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Genetic and Pathogenic Analyses of Colletotrichum Gloeosporioides Isolates from Strawberry and Noncultivated Hosts

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Genetic and Pathogenic Analyses of Colletotrichum Gloeosporioides Isolates from Strawberry and Noncultivated Hosts Population Biology Genetic and Pathogenic Analyses of Colletotrichum gloeosporioides Isolates from Strawberry and Noncultivated Hosts C. L. Xiao, S. J. MacKenzie, and D. E. Legard First author: Department of Plant Pathology, Washington State University, Tree Fruit Research and Extension Center, Wenatchee 98801; and second and third authors: University of Florida, Gulf Coast Research and Education Center, Dover 33527. Current address of D. E. Legard: California Strawberry Commission, Watsonville 95077. Accepted for publication 20 January 2004. ABSTRACT Xiao, C. L., Mackenzie, S. J., and Legard, D. E. 2004. Genetic and patho- tatum isolate were recovered from noncultivated hosts. In crown genic analyses of Colletotrichum gloeosporioides from strawberry and inoculation tests, 18 of the 52 C. gloeosporioides isolates recovered from noncultivated hosts. Phytopathology 94:446-453. noncultivated hosts were pathogenic to strawberry. Phylogenetic analysis using RAPD marker data divided isolates of C. gloeosporioides from Colletotrichum crown rot of strawberry in Florida is caused primarily noncultivated hosts into two separate clusters. One cluster contained 50 of by Colletotrichum gloeosporioides. To determine potential inoculum the 52 isolates and a second cluster contained 2 isolates that were homo- sources, isolates of Colletotrichum spp. from strawberry and various non- thallic in culture. Isolates from strawberry were interspersed within the cultivated plants growing in the areas adjacent to strawberry fields were cluster containing the 50 isolates that were recovered from noncultivated collected from different sites. Species-specific internal transcribed spacer hosts. The results are not inconsistent with the hypothesis that C. gloeo- primers for C. gloeosporioides and C. acutatum were used to identify sporioides isolates obtained from strawberry and noncultivated hosts isolates to species. Random amplified polymorphic DNA (RAPD) mark- adjacent to strawberry fields are from the same population and that non- ers were used to determine genetic relationships among isolates recovered cultivated hosts can serve as potential inoculum sources for Colletot- from noncultivated hosts and diseased strawberry plants. Selected isolates richum crown rot of strawberry. also were tested for pathogenicity on strawberry plants in the greenhouse. In all, 39 C. gloeosporioides and 3 C. acutatum isolates were recovered Additional keyword: anthracnose. from diseased strawberry crowns, and 52 C. gloeosporioides and 1 C. acu- Anthracnose of strawberry collectively refers to diseases of strawberry production seasons is unlikely. Inoculum for the initi- strawberry caused by Colletotrichum spp. (23). Three major ation of Colletotrichum crown rot epidemics in Florida must species of Colletotrichum, C. acutatum (29), C. fragariae (5), and come from sources outside of production fields. Infected trans- C. gloeosporioides (18), are associated with Colletotrichum dis- plants are one potential source and there are some apparent corre- eases on strawberry. Symptoms of Colletotrichum infection in- lations between crown rot epidemics in Florida production fields clude crown rot (15), fruit rot (16,19), root necrosis (10), irregular with the nursery providing transplants (22). Another potential or black leaf spot (18), and spotting of petioles and runners (5,7). source of inoculum is alternate host species growing in the vicin- Necrosis of the crown tissue by Colletotrichum spp., which we ity of strawberry fields. In Florida, strawberry fields generally are will refer to as Colletotrichum crown rot (15,23), typically results surrounded by noncultivated trees, shrubs, and herbaceous plants. in the wilting and death of plants. Although all three Colletot- Our observations indicated that, in some fields, strawberry plants richum spp. pathogenic to strawberry have been isolated from with Colletotrichum crown rot symptoms were aggregated near crowns of collapsed strawberry plants, a recent study has indi- the edge of the field adjacent to noncultivated plants. Therefore, cated that C. gloeosporioides is the primary cause of Colletot- we hypothesized that noncultivated plants can provide sources of richum crown rot in Florida (36). inoculum for strawberry crown rot. C. gloeosporioides isolated from strawberry displays consider- C. gloeosporioides has been found on a broad range of hosts able diversity in random amplified polymorphic DNA (RAPD) (24). The cross-infection potential for strains of C. gloeospo- banding patterns (11,36) and there is little evidence for linkage rioides isolated from different cultivated plants has been re- disequilibrium among loci (36), suggesting that C. gloeospo- ported (1,12). C. gloeosporioides from noncultivated host species rioides from strawberry may derive from a relatively large recom- also has been shown to be pathogenic on black locust (40). bining population. Under west-central Florida climatic condi- C. fragariae, although infrequently isolated from collapsed crowns tions, C. gloeosporioides does not appear to have the capacity to in Florida, has been both reported on a noncultivated host (17) survive on strawberry plant debris in soil during the hot summer and shown to produce disease symptoms on an alternate host (20,37). In Florida, strawberry plants are grown as an annual crop (38). Based upon ribosomal DNA sequence analysis, C. gloeo- in a hill plasticulture system. Soil is fumigated with a mixture of sporioides isolates cluster into two distinct groups (21,28). How- methyl bromide and chloropicrin before bare-root transplants are ever, analysis of more rapidly evolving regions of the genome set in the fall (3). Thus, the carry-over of inoculum between provided by RAPD, restriction fragment length polymorphism, and isozyme analysis have identified genetically distinct sub- groups within groups identified using ribosomal DNA sequence Corresponding author: C. L. Xiao; E-mail address: [email protected] information (14,32). Isolates of C. gloeosporioides recovered from plant material of seven tropical fruit species tend to be more Publication no. P-2004-0317-01R © 2004 The American Phytopathological Society pathogenic on leaves of the plant species from which they are 446 PHYTOPATHOLOGY isolated. Molecular data, however, did not indicate that isolates MATERIALS AND METHODS from different hosts are derived from genetically distinct popu- lations (1). Fungal isolate collections. From 1995 to 1998, isolates of Col- The goal of this study was to determine whether noncultivated letotrichum spp. with morphology consistent with descriptions for hosts adjacent to strawberry fields serve as potential sources of C. gloeosporioides (13,30) were collected from various nonculti- C. gloeosporioides inoculum for infection of strawberry crown. vated hosts growing adjacent to strawberry production fields and The specific objectives were to (i) collect and identify isolates of from diseased strawberry plants in west-central Florida (Table 1). Colletotrichum spp. from noncultivated plant species adjacent to Isolations were made from diseased tissues, such as foliar and strawberry fields; (ii) evaluate C. gloeosporioides isolates from fruit lesions of noncultivated host plants and diseased crowns of noncultivated hosts for their pathogenicity on strawberry; and (iii) strawberry plants. Diseased plant tissues were surface disinfested determine the genetic relatedness of C. gloeosporioides isolates for 5 min with 0.525% sodium hypochlorite and plated onto from noncultivated plant species to isolates recovered from dis- either potato dextrose agar (PDA) amended with neomycin sul- eased strawberry plants. fate (20 mg/liter), chloramphenicol (6.5 mg/liter), tetracycline hy- TABLE 1. Collection sites and host species from which Colletotrichum spp. were recovered 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 S. 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 Bidens bipinnata (sp2) Bidens 1 C. Americana, fruit (sp3) Beauty berry 1 Melia acustralis Sweet (sp8) China berry 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
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