenic and nonpathogenic isolate.

Although isolates with the same genotype occurred on either strawberry or

noncultivated hosts, no identical genotypes were found on both strawberry and

noncultivated hosts. Assuming a randomly mating population, the occurrence of even

two isolates with identical genotypes would be a relatively rare event given the sample

size and polymorphic loci examined in this study. In total, there were seven genotypes

detected more than once in isolates from the nonhomothallic strawberry and

noncultivated host population. This overrepresentation of specific genotypes is

consistent with the important role of clonal reproduction in this species (33). Genetic

bottlenecks created by recent colonizing events in spatially subdivided populations can

also result in overrepresentation of genotypes at specific sites. However, this is unlikely

as the sites from where clonal genotypes were collected contained a substantial amount of

genetic diversity. Genetically isolated pathogen subpopulations can also arise relatively

rapidly from interbreeding fungal populations due to asexual reproduction and may serve

as a mechanism to preserve particularly virulent gene combinations on specific hosts (8).

The data provided from this study does not support the hypothesis that this has occurred

with C. gloeosporioides from either strawberry or noncultivated hosts as clonal isolates

tended to occur at specific sites and not on specific hosts. There was, however, one

genotype that was isolated from six different strawberry crowns at two separate sites

(S2/97 and S14). This was the most common genotype observed and suggests that









selection may be preserving some combinations of genes for pathogencity on strawberry.

Alternatively, the occurrence of this genotype may result from selection for a clone at a

nursery supplying transplants to farms in the area investigated.

Although a defined group of species harboring pathogenic isolates could not be

identified among the nonpathogenic hosts sampled, there was a strong correlation

between sampling site and pathogenicity on strawberry. Because not all of the host

species were present at each sample site, there may be some bias in the tests for

association. This bias might make it difficult to discern whether or not isolates from

particular hosts or particular sites differed in pathogenicity. However, eleven of 19

isolates from site NC 1 that had hosts identical to those found at sites NC2, NC3, NC4

and NC5 were pathogenic to strawberry, whereas only two of the 22 isolates from the

four other sites were pathogenic on strawberry. This result indicates that the analyses

were correct in that pathogenicity correlated primarily with the site from which isolates

were recovered and not the host species from which they were isolated. Variation in

levels of pathogenicity among noncultivated host sampling sites may result from different

levels of migration from strawberry fields, where selection for virulence on strawberry

would likely occur.

Three C. gloeosporioides isolates from mango and four from citrus were included in

the phenogram (Fig. 3-1). The mango isolates examined in this study did not appear to

form a subgroup genetically distinct from C. gloeosporioides isolated from strawberry or

noncultivated hosts. These findings were not expected because a study of C.

gloeosporioides isolates from mangos at locations around the world found the isolates to

be relatively homogeneous and genetically distinct from those recovered from other fruit









species (47). However, that study only included one mango isolate from Florida. The

Florida isolate had a slightly smaller rDNA size compared to the isolates from other sites.

Repeated sampling of C. gloeosporioides from mango in Sri Lanka also revealed a

greater amount of diversity in rDNA and mtDNA restriction fragment length

polymorphisms than was previously thought to exist in that population (2). Also of

interest is that the two mango isolates pathogenic on strawberry in greenhouse

inoculation tests were more closely related to strawberry isolates than they were to the

third mango isolate that was not pathogenic on strawberry. The citrus isolates used in

this study were previously demonstrated to be genetically distinct from strawberry

isolates (92). Whether or not this genetic divergence is due to geographic isolation or

sexual incompatibility was not examined, but test crosses of citrus isolates to apple

reference strains have been unsuccessful (23).

A C. acutatum isolate was also obtained from a noncultivated host in this study. The

isolate was not pathogenic on strawberry and was genetically distinct from strawberry

crown isolates, indicating this strain of C. acutatum is not responsible for diseases on

strawberry.












Table 3-1. Collection sites and host species for Colletotrichum spp.
Species, collection site Host (species code)b
Colletotrichum gloeosporioides


NC1/96


NC1/97









NC2

NC3






NC4
NC5


Quercus spp. (spl2)
Smilax rotundifolia (sp 14)
S. rotundifolia, fruit (spl4B)
Vitis rotundifolia (sp 16)
Callicarpa americana, fruit (sp3)
Dioscorea bulbifera (sp4)
Ipomoea spp. (sp5)
Liquidambar styraciflua (sp6)
Myrica cerifera L. (sp 10)
Parthenocissus quinquefolia (sp 11)
Quercus spp. (spl2)
Smilax rotundifolia, fruit (spl4B)
Urena lobata (sp 15)
Momordica charantia L. (sp9)
Richardia brasiliensi (sp 13)
Magnolia virginiana L. (sp7)
Myrica cerifera L. (sp 10)
Quercus spp. (spl2)
S. rotundifolia (spl4)
S. rotundifolia, fruit (spl4B)
V rotundifolia (sp 16)
Unknown species (spl7)
V rotundifolia (sp 16)
Asclepias spp. (spl)


Common name


Oak
Smilax
Smilax berry
Wild grape
Beauty berry
Air potato
Morning glory
Sweet gum
Wax myrtle
Virginia creeper
Oak
Smilax berry
Caesar weed
Balsamapple
Brazilian pusley
Magnolia, sweet bay
Wax myrtle
Oak
Smilax
Smilax berry
Wild grape

Wild grape
Milkweed


Number of isolates


aSites beginning with NC are areas with noncultivated plants adjacent to strawberry fields and sites beginning with S are
strawberry fields. Numbers followed by "/" represent year sample was collected at sites sampled more than once.
bSpecies codes given in parentheses are combined with site numbers to identify the sample location and host for isolates
displayed in Figure 3-1.


NC1/96












Table 3-1. Continued
Species, collection site


S1
S2/95
S2/97
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
S13
S14
S15
Lake Alfred, FL
Homestead, FL
Colletotrichum acutatum
NC5
S9
S16
S17


Host (species code)b
Bidens bipinnata (sp2)
C. americana-fruit (sp3)
Melia australis Sweet (sp8)
S. rotundifolia (spl4)
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa
Citrus spp.
Mangifera indica

Callicarpa americana, fruit (sp3)
Fragariae x aananssa
Fragariae x aananssa
Fragariae x aananssa


Common name
Bidens
Beauty berry
Chinaberry
Smilax
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Strawberry crown
Citrus
Mango

Beauty berry
Strawberry crown
Strawberry crown
Strawberry crown


Number of isolates
1
1
2
2
1
11
9
1
1
1
1
1
1
1
2
2
1
1
3
2
4
3

1
1
1
1













Table 3-1. Continued
Species, collection site


Host (species code)b


Common name


Number of isolates


Dover, FL Fragariae x annanssa Strawberry Fruit 1
Lake Alfred, FL Citrus spp. Citrus 1












Table 3-2. Isolates of Colletotrichum spp. collected from noncultivated hosts
summarized according to site and pathogenicity on strawberry plants
Number of isolates


Species, collection site Pathogenic Nonpathogenic Total
Colletotrichum gloeosporioides
NC1/96 8 6 14
NC1/97 8 8 16
NC2 1 2 3
NC3 1 9 10
NC4 0 1 1
NC5 0 8 8
All Sites 18 34 52
C. acutatum
NC5 0 1 1
a Sites beginning with NC are areas with noncultivated plants in close proximity to
strawberry fields in west-central Florida. There was a significant association (P =
0.02) between the pathogenicity of the C. gloeosporioides isolates and the site from
which the isolates were collected, based on a Fisher's exact test (P = 0.05).











Table 3-3. Isolates of Colletotrichum spp. collected from noncultivated hosts
summarized according to host species and pathogenicity on strawberry plants
Number of isolates
Fungal species, host species Pathogenic Nonpathogenic Total
Colletotrichum gloeosporioides
Asclepias spp. (spl) 0 2 2
Bidens bipinnata (sp2) 0 1 1
Callicarpa americana, fruit (sp3) 0 2 2
Dioscorea bulbifera (sp4) 2 2 4
Ipomoea spp. (sp5) 0 1 1
Liquidambar styraciflua (sp6) 0 2 2
Magnolia virginiana L. (sp7) 1 1 2
Melia australis Sweet (sp8) 0 2 2
Momordica charantia L. (sp9) 0 2 2
Myrica cerifera L. (sp 10) 2 1 3
Parthenocissus quinquefolia (sp11) 2 1 3
Quercus spp. (spl2) 2 2 4
Richardia brasiliensi (spl 3) 1 0 1
Smilax rotundifolia (sp 14) 0 5 5
S. rotundifolia, fruit (spl4B) 1 4 5
Urena lobata (sp 15) 1 0 1
Vitis rotundifolia (sp 16) 6 4 10
Unknown species (spl7) 0 2 2
All host species combined 18 34 52
C. acutatum
Callicarpa americana, fruit (sp3) 0 1 1
aThere was no significant association (P = 0.23) between the pathogenicity of the C.
gloeosporioides isolates and the host species from which the isolates were collected,
based on a Fisher's exact test (P = 0.05). Species codes given in parentheses are
combined with site numbers to identify the sample location and host for isolates
displayed in Figure 3-1.












Table 3-4. Frequencies of RAPD bands for Colletotrichum gloeosporioides isolates
from strawberry and noncultivated hosts
Host
Noncultivated
Primer, length Strawberry Nonhomothallic Homothallic
(kb) (n=39)a (n=50) (n=2)


(ACTG)4
2.145
1.9
1.55
1.5
1.12
0.6
0.4
(GACA)4
1.5
1.35
1.3
1.2
1.15
0.95
0.9
0.8
0.75
0.5


0.49
0.97
0.00
1.00
0.85
0.92
0.33

0.05
0.95
0.00
0.97
0.03
0.69
0.46
0.13
0.00
1.00


0.52
1.00
0.00
1.00
0.84
0.84
0.38

0.02
0.96
0.00
0.96
0.04
0.74
0.38
0.30
0.02
0.96


0.00
0.00
1.00
0.00
0.00
1.00
1.00

0.00
0.00
1.00
0.00
0.00
0.00
1.00
0.00
0.00
0.00


(TCC)5
2 0.03 0.10 0.00
1.9 0.62 0.62 1.00
1.55 0.23 0.28 0.00
1.15 1.00 0.98 1.00
0.9 0.03 0.02 0.00
0.75 0.13 0.22 0.00
aExact test for population differentiation between strawberry and noncultivated
(nonhomothallic) host isolates over all loci was not significant (P=0.29).
bExact test for population differentiation between strawberry and noncultivated
(nonhomothallic) host isolates was significant at specified loci (P < 0.05).
cExact test for population differentiation between pathogenic and nonpathogenic,
noncultivated (nonhomothallic) host isolates was significant at specified loci (P <
0.05).












Table 3-4. Continued
Host
Noncultivated
Primer, length Strawberry Nonhomothallic Homothallic
(kb) (n=39) (n=50) (n=2)
OPC-2


0.08
0.62
0.03
0.08
1.00
0.03

0.18
0.00
0.21
0.59
0.21
0.18
0.08
0.10
0.74
0.33
0.03
0.38


OPC-5


2.7
2.6
2.5
2.15
2
1.8
1.75
1.65
1.6
1.55
1.4
1.2


0.06
0.64
0.00
0.02
1.00
0.00


0.00
0.00
0.00
0.00
1.00
0.00

0.00
1.00
0.00
1.00
0.00
1.00
0.00
0.00
1.00
0.00
0.00
0.00


0.34
0.00
0.44b
0.82b
0.22
0.16
0.08
0.30b
0.22bc
0.32
0.06
0.22












































Strawberry
and
Noncultivated hosts


Mango

Citrus
Noncultivated hosts
(Homothallic)
Strawberry


C. gloeosporioides



























C. aculalum


Similarity Coefficient




Fig. 3-1. A phenogram using unweighted pair group method with arithmetric averages

showing similarity (Dice) between Colletotrichum gloeosporioides and C. acutatum

isolates from noncultivated plants, strawberry crowns, citrus, and mango. Numbers at

nodes are the percentage of occurrence of the cluster to the right of the branch in 1,000

bootstrapped samples. All bootstrapped values are reported for clusters that are less than

0.50 similar to other isolates or clusters. For all other clusters, only bootstrapped values

greater than 50 are reported. Nonstrawberry C. gloeosporioides isolates with asterisks

(**) indicate they were pathogenic on strawberry in greenhouse inoculation tests.
Isolates are identified in Table 3-1.


2/97
2/97
2/95
5sp '
C3sp7*4
31/97sp4


13
C5spl
C5sp8
15
C5sp2
355sp1
c1/9Gsp12""
C1/96sp14B
1/97sp6
10
in













CHAPTER 4
SELECTION FOR PATHOGENICITY TO STRAWBERRY IN POPULATIONS OF
Colletotrichum gloeosporioides FROM NATIVE PLANTS

Introduction

Colletotrichum crown rot of strawberry causes wilting and collapse of strawberry

plants in production fields and nurseries in Florida. Currently most of the transplants

used for the annual winter production season are propagated in nurseries located in the

northern United States or provinces of Canada. This has greatly decreased the incidence

of crown rot in production fields, although moderate plant losses still occur. At the

present time, the vast majority of Colletotrichum species isolated from diseased

strawberry crowns in Florida are from a nonhomothallic C. gloeosporioides population.

This population is genetically diverse and recombination appears to occur at a relatively

high frequency (92). As shown in chapter 3, C. gloeosporioides can be isolated from

foliar lesions of a broad range of introduced and native noncultivated plant species

growing adjacent to strawberry fields as well as from strawberry petioles where it forms

latent infections (66). Isolates from these sources vary in their ability to cause crown rot

with some being aggressive pathogens on strawberry and others lacking the ability to

produce crown rot symptoms. Also from results reported in chapter 3, a large proportion

of the C. gloeosporioides isolates on noncultivated hosts appear to be from the same

population as those isolated from diseased strawberry crowns, indicating that

noncultivated hosts may provide inoculum for crown rot epidemics. However,

populations of C. gloeosporioides from noncultivated hosts at sites distant from

strawberry production areas have not been studied. Wild strawberry is not known to









occur in the subtropical regions of Florida where commercial strawberries are produced

(26). Thus, sampling of C. gloeosporioides at sites away from strawberry fields can

provide information as to whether isolates on noncultivated hosts are recent migrants

from strawberry or if there is an indigenous population of C. gloeosporioides on

noncultivated hosts and strawberry. In addition, the C. gloeosporioides/strawberry

pathosystem provides a unique system for examination of local selection for

pathogenicity.

In Florida, strawberry production is highly centralized. More than 90% of the area

dedicated to strawberry production is located within Hillsborough County, with most

plantings concentrated in the Dover/Plant City area (5). If the C. gloeosporioides

population on strawberry is derived from a widely dispersed population with a wide host

range, the cultivation of strawberry in a particular area may influence the frequency of

the pathogenic phenotype in the C. gloeosporioides population on noncultivated hosts.

Studies examining mean host resistance and mean pathogen virulence have shown a

positive correlation between these two traits in both natural and agricultural pathosystems

(64,88), suggesting directional selection in the pathogen population for increased

infectivity. Analogously, genes controlling pathogenicity on strawberry may be selected

in C. gloeosporioides populations at sites where strawberries are grown extensively.

In this study we genetically characterized C. gloeosporioides isolates from two

native hosts, oak (Quercus spp.) and wild grape (Vitis spp.), at four locations. Two

locations were immediately adjacent to strawberry fields and two sites were distant from

any commercial strawberry production. Sampling at all sites was conducted at least four

months after strawberry plants had been removed from fields. The genetic data was used









to identify a population to test the hypothesis that there is local selection for

pathogenicity to strawberry in areas where strawberries are grown extensively.

Materials and Methods

Sampling Strategy and Isolate Codes

In Florida, strawberries are grown as an annual crop on raised, plastic mulched

beds. Plants are set in late September and harvested from November to late March. At

the end of the harvest season, plants are chemically destroyed and the field tilled or a

different crop planted on the mulched beds. Sampling was done in late August and early

September 2002, 4 to 5 months after plants were removed from fields, to avoid sampling

migrants from strawberry fields that may have only transiently established themselves on

native hosts. At least 20 isolates identified as C. gloeosporioides based on morphology

(43) were obtained from necrotic lesions on oak (Quercus spp.) or wild grape (Vitis spp.)

leaves at each of four sites (Fig. 4-1). Samples were taken from leaves of different trees

and vines at each site to reduce the possibility of sampling clones. Two sites were

adjacent to commercial strawberry fields in Dover, FL. These sites were 3.5 km apart

within the strawberry production region in Hillsborough County. The native hosts

sampled at these sites were located 9 to 50 m from the edges of fields. Two sites distant

from strawberry production fields were sampled: University of Florida, Citrus Research

and Education Center in Lake Alfred, FL and a residential area in Sarasota, FL. Both

sites contained natural stands of vegetation along edges of roads or citrus groves and in

preserved areas. The Lake Alfred and Sarasota sites are located in Polk and Sarasota

counties respectively. The combined acreage used for strawberry production in these

counties is less than 2% of the acreage used in Hillsborough County. The nearest

commercial strawberry farm was approximately 28 km from the Lake Alfred site and 15









km from the Sarasota site. Native host isolates from the two sites in Dover were

designated D1-oak, D1-grape, D2-oak, and D2-grape. Those from Lake Alfred were

designated LA-grape, LA-oak, and those from Sarasota labeled SS-grape and SS-oak.

The site D2 was also sampled in the study presented in chapter 3, where it was designated

NS1. Site Dl had not been sampled previously. Twenty C. gloeosporioides isolates

from diseased strawberry crowns, seven citrus isolates, and one C. fragariae isolate from

crown tissue were also included in the analysis. Strawberry isolates came from samples

submitted by local growers to the diagnostic laboratory at the University of Florida, Gulf

Coast Research and Education Center in Dover, Florida from 1995 to 2000 and are coded

'strawberry'. Citrus isolates came from sweet orange or tangelo fruit, twigs, or leaves

from plantings near Lake Alfred, Avon Park, Vero Beach, or Frostproof, Florida. These

isolates are coded 'citrus'. C. gloeosporioides isolates from citrus have been shown to be

distinct from those on strawberry (92).

Fungal Isolation

Isolates from strawberry were obtained by placing tissue from necrotic crowns of

wilted strawberry plants directly onto CIM media (CIM 12 g potato dextrose broth, 17 g

agar, 100 mg streptomycin, 250 mg ampicillin, and 8 mg iprodione per L plus 0.02 %

tergitol). For isolations from native hosts, portions of oak and grape leaves with one or

more circular necrotic lesions were surface sterilized with 0.525 % sodium hypochlorite

for 1 min, rinsed in sterile water, and placed on CIM media. Plates were incubated under

fluorescent light for 3 to 5 days and single-spore isolates made from growing colonies.

Cultures were stored at -85 C in 20% glycerol.









DNA Extraction and PCR Amplifications

Two to 3 days after seeding with an agar plug, mycelium was harvested from 50-

mL liquid shake cultures (Emerson media 4 g yeast extract, 15 g soluble starch, 1 g

K2HPO4, and 0.5 g MgSO4-7H20 per L) and dried overnight in a centrifugal evaporator.

DNA was isolated from 60 mg of the dried mycelia. Mycelia powder was suspended in

750 [tl of DNA extraction buffer consisting of 700 mM NaC1, 50 mM Tris(pH 8.0), 10

mM EDTA(pH 8.0), 1% cetyltrimethylammonium bromide, and 1% P-mercaptoethanol

for 2 h with periodic shaking at 650C. Particulate material was pelleted by centrifugation

at 12,000x g for 10 min, the supernatent removed and extracted once with

chloroform:isoamyl alcohol (24:1). Two volumes of 100% ethanol were added to the

aqueous extract and the mixture incubated at room temperature for 10 min. Nucleic acids

were pelleted from the ethanol solution by centrifugation at 12,000x g for 10 min. The

pellet was washed with 100% ethanol and suspended in 400 tl 1 x TE buffer containing

10 [tg/mL RNase for 1 hour at 370C. Ribonuclease was removed from the nucleic acid

solution by extraction with 400 [tl phenol/chloroform/isoamyl alcohol (25:24:1). To the

aqueous extract, 1/10 volume 3 M sodium acetate and 2.5 volumes of ethanol were added

to precipitate the DNA. This solution was incubated at -200C for 1 h and the DNA was

pelleted at 12,000x g for 10 min. The DNA pellet was washed once with 1 mL 80%

ethanol, dried, suspended in 50 to 500 [tl x TE buffer, and stored at -200C.

Isolates were identified to species using a C. g1le,\pe i,,, \ ,e C. fragariae specific

ITS1 primer (5'-GACCCTCCCGGCCTCCCGCC-3') and a conserved universal primer

encoded within the 28S ribosomal subunit (5'-TCCTCCGCTTATTGATATGC-3'). This

primer set amplifies the ITS region from subpopulations within the C. gloeosporioides









species complex as well as that of C. fragariae (83,92). Amplifications from 5 ng of

template DNA were carried out under mineral oil in 20 [tl containing 1 x reaction buffer

(10 mM Tris [pH 8.3], 50 mM KC1, 1.5 mM MgCl2, and 0.001% gelatin), 200 [M dNTP,

1 unit of Taq polymerase, and 10 pmol of each primer/reaction. Cycling parameters

consisted of a 5-min denaturing step at 940C followed by 26 cycles at 940C for 1 min,

600C for 2 min, and 720C for 2 min. RAPD -PCR using four primers (ACTG)4,

(GACA)4, (TCC),, and 5'-GTGAGGCGTC-3' (OPC-2) (Operon Technologies, Alameda,

CA) was used to differentiate C. fragariae from C. gloeosporioides and to identify

subpopulations within isolates identified as C. gloeosporioides. Reactions were carried

out using 5 ng of template DNA under mineral oil in 20 [tl containing Ix reaction buffer

(50 mM Tris [pH 8.3], 0.25 mg/mL bovine serum albumin, 2 mM MgCl2, 0.5% Ficoll,

and 1 mM Tartrazine; Idaho Technology), 200 [LM dNTP, 1 unit of Taq polymerase, and

20 pmol primer/reaction (primers (ACTG)4, (GACA)4, and (TCC),) or 8 pmol OPC-

2/reaction. Cycling parameters consisted of a 5-min denaturing step at 950C, 30 cycles of

1 min at 950C, 1 min at 480C, and 2 min at 720C for primers (ACTG)4 and (GACA)4; 34

cycles of 1 min at 950C, 1 min at 460C, and 1.5 min at 720C for primer (TCC)5 or 38

cycles of 1 min at 950C, 1 min at 350C, and 2 min at 720C for primer OPC-2. Amplified

products were separated in 2% agarose gels made with IxTAE. Samples were

randomized before processing and two separate reactions performed for each

primer/template combination. Bands were scored manually. Bands that were weak and

those that failed to consistently amplify from the same template were excluded from the

analysis.









Pathogenicity Tests

Conidia from 7 to 10-day old grape and oak C. gloeosporioides cultures grown on

PDA were suspended in sterile deionized water, passed through four layers of cheese

cloth, and adjusted to 10 conidia/mL. Approximately 0.1 mL of this solution was

injected directly into the crown of three 'Camarosa' strawberry plants in a greenhouse

during the summer of 2003. Under the warm environmental conditions used for the

assay, pathogenicity could be determined unambiguously since no isolate caused collapse

of an intermediate number of plants. Isolates were categorized as pathogenic on

strawberry if they caused collapse of 3 of 3 plants within 4 weeks and nonpathogenic if

they failed to affect any of the 3 plants inoculated. The repeatability of the assay was

determined from inoculations on subgroups of isolates done during different time periods

between the winter of 2002 and the fall of 2004. Based on theses data only one of 89

isolates would have been classified differently (data not shown).

The validity of the assay was confirmed by topically applying 1 mL of 106/mL

conidia suspension to crowns in ten-plant plots of the cultivar Camarosa under field

conditions. Twelve plots were treated with oak or grape isolates determined to be

pathogenic in greenhouse tests and twelve plots treated with isolates determined to be

nonpathogenic in greenhouse tests. As a positive control, four plots were inoculated with

isolates from diseased strawberry crowns and four plots were inoculated with distilled

water as a negative control. Transplants were set on October 24, 2003 on plastic mulch

covered beds and inoculations conducted on March 12, 2004. Captan 80WP (4.2 kg per

ha) was applied to plants weekly, with applications suspended 2 weeks before plants were

inoculated and subsequently resumed 2 weeks following inoculations. The proportion of









plants collapsed was recorded for each plot 45 days after inoculation, just before water

and nutrient supplies to plants under cultivation were discontinued for the season.

Statistical Analyses

Cluster analysis was performed on Dice similarity coefficients using the

unweighted pair group method with arithmetic averages (UPGMA) with NTSYS, PC

version 2.0 (Exeter Software, Setauket, NY). All scored bands were used for this

analysis. Statistical support for phenogram branches was based on 1,000 replications

using the Winboot bootstrap algorithm (71). Values of theta (0) were estimated using the

method of Weir and Cockerham (99), with confidence intervals generated by

bootstrapping over loci using TFPGA (Utah State University, Logan). Statistical support

for population subdivision was concluded if the lower boundary of the 90% confidence

interval generated by 10,000 bootstrap replications exceeded zero. Theta measures the

correlation of alleles of different individuals in the same population relative to all

populations and is described by the equation (Q q)/( q), where Q is the probability

that two randomly sampled genes within a population are the same allele and q is the

probability that genes randomly selected from different populations are the same allele

(22). Bands absent or fixed in those populations that were being compared were

excluded from this analysis. The effect of proximity to strawberry production, specific

sampling site, and native host species on the incidence of pathogenicity was determined

using logistic analysis performed with the GENMOD procedure in SAS (SAS Institute

Inc., Cary, NC). For this analysis, isolates were categorized as being either close or

distant from the strawberry production area. Effects of specific sampling sites were

included in the model as nested effects within the variable "proximity to strawberry









production" and were compared to one another using specified contrasts. The incidence

of plant collapse in field plots inoculated with isolates determined to be pathogenic or

nonpathogenic in greenhouse experiments were compared using one-way analysis of

variance or unpaired t tests. Proportions were transformed using the arcsine-square root

transformation prior to analysis.

Results

Species Identification and Population Structure

Eighteen isolates from oak and grape lesions collected at Dover site 1 and 24

isolates collected at Dover site 2 produced a positive amplification product with the C.

gloeosporioides/C. fragariae-specific ITS1 primer. From the two sites distant from

strawberry production, Lake Alfred and Sarasota, 22 isolates and 25 isolates,

respectively, were obtained that produced a positive amplification product with the

species-specific ITS1 primer. Cluster analysis using data from 38 RAPD bands grouped

isolates from grape and oak into 4 distinct clusters with similarity among isolates within

the cluster greater than 0.65 and similarity between clusters less than 0.35 (Fig. 4-2). The

vast majority (82 of 89) of isolates from these native hosts formed a large cluster along

with 20 C. gloeosporioides isolates from diseased strawberry crowns. This cluster

occurred in 89% of bootstrapped trees. Two homothallic isolates from Sarasota and one

from Dover site 1 formed a distinct cluster, an isolate from an oak lesion in Lake Alfred

clustered with a C. fragariae isolate, and three isolates from Lake Alfred clustered among

six citrus isolates obtained from groves at various locations in Florida. Interestingly, one

of the seven citrus isolates used as a control clustered among isolates from strawberry

crown. The cluster containing only homothallic isolates and the cluster with isolates









related to citrus each occurred in 99% of bootstrapped trees. The cluster that included the

C. fragariae isolate occurred in 100% of bootstrapped trees.

Of the 38 RAPD bands scored, nine were unique to the cluster defined by isolates

of C. gloeosporioides from crown tissue, six were unique to the cluster defined by

isolates from citrus, four were unique to the homothallic isolate cluster and six were

unique to the cluster containing C. fragariae (Table 4-1.). Within the cluster that

included isolates from crown tissue, there were only two bands that were amplified from

native hosts only and these occurred at an overall frequency within these populations of

less than 0.04.

When only isolates that clustered with those from crown rot were examined, there

was no evidence for population differentiation between isolates from oak and grape hosts

at any of the four sites (Table 4-2). In a hierarchal analysis in which native host isolates

from all sites were used and sampling site was included in the analysis to delimit

subpopulations, there was also no evidence for population subdivision based on host (0 =

-0.029, 90% C. I. = -0.054 0.000). When sampling sites were examined in pairwise

tests for population differentiation, there was evidence for population subdivision

between isolates taken from the Sarasota sampling site and samples from Lake Alfred,

Dover, and diseased crowns (Table 4-3). There was no evidence for population

subdivision for isolates from any of the other samples (Lake Alfred, Dover 1, Dover 2

and the population from diseased crowns). Although there was evidence for population

subdivision between the Sarasota sampling site and all others, there were no bands unique

to this population nor did it lack any bands present in all of the other populations

examined.









Pathogenicity

In greenhouse tests, a higher proportion of isolates from the Dover sites located

adjacent to strawberry fields were pathogenic to strawberry than isolates from the sites

distant from strawberry fields (P = 0.002; Tables 4-4 and 4-5). When the Sarasota

population was excluded from the analysis, the proportion of isolates pathogenic on

strawberry was still greater at the Dover sites (P = 0.03). Isolates from oak did not differ

in degree of pathogenicity from grape isolates. There was also no significant difference

in the proportion of pathogenic isolates at the two Dover sites located in close proximity

to strawberry fields (Contrast P = 0.59), nor a difference in the proportion of pathogenic

isolates at the Lake Alfred and Sarasota sites distant from strawberry production

(Contrast P = 0.76). Homothallic C. gloeosporioides isolates from native hosts and C.

gloeosporioides isolates from native hosts that clustered with citrus isolates did not cause

crown rot symptoms. The single isolate from Lake Alfred that clustered with a C.

fragariae isolate also caused collapse of plants when injected into crown tissue, produced

tapered conidia, and possessed setae that functioned as phialides. These traits are

characteristic of C. fragariae (43).

In the field experiment using both nonpathogenic and pathogenic isolates identified

by direct injection of inoculum into crown tissue, plots topically inoculated with

pathogenic isolates had a higher incidence of crown rot (38.3% vs. 3.3%, P < 0.001

unequal variances assumed, n = 12 per treatment). The relatively low incidence of crown

rot in plots inoculated with isolates determined to be nonpathogenic in greenhouse tests

was not statistically significantly different from plots sprayed with water (3.3% vs. 2.5%,

P = 0.783, n = 12 and 4), suggesting that inoculation of plots with nonpathogenic isolates









did not increase disease incidence above that caused by natural sources of inoculum.

Incidence of plant collapse was not different in the field for pathogenic isolates from the

four different sites (F = 0.36, P = 0.781, n = 3 per site), nor was the percentage of plants

collapsed for plots treated with native host isolates different from plots treated with

isolates from diseased crown (38.3% vs. 60%, P = 0.174, n = 12 and 4).

Discussion

Colletotrichum isolates from native grape and oak leaves producing a positive PCR

product with C. gloeosporioides/C. fragariae-specific ITS1 primers fell into four separate

clusters based on RAPD marker data. The majority of the isolates from all sites sampled

fell into the same cluster as C. gloeosporioides isolates from diseased strawberry crowns.

However, one isolate clustered with C. fragariae, another pathogen causing strawberry

crown rot, and three isolates grouped with C. gloeosporioides from citrus. For the oak

and grape isolates that grouped with isolates from diseased crowns, band frequencies

were not significantly different between the crown rot population and the population

distant from strawberry production in Lake Alfred. This supports the hypothesis that the

population of C. gloeosporioides on strawberry is derived from a population already

present on hosts in Florida. Although band frequencies in the Sarasota population were

different from the crown rot population, no unique bands were found in the Sarasota

population and the phenogram constructed from RAPD data provided no evidence that

the population is monophyletic. This suggests that differences in band frequencies are

due to restricted gene flow combined with genetic drift rather than a speciation event.

Fixation indices may also be estimated using the statistic GST, which differs from 0 as a

function of the number of individuals in each sample population and the number of









sample populations used in the calculation (22). This statistic has been used in the past to

examine genetic differentiation in pathogen populations. For comparison to other

studies, pairwise GST estimates between the Sarasota population and the other four

sampled populations were estimated and ranged from 0.11 to 0.18. These values are in

the range of estimates for other fungal populations using RAPD, amplified fragment

length polymorphism (AFLP), or RFLP markers (42,67,74). However, in only one study

where population structure of the chestnut blight fungus Cryphonectriaparasitica was

examined were distances between populations comparable to those between Sarasota and

the other sites (67). No population subdivision was evident between oak and grape hosts

at any location. In the study presented in chapter 3, band frequencies were not

significantly different between a group of isolates from numerous noncultivated hosts and

strawberry crowns. However, due to limited sampling, any host specificity within the

noncultivated host population could not be examined. The observation of limited

subdivision by host in areas where host species occur in close proximity further supports

the hypothesis that the C. gloeosporioides population found on both native hosts and

strawberry has a broad host range. Similar results were observed in a study examining

host specificity of C. gloeosporioides isolates classified as endophytes collected from the

foliage of trees in a tropical forest (60).

The three homothallic C. gloeosporioides isolates obtained from the Dover and

Sarasota sites were compared to historical homothallic strawberry isolates (Florida

isolates 311-1 and 329-1, C. M. Howard) (38) using RAPD markers and the two groups

do not appear to be from the same population (data not shown). In chapter 3, homothallic

isolates distinct from historical strawberry isolates were also collected from noncultivated









hosts. Although many of the bands scored for isolates in the study presented here and in

chapter 3 were the same, several were not. There also may have been differences in

calculated band migration rates. For these reasons, the relatedness of isolates collected in

the two separate studies cannot be determined by a simple analysis of marker frequencies

reported in tables. However, a side by side comparison of bands amplified from isolates

obtained in these two studies using the primer OPC-2 indicates that they are from the

same population (data not shown). The single C. fragariae isolate was obtained in Lake

Alfred, FL approximately 28 km from a commercial strawberry farm. This species was

previously shown to colonize Cassia obtusifolia growing in and around a strawberry

nursery (50), but has never been reported away from a strawberry field. Its occurrence on

oak at a considerable distance from any strawberry production suggests that C. fragariae

may have a wider host range than previously believed and that it may be indigenous to

Florida. In Louisiana, C. fragariae is responsible for most strawberry crown rot

epidemics (79). Contaminated stock appears to be the major source of inoculum for these

epidemics, as crown rot is not observed in production fields using disease free transplants

(65). However, this does not exclude the possibility that a native host provided the initial

inoculum for the population that persists on strawberry. Also, native hosts may still play

a role in disease caused by C. fragariae as runners taken from plants that were free of

disease after one production season develop crown rot symptoms in subsequent years

(65).

Isolates of C. gloeosporioides from grape and oak that were closely related to citrus

isolates were only observed in Lake Alfred. The grape vines and oak trees sampled at

this site were next to a citrus grove and immigration of spores from citrus hosts would









likely be high in this area. The fact that only a small proportion of native host isolates

from this site clustered with isolates from citrus underscores the specific interaction

between the population on citrus and its host. However, the finding of a few citrus type

isolates at the Lake Alfred site also demonstrates that, in the presence of a sufficient

amount of inoculum from an outside source, alternate hosts can be colonized.

In greenhouse tests, the proportion of isolates from native hosts closely related to

those on strawberry that were pathogenic on strawberry was greater at sampling sites

close to strawberry fields. This provides support for the hypothesis that local selection

for pathogenicity on strawberry occurs where this host is grown in abundance.

Experimental studies of pathogen evolution have been conducted in pathosystems where

the fungal pathogen displays a high degree of host specificity, such as the Hordeum

vulgare-Rhynchosporium secalis pathosystem and the wild Linum marginale-

Melampsora lini pathosystem (56,58,64,88). Within these pathosystems, pathogenic

variation is governed by gene-for-gene interactions (15,56). Evidence that the biological

relationship between C. gloeosporioides and strawberry is different from that found in

these other pathosystems comes from research showing that isolates indistinguishable

from those on strawberry can be found on numerous hosts. In addition, there is in no

differential interaction between C. gloeosporioides isolates and strawberry cultivars as

demonstrated in the following chapter. No microscopic studies investigating the

infection of strawberry crown tissue by C. gloeosporioides have been conducted. The

best available information on whether the interaction of C. gloeosporioides and

strawberry is necrotrophic or biotrophic comes from a study examining C. fragariae on

stolons. In this study, there was only a brief biotrophic phase before the pathogen entered









an extended necrotrophic stage (25). There is also good evidence that C. gloeosporioides

forms infections in strawberry petioles, citrus twigs, and citrus fruit that remain quiescent

until infected tissue senesces (12,66). Taken together, these studies suggest that C.

gloeosporioides does not form a biologically intimate relationship with its host and gene-

for-gene relationships do not play a major role. Because C. gloeosporioides has a broad

host range and likely uses more than one pathogenic strategy to invade its host, it would

be hard to identify an overriding factor shaping the evolution of this species in a natural

environment. However, this may not be the case in agricultural areas where the presence

of a large, genetically uniform host population at a specific site would select for

individuals that can grow on the overrepresented host. This would be consistent with

research showing races immune to specific host resistance genes are overrepresented in

samples from areas where resistance genes are deployed (98). It is not clear whether the

isolates collected in this study were actually pathogens on oak and grape leaves. Isolates

from these hosts came from typical anthracnose type lesions, suggesting that they were

pathogens. However, it is also possible that isolates were growing as saprophytes on

lesions caused by another pathogen, insect damage, or injuries. In greenhouse

inoculations of oak leaves, isolates were recovered from inoculated leaves, but produced

necrotic symptoms only if the tissue was first wounded (data not shown).

One of the limitations of the crown injection assay may be that resistance to

penetration by the pathogen may be circumvented (54). For this reason, topical

inoculations of strawberry plots in the field were examined. The field trial indicated that

isolates classified as pathogens in greenhouse tests caused plant collapse and those

classified as nonpathogens did not. The field experiment also provided quantitative data









to differentiate pathogenic isolates. The aggressiveness of pathogenic isolates from

native hosts was not statistically different between sampling sites, nor was aggressiveness

of these native host isolates statistically different from crown isolates. However, the

comparison of isolates from different sites and hosts suffered from high variability in the

number of collapsed plants observed per plot as well as small sample sizes. The mean

percentage of plants killed per plot for isolates from diseased crown tissue was 60%,

whereas only 38% of plants in plots inoculated with pathogenic isolates from native hosts

were killed. These numbers were not significantly different from one another, but it

would not be inconsistent with the data on the incidence of pathogenicity that isolates

from crown and pathogenic isolates from native hosts would differ from one another

using a more quantitative measure for pathogenicity.

In summary, there was no conclusive evidence that C. gloeosporioides isolates

from diseased strawberry crowns in Florida are genetically distinct from the C.

gloeosporioides population broadly distributed on oak and wild grape hosts both close to

and distant from commercial strawberry fields. Isolates pathogenic to strawberry were

also broadly distributed on these native hosts, although they occurred at a higher

frequency at sites close to strawberry fields. The results observed in this study are

consistent with earlier work indicating that native plants can serve as a source of

inoculum for crown rot epidemics. The high incidence of Collectotrichum crown rot that

occurs in summer nurseries located in Florida is one of the reasons why transplants used

for commercial fruit production in Florida are purchased from nurseries located at higher

latitudes. The current study suggests that over the long term using transplants from

nurseries not located in Florida will likely reduce the amount of initial inoculum in






82


growers fields but will not prevent epidemics caused by introduced isolates, since C.

gloeosporioides isolates pathogenic to strawberry are present away from strawberry fields

and the frequency of pathogenic isolates appears to respond to selective pressures.














Table 4-1. Frequencies of randomly amplified polymorphic DNA bands from Colletotrichum gloeosporioides (C.g.) and
Colletotrichum fragariae (C.f) isolates
Cluster
C.g. C.g. C.g.
Strawberry Citrus Homothallic C.f
D1 D2 LA SS Strawberry LA Citrus SS &D1 LA Strawberry
Primer, length (kb) (n = 17) (n = 24) (n = 23) (n = 20) (n = 20) (n = 3) (n =6) (n = 3) (n = 1) (n = 1)
(ACTG)4
2.145 0.35 0.46 0.89 0.74 0.65 1.00 1.00 0.00 0.00 0.00
1.9 1.00 1.00 1.00 0.91 1.00 0.00 0.00 1.00 0.00 0.00
1.85 0.00 0.00 0.00 0.00 0.00 1.00 1.00 0.00 0.00 0.00
1.6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00
1.5 1.00 1.00 1.00 1.00 1.00 0.33 1.00 0.00 0.00 0.00
1.45 0.00 0.00 0.00 0.00 0.00 0.67 0.00 0.00 0.00 0.00
1.3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 0.00 0.00
1.1 0.71 0.83 0.67 0.22 0.80 0.00 0.00 0.00 0.00 0.00
(GACA)4
1.6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00
1.45 0.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
1.4 0.12 0.04 0.17 0.70 0.05 0.00 0.00 0.00 0.00 0.00
1.35 1.00 1.00 0.94 1.00 0.95 0.00 0.00 0.00 0.00 0.00
1.25 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 0.00 0.00
1.2 0.88 0.96 0.89 0.87 0.90 0.33 0.00 0.00 0.00 0.00
1.1 0.06 0.04 0.11 0.13 0.05 0.67 1.00 0.00 1.00 1.00
0.95 0.65 0.50 0.61 0.87 0.55 0.00 0.00 0.00 0.00 0.00
0.9 0.47 0.63 0.56 0.74 0.60 1.00 1.00 1.00 1.00 1.00
0.8 0.24 0.21 0.11 0.04 0.05 0.00 0.00 0.00 0.00 0.00
0.55 0.00 0.00 0.06 0.09 0.00 1.00 1.00 1.00 0.00 0.00
0.5 1.00 0.96 1.00 0.96 1.00 1.00 1.00 0.00 1.00 1.00
aClusters correspond to the four groups of isolates identified in fig. 4-2.
bIsolates from native hosts oak and grape are identified by site code Dl, D2, LA, or SS. Isolates from cultivated hosts used as controls are referred to by
the name of the host.
















Table 4-1. Continued
Cluster
C.g. C.g. C.g.
Strawberry Citrus Homothallic C.f
D1 D2 LA SS Strawberry LA Citrus SS &D1 LA Strawberry
Primer, length (kb) (n = 17) (n = 24) (n = 23) (n = 20) (n = 20) (n = 3) (n =6) (n = 3) (n = 1) (n = 1)
(TCC)5


2.0
1.95
1.9
1.55
1.1
1.05
0.75
0.7
OPC-2
2.4
2.1
1.9
1.85
1.7
1.3
1.1
1.08
1.05
0.7


0.00
0.00
0.59
0.00
0.59
0.00
0.24
0.00

0.00
0.00
0.65
0.00
0.00
0.00
1.00
0.00
0.00
0.00


0.28
0.00
0.44
0.00
0.50
0.00
0.39
0.00

0.00
0.00
0.67
0.00
0.00
0.00
1.00
0.00
0.00
0.00


0.05
0.00
0.60
0.00
0.40
0.00
0.25
0.00

0.00
0.00
0.50
0.00
0.00
0.00
1.00
0.00
0.00
0.00


0.00
0.00
0.33
1.00
0.00
0.00
0.00
0.00

0.00
0.00
0.00
0.67
1.00
1.00
0.00
0.00
0.00
1.00


0.00
1.00
0.00
0.00
0.00
1.00
0.00
1.00

0.00
1.00
0.00
0.00
0.00
1.00
0.00
0.00
1.00
0.00


0.00
1.00
0.00
0.00
0.00
1.00
0.00
1.00

0.00
1.00
0.00
0.00
0.00
1.00
0.00
0.00
0.00
0.00






85



Table 4-2. Estimates of 0 for pairwise comparisons of Colletotrichum
gloeosporioides isolates from oak and grape hosts at four sites
Site 0 90% Confidence interval 6b
Dover 1 -0.094 -0.114 -0.069
Dover 2 0.031 -0.032 0.091
Lake Alfred 0.056 -0.032 0.157
Sarasota -0.017 -0.057- 0.040
aThirteen to 15 bands were used to estimate 0.
bThe 90% confidence interval was determined from 10,000 bootstrap replications.













Table 4-3. Pairwise estimates of 0 (above diagonal) for Colletotrichum gloeosporioides populations at four sites and the
estimated 90% confidence interval for 0 (below diagonal)

Dover 1 Dover 2 Lake Alfred Sarasota Strawberry crown
Dover 1 -0.021a -0.031 0.218* -0.005
Dover 2 -0.032 -0.010b 0.030 0.275* -0.007
Lake Alfred -0.039 0.129 -0.017 0.094 -0.168* -0.004
Sarasota 0.116 0.310 0.141 0.396 0.087 0.239 -0.281*
Strawberry crown -0.032 0.028 -0.027 0.014 -0.025 0.023 0.131 0.373
aFifteen to 17 bands were used to estimate 0. The lower boundary of the 90% confidence interval for estimates followed by an
asterisk is greater than zero.
bNinety percent confidence limits were determined from 10,000 bootstrap replications.






87



Table 4-4. Percentage of isolates pathogenic on strawberry from oak (Quercus spp.)
and grape (Vitis spp.) lesions at four sites
Number of isolates Pathogenic
Site Host Path Nonpath (%)
Dover 1 Quercus spp. 4 4 50.0
Vitis spp. 3 6 33.3
Dover 2 Quercus spp. 6 7 46.2
Vitis spp. 6 5 54.6
Lake Alfred Quercus spp. 2 9 18.2
Vitis spp. 1 6 14.3
Sarasota Quercus spp. 2 10 16.7
Vitis spp. 1 10 9.1
apath = pathogenic, Nonpath = nonpathogenic






88




Table 4-5. Likelihood ratio statistics examining the effect of local strawberry
production, specific sampling site and native host species on the proportion of
native host isolates pathogenic to strawberry.
Source of variance df X2 P > X2
Proximity to strawberry production 1 9.41 0.002
Site(proximity to strawberry production) 3 0.38 0.828
Host 1 0.17 0.677
proximity to strawberry production was classified as either near or distant.















?-Qe 7.j Lake Alfred
Dover -
Sarasota



50 km




Fig. 4-1. Map showing the three locations where Colletotrichum gloeosporioides isolates
were sampled from lesions on oak and grape leaves. There were two separate sampling
sites in Dover. Hillsborough County, the main strawberry-growing county in Florida, is
shaded. Map template provided by the Department of Geography, Geology, and
Anthropology, Indiana State University, Terre Haute 47809.