Ecology and Population Biology Etiology and Population Genetics of Colletotrichum spp. Causing Crown and Fruit Rot of Strawberry A. R. Ureña-Padilla, S. J. MacKenzie, B. W. Bowen, and D. E. Legard First, second, and fourth authors: University of Florida, Gulf Coast Research and Education Center, Dover 33527; and third author: Department of Fisheries and Aquatic Sciences, University of Florida, Gainesville 32611. Accepted for publication 5 June 2002. ABSTRACT Ureña-Padilla, A. R., MacKenzie, S. J., Bowen, B. W., and Legard, D. E. from the genetic analysis identified several primary lineages. One lineage 2002. Etiology and population genetics of Colletotrichum spp. causing included isolates of C. acutatum from fruit and was characterized by low crown and fruit rot of strawberry. Phytopathology 92:1245-1252. diversity. Another lineage included isolates of C. gloeosporioides from crowns and was highly polymorphic. The isolates from strawberry form- Isolates of Colletotrichum spp. from diseased strawberry fruit and ed distinctive clusters separate from citrus isolates. Evaluation of linkage crowns were evaluated to determine their genetic diversity and the etiol- disequilibrium among polymorphic loci in isolates of C. gloeosporioides ogy of the diseases. Isolates were identified to species using polymerase from crowns revealed a low level of disequilibrium as would be expected chain reaction primers for a ribosomal internal transcribed spacer region in sexually recombining populations. These results suggest that epi- and their pathogenicity was evaluated in bioassays. Isolates were scored demics of crown rot are caused by Glomerella cingulata (anamorph C. for variation at 40 putative genetic loci with random amplified polymor- gloeosporioides) and that epidemics of fruit rot are caused by C. phic DNA and microsatellite markers. Only C. acutatum was recovered acutatum. from diseased fruit. Nearly all isolates from crowns were C. gloeospo- rioides. In crown bioassays, only isolates of C. gloeosporioides from Additional keywords: arbitrarily primed polymerase chain reaction, strawberry caused collapse and death of plants. A dendrogram generated Fragaria × ananassa, random amplified polymorphic DNA. Anthracnose fruit rot and Colletotrichum crown rot are entiation based on morphological characteristics may be uncertain important diseases of strawberry worldwide (9,13,23,24). Symp- (7,37). This situation has lead to numerous cases of incorrect toms of anthracnose fruit rot include sunken necrotic lesions with species identification (1,37,39). In California, a group of historical abundant conidia in acervuli (4). In contrast, Colletotrichum isolates from apple and peach originally identified as G. cingulata crown rot is characterized by reddish-brown necrotic areas in the were later reclassified as C. acutatum (19). In Florida, postbloom crown, which ultimately cause wilting and death of the plant. In fruit drop of citrus was originally identified as being caused by some locations, up to 80% of transplants were infected with Col- slow growing strains of C. gloeosporioides before species-specific letotrichum spp. before planting (3) or were killed by Colletot- polymerase chain reaction (PCR) primers revealed that C. richum crown rot (23). In the United States, losses attributed to acutatum was the correct pathogen (1,5). Construction of species- fruit rot have been greater than 30% when warm and humid specific primers, especially from the ribosomal DNA internal conditions favor serious epidemics (23,42). transcribed spacer (ITS) region, has been proposed as the most Colletotrichum acutatum, C. fragariae, and C. gloeosporioides efficient and reliable system for detection and differentiation of (teleomorph Glomerella cingulata) are the three main species of Colletotrichum spp. (15,36). However, strains of C. fragariae are Colletotrichum that cause strawberry diseases (23). The sexual still difficult to distinguish from those of C. gloeosporioides using state of Colletotrichum spp. does not have a recognized role in morphological characters or ITS primers (6,36). Molecular evi- strawberry epidemics and its role in the disease cycle has not been dence from mitochondrial DNA, ribosomal DNA, and random studied (23,25). However, Glomerella spp. do play a role in the amplification of polymorphic DNAs (RAPDs) suggests a relative- epidemiology of diseases affecting other annual and perennial ly close genetic relationship between C. fragariae and C. gloe- crops including other members of the family Rosaceae (37). On osporioides (36,37). apple, where C. gloeosporioides and G. cingulata produce a dis- The biology and epidemiology of Colletotrichum spp. on straw- ease called bitter rot, ascospores contribute the primary inoculum berry is unclear (23,24). In Florida, clonally produced strawberry for epidemics (38). The sexual stage for C. acutatum (G. acutata) plants are grown as an annual winter crop. Green-top transplants has also been described recently (18). from nurseries in the northeastern United States or southeastern Species identification for isolates in the genus Colletotrichum Canada are planted in the fall on fumigated plastic-mulched raised has traditionally been based on the shape and size of conidia, beds. Fruit are typically harvested from middle November to early production of perithecia, morphology of setae (if present), and April. Although the sources of primary inoculum for Colletot- colony growth characteristics (19,34,37). However, these charac- richum epidemics in Florida are uncertain (23,24), transplants are teristics can be highly variable in Colletotrichum spp. and differ- the likely source (16,23). Transplants infected with Colletotri- chum spp. have been reported in California (10), Israel (13), and Switzerland (3). In temperate regions, inoculum from plant debris Corresponding author: D. E. Legard; E-mail address: [email protected] may initiate epidemics each spring (10,42). However, in the south- Florida Agricultural Experiment Station Journal Series No. R-08871 eastern United States, Colletotrichum spp. do not survive between strawberry seasons in plant debris (41) or in soil (22). Publication no. P-2002-0830-02R Evaluation of the genetic structure and relationships among © 2002 The American Phytopathological Society lineages of Colletotrichum spp. infecting strawberry should pro- Vol. 92, No. 11, 2002 1245 vide insights into their biology and the etiology of the diseases. were collected from different locations and different seasons and For asexually reproducing fungi such as C. acutatum and C. were representative samples from epidemics of fruit rot and crown gloeosporioides, detection of subdivisions among populations rot in the Plant City-Dover strawberry production region in west within a particular area can indicate the source or sources of initial central Florida. Isolates from a majority of commercial cultivars inoculum, reveal host or tissue specificity, or test assumptions grown in the region during the late 1990s are represented in this about the role of the teleomorphic state in the epidemiology of the collection. One field was sampled for crown rot three times in the diseases (32,35). The purpose of this study was to characterize same season (Table 1; samples Crown-9b, Crown-9c, and Crown- intra- and interspecies isolates responsible for fruit rot and crown 9d) to obtain a large collection of isolates from one farm for popu- rot epidemics in Florida, to evaluate the genetic relationships lation genetic analysis. A large sample of anthracnose fruit rot among isolates of these pathogens, and to provide insights into the isolates from a single farm was also collected for genetic compari- biology of the pathogens and etiology of Colletotrichum crown rot sons (Fruit-5a). Six isolates of C. fragariae (Table 1) from straw- and anthracnose fruit rot. A preliminary report of this research has berry crown rot lesions previously isolated and characterized were been made (40). included to confirm differences between this species and C. gloeosporioides in the genetic analysis. Ten isolates of C. gloeo- MATERIALS AND METHODS sporioides and three isolates of C. acutatum from nonstrawberry hosts (Table 2) were included in the study as putative nonpatho- Isolates. Monoconidial isolates of Colletotrichum spp. causing genic controls in the bioassays. The five isolates from citrus (four anthracnose fruit rot (44 isolates) and Colletotrichum crown rot C. gloeosporioides and one C. acutatum) were also included in the (132 isolates) of strawberry were evaluated (Table 1). The isolates genetic analysis to provide out-group information within those species. Cultures were stored in 20% glycerol in a freezer at –85°C. TABLE 1. Isolates of Colletotrichum spp. from strawberry used for genetic Mycelium production and DNA extraction. Isolates were analysis grown on potato dextrose agar (PDA) at 24°C under continuous Year Number fluorescent light for 5 to 7 days. Conidial suspensions were Tissuea Source locationb collected of isolates Speciesc prepared from the cultures in sterile distilled water and 5 ml of the Crownà Crown-1 1999 1 C. gloeosporioides suspension, which was transferred with sterile pipettes to 250-ml Crown-2 1997 1 C. gloeosporioides flasks containing 100 ml of Emerson Media (4 g of yeast extract, Crown-3 1996 1 C. gloeosporioides 15 g of soluble starch, 1 g of potassium phosphate, 0.5 g of mag- Crown-4 1995 1 C. gloeosporioides nesium sulfate, and distilled water up to 1 liter). Cultures were Crown-5b 1997 1 C. gloeosporioides