Molecular analyses of colletotrichum species from almond and other fruits

ABSTRACT Isolates of Colletotrichum spp. from almond, avocado, and strawberry from Israel and isolates of the pink subpopulation from almond from the United States were characterized by various molecular methods and compared with morphological identification. Taxon-specific primer analysis grouped the avocado isolates within the species C. gloeosporioides and the U.S. almond and Israeli strawberry isolates within the species C. acutatum. However, the Israeli almond isolates, previously identified morphologically as C. gloeosporioides, reacted with C. acutatum-specific primers. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses determined that each population from almond and strawberry was distinct and clonal. Sequence analysis of the complete internal transcribed spacer (ITS) region (ITS 1-5.8S-ITS 2) revealed a similarity of between 97.03 and 98.72% among almond isolates from Israel, C. acutatum almond isolates from the United States, and C. acutatum strawberry isolates from Israel. Similarity of the above populations to that of C. gloeosporioides of avocado was between 92.42 and 92.86%. DNA sequence analysis of the entire ITS region supported the phylogeny inferred from the ITS 1 tree of 14 different Colletotrichum species. Although morphological criteria indicated that the Israeli isolates from almond are unique, this population was grouped within the C. acutatum species according to molecular analyses.     

Characterization of Colletotrichum acutatum Isolates Causing Anthracnose of Almond and Peach in California

ABSTRACT The causal organism responsible for the recent outbreak of almond and peach anthracnose in California was identified and characterized as Colletotrichum acutatum. Isolates of C. acutatum from almond were found to be similar to California strawberry isolates and South Carolina peach and apple isolates of C. acutatum based on conidial morphology, temperature relationships, fungicide sensitivity, and polymerase chain reaction (PCR) methods using DNA species-specific primers. On almond, blossoms and immature or mature fruit were affected by the disease, causing direct losses of crop. On peach, the disease was observed only on mature fruit. Pathogenicity of almond and peach isolates of C. acutatum was demonstrated on wound- and nonwound-inoculated almond or peach fruit by fulfilling Koch's postulates. Conidial morphology of isolates was variable, depending on the medium or substrate used to culture the isolates. Isolates of C. acutatum from strawberry, almond, and peach were grouped together based on a similar response to temperature, with an optimal growth rate at 25 degrees C (generally less than 10 mm/day), whereas isolates of C. gloeosporioides from citrus and papaya had an optimal growth rate at 30 degrees C (generally greater than 10 mm/day). In fungicide disk assays, isolates of C. acutatum from strawberry, peach, and apple, as well as almond and peach isolates from California, were less sensitive to benomyl at 300, 600, or 1,200 mug/ml. In contrast, C. gloeosporioides isolates from citrus and papaya were very sensitive to benomyl at all concentrations evaluated. All isolates of both species were sensitive to captan (300, 600, or 1,200 mug/ml). Oligonucleotide primers were synthesized for C. acutatum, C. fragariae, or C. gloeosporioides using published DNA sequences from the internal transcribed spacer 1 region of ribosomal DNA. Thirty-two Colletotrichum isolates from almond fruit produced DNA products with a C. acutatum primer (CaInt-2) that matched products and approximate molecular weight of known C. acutatum isolates. No PCR products were produced with primers for C. gloeosporioides or C. fragariae. Isolates from citrus and papaya produced DNA products only with primers from C. gloeosporioides or C. fragariae. Thus, worldwide, anthracnose of almonds may be caused by either C. gloeosporioides, as previously reported, or by C. acutatum, as indicated in this study.     

Pathogenic and Nonpathogenic Lifestyles in Colletotrichum acutatum from Strawberry and Other Plants

ABSTRACT Anthracnose is one of the major fungal diseases of strawberry occurring worldwide. In Israel, the disease is caused primarily by the species Colletotrichum acutatum. The pathogen causes black spot on fruit, root necrosis, and crown rot resulting in mortality of transplants in the field. The host range and specificity of C. acutatum from strawberry was examined on pepper, eggplant, tomato, bean, and strawberry under greenhouse conditions. The fungus was recovered from all plant species over a 3-month period but caused disease symptoms only on strawberry. Epiphytic and endophytic (colonization) fungal growth in the different plant species was confirmed by reisolation from leaf tissues and by polymerase chain reaction (PCR)-specific primer amplification. C. acutatum was also isolated from healthy looking, asymptomatic plants of the weed genera Vicia and Conyza. Isolates that were recovered from the weeds caused disease symptoms on strawberry and were positively identified as C. acutatum by PCR. The habitation of a large number of plant species, including weeds, by C. acutatum suggests that, although it causes disease only on strawberry and anemone in Israel, this fungus can persist on many other plant species. Therefore, plants that are not considered hosts of C. acutatum may serve as a potential inoculum source for strawberry infection and permit survival of the pathogen between seasons.     

Characterization and pathogenicity of Colletotrichum species associated with anthracnose on chilli (Capsicum spp.) in Thailand

Fungal isolates from chilli ( Capsicum spp.) fruits in Thailand that showed typical anthracnose symptoms were identified as Colletotrichum acutatum , C . capsici and C . gloeosporioides . Phylogenetic analyses from DNA sequence data of ITS rDNA and β-tubulin ( tub 2) gene regions revealed three major clusters representing these three species. Among the morphological characters examined, colony growth rate and conidium shape in culture were directly correlated with the phylogenetic groupings. Comparison with isolates of C . gloeosporioides from mango and C . acutatum from strawberry showed that host was not important for phylogenetic grouping. Pathogenicity tests validated that all three species isolated from chilli were causal agents for chilli anthracnose when inoculated onto fruits of the susceptible Thai elite cultivar Capsicum annuum cv. Bangchang. Cross-infection potential was shown by C . acutatum isolates originating from strawberry, which produced anthracnose on Bangchang. Interestingly, only C . acutatum isolates from chilli were able to infect and produce anthracnose on PBC 932, a resistant genotype of Capsicum chinense . This result has important implications for Thai chilli breeding programmes in which PBC 932 is being hybridized with Bangchang to incorporate anthracnose resistance into chilli cultivars.     

Genetic diversity and pathogenic variability among isolates of colletotrichum species from strawberry

ABSTRACT Ninety-five isolates of Colletotrichum including 81 isolates of C. acutatum (62 from strawberry) and 14 isolates of C. gloeosporioides (13 from strawberry) were characterized by various molecular methods and pathogenicity tests. Results based on random amplified polymorphic DNA (RAPD) polymorphism and internal transcribed spacer (ITS) 2 sequence data provided clear genetic evidence of two subgroups in C. acutatum. The first subgroup, characterized as CA-clonal, included only isolates from strawberry and exhibited identical RAPD patterns and nearly identical ITS2 sequence analysis. A larger genetic group, CA-variable, included isolates from various hosts and exhibited variable RAPD patterns and divergent ITS2 sequence analysis. Within the C. acutatum population isolated from strawberry, the CA-clonal group is prevalent in Europe (54 isolates of 62). A subset of European C. acutatum isolates isolated from strawberry and representing the CA-clonal and CA-variable groups was assigned to two pathogenicity groups. No correlation could be drawn between genetic and pathogenicity groups. On the basis of molecular data, it is proposed that the CA-clonal subgroup contains closely related, highly virulent C. acutatum isolates that may have developed host specialization to strawberry. C. gloeosporioides isolates from Europe, which were rarely observed were either slightly or nonpathogenic on strawberry. The absence of correlation between genetic polymorphism and geographical origin in Colletotrichum spp. suggests a worldwide dissemination of isolates, probably through international plant exchanges.     

Identification of Subpopulations of Colletotrichum acutatum and Epidemiology of Almond Anthracnose in California

ABSTRACT In recent years, almond anthracnose has developed into a major problem for the California almond industry. The identification of the causal pathogen as Colletotrichum acutatum was confirmed using species-specific primers and restriction fragment length polymorphisms of ribosomal DNA in comparative studies with isolates of C. acutatum from strawberry and C. gloeosporioides from citrus. Two distinct clonal subpopulations among the almond isolates of C. acutatum were identified. These two subpopulations differed in their colony appearance (pink versus gray cultures), conidial morphology, virulence in laboratory inoculation studies, temperature relationships for growth, and molecular fingerprints using random and simple-repeat primers in polymerase chain reactions. Both subpopulations were commonly isolated from the same orchard or even the same fruit. In other orchards, one subpopulation predominated over the other subpopulation. Using random, simple-repeat, and species-specific primers, isolates of the almond anthracnose pathogen from Israel were very similar to the California isolates that produce gray colonies. In addition to fruit, the pathogen was isolated from blighted blossoms, water-soaked or necrotic leaf lesions, symptomless peduncles, and spurs and wood from branches showing dieback symptoms, indicating that the amount of tissue that may be infected is more extensive than previously considered. Overwintering fruit mummies were identified as inoculum sources for early spring infections. Growth studies using almond kernels with different moisture contents indicated that postharvest damage of stored kernels likely originates from preharvest field infections.     

Use of Green Fluorescent Protein-Transgenic Strains to Study Pathogenic and Nonpathogenic Lifestyles in Colletotrichum acutatum

ABSTRACT Colletotrichum acutatum, which causes anthracnose disease on strawberry, can also persist on several other plant species without causing disease symptoms. The genetic and molecular bases that determine pathogenic and nonpathogenic lifestyles in C. acutatum are unclear. We developed a transformation system for C. acutatum by electroporation of germinating conidia, and transgenic isolates that express the green fluorescent protein (GFP) were produced. Details of the pathogenic and nonpathogenic lifestyles of C. acutatum were determined by using GFP-transgenic isolates. Major differences between colonization-mediating processes of strawberry and of other plants were observed. On the main host, strawberry, the germinating conidia formed branched, thick hyphae, and large numbers of appressoria were produced that were essential for plant penetration. In strawberry, the fungus developed rapidly, filling the mesophyll with dense mycelium that invaded the cells and caused necrosis of the tissue. In nonpathogenic interactions on pepper, eggplant, and tomato, the conidia germinated, producing thin, straight germ tubes. Appressoria were produced but failed to germinate and penetrate leaf tissue, resulting in epiphytic growth without invasion of the plant. Penetration of the plant occurred only several days after inoculation and was restricted to the intercellular spaces of the first cell layers of infected tissue without causing any visible damage. Much of the new fungal biomass continued to develop on the surface of inoculated organs in the nonpathogenic interaction. The differences in fungal development on strawberry compared with the other plant species suggest that signal molecules, which may be present only in strawberry, trigger appressorial germination and penetration of the primary host.     

PCR-based detection of Colletotrichum acutatum on strawberry

An oligonucleotide primer ( Ca Int 2) was synthesized from the variable internal transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) from Colletotrichum acutatum . PCR with primers Ca Int2 and ITS4 (from a conserved sequence of the rDNA) amplified a 490 bp fragment from several isolates of C. acutatum but not from other members of the genus Colletotrichum . Amplification of this fragment was achieved from 100 fg of fungal DNA. These primers amplified a fragment of the same size from DNA extracted from strawberry tissues infected by C. acutatum . Southern hybridization analysis confirmed the 490 bp fragment from C. acutatum DNA and infected strawberry to be identical. The species-specific primer ( Ca Int2) developed in this work could be used for the accurate identification of C. acutatum and its detection on other host plants.     

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

ABSTRACT Colletotrichum crown rot of strawberry in Florida is caused primarily by Colletotrichum gloeosporioides. To determine potential inoculum sources, isolates of Colletotrichum spp. from strawberry and various noncultivated plants growing in the areas adjacent to strawberry fields were collected from different sites. Species-specific internal transcribed spacer primers for C. gloeosporioides and C. acutatum were used to identify isolates to species. Random amplified polymorphic DNA (RAPD) markers were used to determine genetic relationships among isolates recovered from noncultivated hosts and diseased strawberry plants. Selected isolates also were tested for pathogenicity on strawberry plants in the greenhouse. In all, 39 C. gloeosporioides and 3 C. acutatum isolates were recovered from diseased strawberry crowns, and 52 C. gloeosporioides and 1 C. acutatum isolate were recovered from noncultivated hosts. In crown inoculation tests, 18 of the 52 C. gloeosporioides isolates recovered from noncultivated hosts were pathogenic to strawberry. Phylogenetic analysis using RAPD marker data divided isolates of C. gloeosporioides from noncultivated hosts into two separate clusters. One cluster contained 50 of the 52 isolates and a second cluster contained 2 isolates that were homothallic in culture. Isolates from strawberry were interspersed within the cluster containing the 50 isolates that were recovered from noncultivated hosts. The results are not inconsistent with the hypothesis that C. gloeosporioides isolates obtained from strawberry and noncultivated hosts adjacent to strawberry fields are from the same population and that noncultivated hosts can serve as potential inoculum sources for Colletotrichum crown rot of strawberry.     

Development of protocols for detection of Colletotrichum acutatum and monitoring of strawberry anthracnose using real-time PCR

Real-time PCR (TaqMan®) assays were developed for the specific detection and discrimination of Colletotrichum spp., C. acutatum and C. gloeosporioides causing anthracnose in strawberry using the most divergent area of the internal transcribed spacers (ITS1 and ITS2) and 5·8S ribosomal RNA (rRNA) gene region. The specificity of the new assays was tested using DNA from six species of Colletotrichum and nine fungal species commonly found associated with strawberry material, and additionally by comparing the sequences with those from databases using a blast search. The sequences only showed identity with homologous sequences from the desired target organisms. The new assays were 10–100 times more sensitive than conventional PCR methods previously published for the diagnosis of strawberry anthracnose. When real-time PCR was compared with ELISA methods, PCR improved the sensitivity of the identification by obtaining positive results for samples of strawberry plant material that tested negative with ELISA. The development of C. acutatum was monitored using artificially infected strawberry crowns from two strawberry cultivars (Camarosa and Ventana) and a real-time PCR assay specific for this species between January and June 2006. The amount of C. acutatum detected using real-time PCR varied significantly by month ( P  < 0·001), but not by cultivar ( P  = 0·394). The new assays were shown to be useful tools for rapid detection and identification of these pathogens and to allow rapid and accurate assessment of the casual agents of anthracnose in strawberry.     

Genetic Diversity Within Colletotrichum acutatum sensu Simmonds

ABSTRACT Isolates of Colletotrichum acutatum from several hosts were characterized by various molecular methods in comparison with morphological identification. Species-specific primer analysis was reliable for grouping C. acutatum isolates to their designated species. Arbitrarily primed polymerase chain reaction and A+T-rich DNA analyses identified four subgroups within C. acutatum. Subgroup I contained U.S. isolates from almond, apple, peach, and pecan, subgroup II contained isolates from anemone, olive, and strawberry, subgroup III contained isolates from almond (Israel) and strawberry (Spain), and subgroup IV contained a single isolate from anemone (the Netherlands). Likewise, sequence analysis of the internal transcribed spacer (ITS) 2 region alone or the complete ITS (ITS 1-5.8S-ITS 2) region grouped the isolates into the same four subgroups. Percent similarity of the complete ITS region within each cluster ranged from 99.6 to 100.0, 99.8 to 100.0, and 98.6% among subgroups I, II, and III, respectively. DNA sequence analysis of the ITS 2 region alone or the entire ITS 1-2 region was more informative than that of the ITS 1 region, which could only group the isolates into two main clusters. The molecular methods employed for studying genetic variation in populations of C. acutatum determined that this species is diverse, indicating that isolates within populations of each subgroup are not host specific.     

Etiology and Population Genetics of Colletotrichum spp. Causing Crown and Fruit Rot of Strawberry

ABSTRACT Isolates of Colletotrichum spp. from diseased strawberry fruit and crowns were evaluated to determine their genetic diversity and the etiology of the diseases. Isolates were identified to species using polymerase chain reaction primers for a ribosomal internal transcribed spacer region and their pathogenicity was evaluated in bioassays. Isolates were scored for variation at 40 putative genetic loci with random amplified polymorphic DNA and microsatellite markers. Only C. acutatum was recovered from diseased fruit. Nearly all isolates from crowns were C. gloeosporioides. In crown bioassays, only isolates of C. gloeosporioides from strawberry caused collapse and death of plants. A dendrogram generated from the genetic analysis identified several primary lineages. One lineage included isolates of C. acutatum from fruit and was characterized by low diversity. Another lineage included isolates of C. gloeosporioides from crowns and was highly polymorphic. The isolates from strawberry formed distinctive clusters separate from citrus isolates. Evaluation of linkage disequilibrium among polymorphic loci in isolates of C. gloeosporioides from crowns revealed a low level of disequilibrium as would be expected in sexually recombining populations. These results suggest that epidemics of crown rot are caused by Glomerella cingulata (anamorph C. gloeosporioides) and that epidemics of fruit rot are caused by C. acutatum.     

Quantitative detection and monitoring of Colletotrichum acutatum in strawberry leaves using real-time PCR

Real-time PCR assays for Colletotrichum acutatum , one of the most important pathogens of strawberry worldwide, were developed using primers designed to the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) and the β-tubulin 2 gene. Using TaqMan technology, the ITS-based assay could reliably detect as little as 50 fg genomic DNA, 100 copies of target DNA, or 25 conidia. The β-tubulin-based assay was c . 66 times less sensitive, and therefore less suitable for detection purposes. The TaqMan-ITS assay recognized all C. acutatum isolates tested from various intraspecific molecular groups, while no amplification was observed with several other Colletotrichum species or other strawberry pathogens, indicating the specificity of this assay. Detection and quantification of C. acutatum was demonstrated in artificially and naturally infected strawberry leaves. First, C. acutatum was detected in plant mixes of which only 0·001% of the tissue was infected by C. acutatum . Secondly, real-time PCR analysis of leaf samples taken at various times after inoculation indicated that the assay allowed monitoring of growth progression of C. acutatum . This real-time PCR-mediated monitoring of the pathogen was well-correlated with microscopic data, and confirmed that leaf age may play a role in the extent of C. acutatum infection. Finally, the assay allowed detection of C. acutatum in naturally infected and symptomless strawberry leaves collected from production fields and planting material.     

Molecular identification of Colletotrichum gloeosporioides causing yam anthracnose in Nigeria

Four forms of Colletotrichum representing three distinct virulence phenotypes were found associated with foliar anthracnose of yam in Nigeria: the highly virulent (= severity of disease) slow-growing grey (SGG); the moderately virulent fast-growing salmon (FGS); the weakly virulent fast-growing grey (FGG); and the moderately virulent fast-growing olive (FGO) morphotype. Isolates of the four forms were identified as C. gloeosporioides , based on morphology. The reaction of monoconidial cultures on casein hydrolysis medium (CHM), PCR-RFLP and sequence analysis of the internal transcribed spacer region of the ribosomal DNA (ITS1-5·8S-ITS2) were used to establish the identity of the yam anthracnose pathogen(s). All yam isolates were distinguished from C. acutatum by the absence of protease activity on CHM. On ITS PCR and enzymatic digestion of PCR products, all FGS, FGO and SGG isolates produced RFLP patterns identical to those of C. gloeosporioides reference isolates, while FGG isolates revealed unique ITS RFLP banding patterns. Sequence analysis of the ITS1 region and of the entire ITS region revealed that SGG, FGS and FGO isolates were highly similar (98–99% nucleotide identity) and showed 97–100% identity to C. gloeosporioides . Less than 93% similarity of these fungal isolates to reference C. acutatum and C. lindemuthianum isolates was observed. The molecular study confirmed that foliar anthracnose of yam is caused by C. gloeosporioides . While a high similarity was found among most C. gloeosporioides fungi from yam, isolates of the FGG form did not cluster with any previously described Colletotrichum species, and probably represent a distinct species.     

Genetic diversity,anastomosis groups and virulence of <Emphasis Type="Italic">Rhizoctonia</Emphasis> spp. from strawberry

Virulent Rhizoctonia spp. isolated from strawberry in Israel belonged to anastomosis groups (AG) of: binucleate Rhizoctonia (BNR) AG-A, AG-G, AG-K and AG-F, and to multinucleate Rhizoctonia (MNR) AG 4 subgroup HG-I. In addition, a soil isolate of AG 4 subgroup HG-III was also found to be virulent on strawberry. None of the Israeli isolates obtained in the present study belonged to BNR AG-I, or other MNR AGs. In the cluster analysis of rDNA-ITS sequences, all of the isolate sequences consistently clustered according to their known AGs and subgroups. One AG-F cluster included sequences of 10 strawberry isolates, while another AG-F cluster included sequences of two isolates submitted to GenBank. Additional work is needed to determine whether the isolates of these two clusters may belong to different AG-F subgroups. The current virulence bioassay used for Rhizoctonia spp. isolates on strawberry is based on inoculation of stolon-derived daughter plants with the isolates and estimation of the reduction in plant biomass, rather than on specific distinct disease severity symptoms. The duration of this test is relatively long (ca. 5 weeks or more) and the availability of daughter plants from runners is naturally limited to a certain season. Among the possible alternative methods evaluated in the present study (inoculation of fruits or seedlings developed from germinated strawberry seeds), the method based on seedlings was best. This method has a potential to replace the currently used stolon-daughter plant inoculation bioassay for testing virulence of strawberry root pathogens. This is the first report indicating that Rhizoctonia spp. isolates that belong to AG-F, AG-K, AG 4 HG-I and AG 4 HG-III are virulent to strawberry.     

Inheritance of Resistance to Colletotrichum acutatum in Fragaria x ananassa

ABSTRACT Anthracnose, caused by Colletotrichum acutatum, is a major disease of the octoploid cultivated strawberry, Fragaria x ananassa The inheritance of high and intermediate level plant resistances to C. acutatum, pathogenicity group 2, was investigated in an 8 x 8 factorial design. A single dominant gene (Rca2) controlled the high-level resistance, although minor genes may also contribute to resistance in cultivars such as Belrubi. The intermediate level of resistance was quantitative and controlled by minor genes. Analysis of 26 genotypes and cultivars from Fragaria spp. showed that the dominant gene was not rare in the germ plasm of F. x ananassa and that anthracnose resistance was also present in other species of Fragaria. These findings have important implications for anthracnose resistance breeding.     

Occurrence and development of Colletotrichum acutatum on symptomless blueberry bushes

The anthracnose fungus Colletotrichum acutatum was detected in symptomless blueberry bushes ( Vaccinium spp.) in a Japanese blueberry field. Naturally diseased bushes and their apparently healthy neighbours were selected, and C. acutatum was isolated from the symptomless tissues of each bush from February 2000 to January 2002. Analysis of the diseased bushes during the dormant period revealed that the fungus was able to survive on symptomless tissues, such as shoot bark and bud scales. Furthermore, C. acutatum was consistently isolated from symptomless leaves and shoots of several surrounding symptomless bushes. Arbitrarily primed PCR (ap-PCR) analyses of the fungal isolates obtained from the diseased and symptomless bushes revealed that most C. acutatum isolates were genotypically identical, regardless of their origins. Inoculation tests using leaves of various blueberry cultivars suggested that the presence or absence of symptoms on each bush can not always be explained by differences in cultivar susceptibility, and other factors may be associated with the appearance of symptoms.     

Germination and Sporulation of Colletotrichum acutatum on Symptomless Strawberry Leaves

ABSTRACT The germination and sporulation of Colletotrichum acutatum were characterized over time on strawberry leaves (cv. Tristar) and plastic coverslips incubated at 26 degrees C under continuous wetness. Conidia germinated within 3 h after inoculation and formed melanized appressoria with pores by 9 h after inoculation. Host penetration was not observed up to 7 days after inoculation. Production of secondary conidia on conidial and hyphal phialides began within 6 h after inoculation. Secondary conidiation was responsible for up to a threefold increase in the total number of conidia within 7 days after inoculation. Primary conidia and hyphae began to collapse 48 h after inoculation, whereas melanized appressoria remained intact. These findings suggest that appressoria and secondary conidia of C. acutatum produced on symptomless strawberry foliage may be significant sources of inoculum for fruit infections.     

Genetic polymorphism and virulence of Colletotrichum gloeosporioides isolated from strawberry (Fragaria × ananassa Duchesne)

To analyze genetic relationships among Colletotrichum gloeosporioides isolates; 34 isolates were collected from strawberries all over Japan, but primarily from Chiba Prefecture, and 20 were isolated from hosts other than strawberry. These isolates were assayed for virulence on strawberries and subjected to a fingerprint (FP) analysis by repetitive extragenic palindromic-PCR. Most of the isolates that were highly virulent to strawberries had relatively similar FPs even though they were isolated from various host plants (strawberry, cyclamen, nipplefruit and Japanese pear) in different regions. A cluster analysis revealed that the highly virulent and the weakly virulent isolates tended to form individual clusters, indicating that the highly virulent strains of C. gloeosporioides, which were genetically close to isolates with similar FPs and distinguishable from the weakly virulent strains, were responsible for strawberry anthracnose in Japan.     

Phylogenetic relationships and pathogenicity of Colletotrichum acutatum isolates from grape in subtropical Australia

The identity of Colletotrichum acutatum as the causal pathogen of grape ripe-rot, which causes yield loss and a bitter taint that lowers wine quality in Australian subtropical wine-grape regions, was confirmed using species-specific primers. Cultural, morphological and molecular methods (RAPD-PCR and sequencing of parts of the 5·8S-ITS regions and the β-tubulin-2 gene) were used to determine the phylogenetic relationships of Australian C. acutatum isolates from wine grapes and other horticultural crops. A combination of RAPD-PCR and β-tubulin-2 gene data showed that all wine-grape ripe-rot isolates from northern regions of New South Wales (NSW) and Queensland belong to a proposed new C. acutatum group (A9), together with isolates from Australian strawberry, mango, blueberry and olive. The 5·8S-ITS sequences for these grape pathogens were identical to published sequences for an isolate from Cyclamen (the Netherlands) and differed by 1 bp from isolates from Capsicum (Taiwan) and orange (Costa Rica). The grape ripe-rot isolates from the Shoalhaven Valley (southern NSW) were clustered within two other C. acutatum groups: A2 and A5. In vitro infection studies showed that Australian C. acutatum isolates from almond, blueberry, chilli, grape, mango, olive, strawberry and tomato were able to infect grape and could also infect blueberry and strawberry, indicating a lack of host specificity. This lack of host specificity, the genetic similarity with non-grape isolates, and the fact that many of the non-grape hosts were isolated from wine-growing regions, suggest the potential for cross-infection between grape and other horticultural crops.