Two subpopulations of Colletotrichum acutatum are responsible for anthracnose in strawberry and leatherleaf fern in Costa Rica

Strawberry, Fragaria × ananassa, and leatherleaf fern, Rumohra adiantiformis, are two important crops in Costa Rica. One of the most severe diseases affecting these crops is anthracnose, caused by members of the fungal genus, Colletotrichum (teleomorph; Glomerella). Eighty single-spore isolates from strawberry and leatherleaf fern were identified as Colletotrichum acutatum by species-specific PCR, and were further characterised by Universally Primed PCR (UP-PCR) fingerprinting analysis, and sequence analysis of the ribosomal internal transcribed spacer (ITS) region. Morphological differences, genotypic variation revealed by UP-PCR fingerprinting analysis, and a single sequence polymorphism within the ITS2 region were found between the isolates from strawberry and leatherleaf fern, respectively. The UPGMA cluster analysis of the fingerprints clearly separated the isolates derived from strawberry and leatherleaf fern into two different clusters. Pathogenicity assays on detached strawberry fruits confirmed the apparent difference between the two groups of isolates. It is therefore suggested that the pathogens responsible for strawberry anthracnose fruit rot and leatherleaf fern anthracnose in Costa Rica, belong to two distinct subpopulations of C. acutatum.     

Phenotypic and genetic characterization of Colletotrichum isolates from Belgian strawberry fields

Colletotrichum isolates (457) were collected from strawberry plant tissues with and without typical anthracnose symptoms and from symptomless weeds in 19 Belgian strawberry fields. The isolates were characterized based on genetic, morphological and pathological features. Isolates were classified according to rDNA‐ITS sequencing: 97% of 211 representative isolates were C. acutatum, 2%C. gloeosporioides and 1%C. coccodes. The C. acutatum isolates belonged to the intraspecific groups A2 (33%), A3 (5%), A4 (50%), A5 (3%) and A7 (6%). Differences in spore morphology, growth rate and colony colour of a selection of 146 isolates confirmed the genetic grouping. Multiple Colletotrichum genotypes were detected in the same field. There was no association between the most common genotypes and geographic origin, presence or absence of symptoms, nor plant species or plant part. Representative Belgian Colletotrichum isolates were used in pathogenicity tests, together with European and American reference isolates. The C. acutatum A2 and the Belgian C. gloeosporioides isolates were the most aggressive on fruits, followed by C. acutatum A3, A4, A5, A7 and C. coccodes isolates. When inoculated into crowns, C. acutatum A2, A5 and American C. gloeosporioides isolates were the most aggressive, followed by C. acutatum A3 isolates. The A4 and A7 isolates and all European C. gloeosporioides isolates were non‐pathogenic on crowns. These data indicate that an unusually diverse Colletotrichum population is present in Belgium. The traditional differentiation between C. acutatum and C. gloeosporioides as causal agents of fruit and crown rot, respectively, proved not to be valid in Belgian strawberry fields.     

Latent entry and spread of Colletotrichum acutatum (species complex) in strawberry fields

Anthracnose, caused by Colletotrichum acutatum (species complex), has become a troublesome problem in strawberry production worldwide. This paper reports (i) an optimized sampling method combined with a real‐time PCR technique to detect the latent presence of C. acutatum in cold‐stored strawberry plants used as planting material in several European countries, and (ii) a study of the spread of C. acutatum following a point inoculation under field conditions. Screening of different parts of planting material suggested that C. acutatum is most likely to be present on runners and old petioles. In addition, in seven out of nine batches of planting material from different nurseries, latent infection by C. acutatum was detected in at least one of five replicate samples. Field experiments in 2009 and 2010 showed extensive latent within‐field spread of the pathogen on strawberry leaves, with a within‐row dispersal distance up to at least 1·75 m in 1 week. A straw ground cover between the rows did not decrease C. acutatum spread, probably because introduced (and/or subsequent) inoculum was confined to the plant bed (within the row) and was not present between the beds. Moreover, the number of C. acutatum spores on the symptomless leaves, as estimated using a real‐time PCR method, was significantly (P < 0·05) correlated with the incidence of fruit rot at harvest and post‐harvest (r = 0·56–0·66). These results illustrate the importance of detecting latent infections in planting material and strawberry leaves in the field.     

Cross-infection of subtropical and temperate fruits byColletotrichumspecies from various hosts

Forty-two isolates ofColletotrichum gloeosporioidesfrom almond, apple, avocado, mango, pecan, and eight isolates ofC. acutatumfrom apple, peach and pecan were compared by molecular analyses and a pathogenicity assay in order to determine genetic variability and host specificity. Polymerase chain reaction (PCR) amplification of genomic DNA using four different primers andHaeIII digestion patterns of genomic DNA (A+T-rich DNA) grouped theC. acutatumisolates separately from theC. gloeosporioidesisolates. Based on arbitrarily primed PCR (ap-PCR), intraspecies similarity among the isolates ofC. acutatumandC. gloeosporioidesranged from 78 to 93% and from 0 to 38%, respectively. Similarity between the isolates ofC. acutatumandC. gloeosporioidesranged from 0 to 26.5%. A+T-rich DNA grouped theC. acutatumisolates separately from those ofC. gloeosporioides, corresponding to ap-PCR analyses. Artificial inoculations with nine representative isolates on almond, apple, avocado, mango and nectarine fruit showed a variation in levels of infection. TheC. gloeosporioidesisolates from almond grew more slowly, causing significantly smaller lesions on all inoculated fruit than the other isolates. TheC. acutatumisolates from apple and peach caused similar levels of infection on all fruit, but differed significantly from theC. gloeosporioidesisolate from apple. Variation in lesion size was also observed with isolates ofC. gloeosporioidesfrom apple, avocado and mango for most fruit inoculations.     

Benomyl sensitivity assays and species-specific PCR reactions highlight association of two <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis> types and <Emphasis Type="Italic">C. acutatum</Emphasis> with rumple disease on Primofiori lemons

Rumple is a serious peel collapse of Primofiori lemons in the southeast of Spain with an unresolved aetiology. Symptoms typically occur on fruits at ripening under wet conditions as dark sunken lesions producing premature fruit drop and damaged fruits unacceptable for fresh commercialization. A total of 16 Colletotrichum spp. isolates established from rumple-affected lemons collected during the autumn of 2007 from two different orchards were characterized by molecular and phenotypic assays and compared with reference isolates. Species-specific PCR reactions using β-tubulin 2 nucleotide sequences showed Colletotrichum gloeosporioides to predominate (81.5%) with limited occurrence of C. acutatum (18.75%). Among the C. gloeosporioides isolates, five (38.5%) showed benomyl resistance and eight (61.5%) were highly sensitive to the fungicide. The limited occurrence of C. acutatum could be related to factors such as the presence of both species on the same fruit, unfavourable meteorological conditions and low disease incidence. This work reveals an association of C. gloeosporioides and C. acutatum isolates with rumple disease of lemons and expands the range of C. acutatum on citrus.     

Tracing Latent Infection of Colletotrichum acutatum on Strawberry by PCR

Colletotrichum acutatum, a quarantine organism on strawberries in the EU, was found in Finland for the first time in 2000. Concern about rapid, unnoticeable spread of this pathogen has necessitated studies to find methods with which the quiescent fungus infection can be detected in imported, cold-stored strawberry plant material. Successful detection of C. acutatum in strawberry tissues by polymerase chain reaction (PCR) is dependent on the method of DNA extraction used. Good-quality nucleic acid, free of PCR inhibitors, was successfully prepared by slightly modifying the DNA extraction method of a commercially available kit. Species-specific primers, previously described in the literature, were successfully used in the PCR reaction. C. acutatum was detected by PCR both on symptomatic and asymptomatic plant parts and in artificially and naturally infected strawberry tissues. Positive PCR results were obtained from ripe and unripe berries, runners, petioles and different parts of crowns. The data demonstrate that the PCR technique can be used to detect C. acutatum in strawberry tissue even in plant parts that do not show visible symptoms.     

Colletotrichum acutatum interactions with unripe and ripe strawberry fruits and differential responses at histological and transcriptional levels

Microscopic investigations were conducted into the interaction of Colletotrichum acutatum on white and red strawberry (Fragaria ×ananassa) fruit surfaces. The results showed that, whilst the early interaction events were similar in both white and red fruits, after 24 h fungal colonization dramatically varied: in white fruits C. acutatum became quiescent as melanized appressoria, but on red fruits it displayed subcuticular necrotrophic invasion. A microarray analysis of white and red strawberries after 24 h of interaction with C. acutatum was performed, in order to reveal differences in gene expression possibly related to the different susceptibility of unripe and ripe fruits. Epi/catechin‐related genes and fatty acid metabolism genes, involved in the production of quiescence‐related molecules such as flavan‐3‐ols, proanthocyanidins and antifungal dienes, were found to be regulated during strawberry ripening, supporting a role for these molecules as preformed defence mechanisms. Besides several genes commonly regulated upon pathogen interaction, different genes were specifically transcribed only in white or red challenged fruits; a number of these, such as those coding for lectin and polyphenol oxidase, possibly account for specific pathogen‐induced responses. The putative biological role of these genes in the different susceptibility of fruits to C. acutatum is discussed.     

云南葡萄产区葡萄炭疽病病原鉴定及致病力分析

为了明确引起云南葡萄产区炭疽病的病原种类,利用形态鉴定和特异性引物分子检测相结合的方法对从云南省主要葡萄产区采集的60株炭疽病菌菌株进行了鉴定。葡萄炭疽病菌菌株的菌落形态和生长速率与对照菌株尖孢炭疽菌Colletotrichum acutatum差异不明显,但其分生孢子大小显著小于尖孢炭疽菌,附着胞深褐色,球形或不规则形。胶孢炭疽菌Colletotrichum gloeosporioides特异性引物CgInt/ITS4从供试葡萄炭疽病菌菌株基因组DNA中扩增出1条约500 bp的特异性条带,而尖孢炭疽菌特异性引物CaInt2/ITS4对葡萄炭疽病菌无扩增条带。研究表明,引起云南葡萄主产区炭疽病的病原为胶孢炭疽菌;供试胶孢炭疽菌对红提葡萄均有致病力,但菌株致病力差异较大,对番茄和草莓存在交叉侵染的能力,且对多菌灵的敏感性较尖孢炭疽菌高。     

Comparison of six inoculation techniques withColletotrichum acutatum on cold stored strawberry plants and screening for resistance to this fungus in French strawberry collections

Six inoculation techniques were compared for their ability to evaluate resistance toColletotrichum acutatum of five strawberry cultivars. Inoculation by dipping the whole cold stored plants in a suspension of conidia adjusted to 2.10⁶ conidia ml^–1 made it possible to screen cultivars resistant to crown rot at 28 days after inoculation. Using the dipping technique, 44 strawberry cultivars were evaluated for their resistance to one strain ofC. acutatum, 1267b. Twelve of them did not show wilt symptoms and could be classified as resistant. When another strain ofC. acutatum, 494a, was inoculated to seven cultivars, all of them including Dover, resistant to 1267b, showed wilt symptoms. This result showed the importance of investigations on genotype × isolate interactions to conduct an efficient breeding programme for screening resistance toC. acutatum.     

Induction of a defense response in strawberry mediated by an avirulent strain of <Emphasis Type="Italic">Colletotrichum</Emphasis>

In the strawberry crop area of Tucumán (north-west Argentina) the three species of Colletotrichum causing anthracnose disease (C. acutatum, C. fragariae and C. gloeosporioides) were detected. Among all isolates characterized, one of them identified as C. acutatum (M11) and another as C. fragariae (F7) were selected due to their conspicuous interaction with the strawberry cultivar Pájaro. Whereas isolate M11 produced a strong compatible interaction in cv. Pájaro with clear disease symptoms (DSR = 5.0), the isolate F7 brought about a typical incompatible interaction (DSR = 1.0). When plants of cv. Pájaro were inoculated with F7 prior to the inoculation with M11, the former avirulent strain prevented the growth of the latter virulent pathogen. Experimental evidence indicated that the time elapsed between the first inoculation with the avirulent pathogen and the second inoculation with the virulent one was crucial to inhibit the growth of the latter. The growth of F7 on the plant without provoking damage and the fact that there was no in vitro antagonistic effect between the pathogens, suggests that the avirulent strain triggers a plant defensive response against M11. The defense response was further confirmed by the detection of an early oxidative burst occurring within 4 h after the first inoculation and by the observation of anatomical changes associated with defense mechanisms that lasted 50 days after the inoculation with F7. Results obtained support the hypothesis that the plant resistance against the virulent strain M11 is elicited by one or more diffusible(s) compound(s) produced by the avirulent strain F7.     

Identification of <Emphasis Type="Italic">Colletotrichum</Emphasis> species associated with anthracnose disease of coffee in Vietnam

Twenty-three isolates of Colletotrichum gloeosporioides, five isolates of C. acutatum, two isolates of C. capsici and six isolates of C. boninense associated with anthracnose disease on coffee (Coffea spp.) in Vietnam were identified based on morphology and DNA analysis. Phylogenetic analysis of DNA sequences from the internal transcribed spacer region of nuclear rDNA and a portion of mitochondrial small subunit rRNA were concordant and allowed good separation of the taxa. We found several Colletotrichum isolates of unknown species and their taxonomic position remains unresolved. The majority of Vietnamese isolates belonged to C. gloeosporioides and they grouped together with the coffee berry disease (CBD) fungus, C. kahawae. However, C. kahawae could be distinguished from the Vietnamese C. gloeosporioides isolates based on ammonium tartrate utilization, growth rate and pathogenicity. C. gloeosporioides isolates were more pathogenic on detached green berries than isolates of the other species, i.e. C. acutatum, C capsici and C. boninense. Some of the C. gloeosporioides isolates produced slightly sunken lesions on green berries resembling CBD symptoms but it did not destroy the bean. We did not find any evidence of the presence of C. kahawae in Vietnam.     

Isolation and pathogenicity of Colletotrichum spp. causing anthracnose of strawberry in south west Spain

Anthracnose, caused by Colletotrichum spp., is a major disease of cultivated strawberry, Fragaria × ananassa. This study identifies the Colletotrichum spp. which causes strawberry anthracnose in the southwest of Spain. A survey of the region was carried out, and the strains isolated were identified as C. acutatum by using the polymerase chain reaction (PCR) with genus and species-specific primers, demonstrating that this species is currently the causal agent of strawberry anthracnose in the studied region. The pathogenicity of C. acutatum and C. gloeosporioides strains was evaluated on two principal strawberry cultivars (cvs Camarosa and Ventana) under field conditions, the latter being more pathogenic than the former.     

Molecular genetic variability of Italian binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> spp. isolates from strawberry

Fifty-eight binucleate Rhizoctonia isolates were collected over six years from strawberry plants displaying symptoms of black root rot in Italy. Almost all isolates were able to produce necrosis on strawberry roots, most of them also showed this ability on faba bean and, with lower frequency, on a crucifer and a cereal crop used in rotation with strawberry in Italy. The sequence alignment of Internal Transcribed Spacer (ITS) regions of 51 binucleate Rhizoctonia were analyzed and compared with a set of eight sequences representative of Rhizoctonia isolate Anastomosis Groups (AG) already found to be pathogenic on strawberry (AG-A, AG-G, AG-I and AG-F). The neighbour-joining tree, based on ITS region sequences, divided Italian strawberry Rhizoctonia isolates into two main clusters corresponding to AG-A and AG-G. The results were confirmed by hyphal anastomosis tests. The clustering obtained with the phylogenetic tree was also confirmed using PCR-Restriction Fragment Length Polymorphism of 28S rDNA to compare some isolates, defined as AG-A and AG-G on the basis of ITS region sequence analysis, with representative AG isolates pathogenic on strawberry. The AG-A and AG-G Rhizoctonia spp. were widespread in Italian strawberry-growing areas, although with different relative frequencies: AG-G was most frequent in northern (latitude 44°N) and AG-A in southern (latitude 39–40°N) Italy. Analysis of MOlecular VAriance, based on geographic location, showed that Rhizoctonia molecular variations between northern and southern Italy accounted for 36.6% of the total, but most of the variations (61%) occurred within each of the four geographical regions from where the isolates originated.     

Simple diagnosis using ethanol immersion of strawberry plants with latent infection by Colletotrichum acutatum, Dendrophoma obscurans, and Fusarium oxysporum f. sp. fragariae

Simple diagnosis by ethanol immersion (SDEI) to detect Glomerella cingulata was used to detect three other fungi that also cause latent infection of strawberry plants. Signs on strawberry leaves with asymptomatic latent infection by Colletotrichum acutatum became visible using SDEI. Salmon-pink conidial masses were produced in the acervuli on the treated leaves 5 days after incubation at 28°C. In the case of Dendrophoma obscurans, pycnidia with amber conidial masses formed 5 days after incubation at 28°C. The pycnidia were observed mainly on the ribs, and conidial masses exuded from the ostiole. These macroscopic conidial masses were similar to those of G. cingulata and C. acutatum. When water was dripped onto a lesion caused by D. obscurans, the pycnidia exuded white filamentous conidial masses, making the distinction of D. obscurans from G. cingulata or C. acutatum. On petioles with latent infection by Fusarium oxysporum f. sp. fragariae, white aerial hyphae grew out from the vascular tissues on the cut surface 3 days after incubation at 28°C and were easily observed by eye or with a loupe. Thus, SDEI was also useful for diagnosing latent infection of strawberry plants by C. acutatum, D. obscurans, and F. oxysporum f. sp. fragariae.     

Lack of host specificity of Colletotrichum spp. isolates associated with anthracnose symptoms on mango in Brazil

The aim of the present study was to analyse the genetic and pathogenic variability of Colletotrichum spp. isolates from various organs and cultivars of mango with anthracnose symptoms, collected from different municipalities of São Paulo State, Brazil. Colletotrichum gloeosporioides isolates from symptomless citrus leaves and C. acutatum isolates from citrus flowers with post‐bloom fruit drop symptoms were included as controls. Sequencing of the ITS region allowed the identification of 183 C. gloeosporioides isolates from mango; only one isolate was identified as C. acutatum. amova analysis of ITS sequences showed larger genetic variability among isolates from the same municipality than among those from different populations. fAFLP markers indicated high levels of genetic variability among the C. gloeosporioides isolates from mango and no correlation between genetic variability and isolate source. Only one C. gloeosporioides mango isolate had the same genotype as the C. gloeosporioides isolates from citrus leaves, as determined by ITS sequencing and fAFLP analysis. Pathogenicity tests revealed that C. gloeosporioides and C. acutatum isolates from either mango or citrus can cause anthracnose symptoms on leaves of mango cvs Palmer and Tommy Atkins and blossom blight symptoms in citrus flowers. These outcomes indicate a lack of host specificity of the Colletotrichum species and suggest the possibility of host migration.     

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.     

A rapid qualitative molecular method for the identification of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> and <Emphasis Type="Italic">C. gloeosporioides</Emphasis>

Identification of the causal agent for anthracnose caused by C. acutatum and C. gloeosporioides based on morphological and cultural criteria is problematic as both are morphologically and genetically diverse. To evaluate a qualitative molecular method to readily distinguish between these two species, Restriction fragment length polymorphisms (RFLP) of a 1-kb intron of the glutamine synthetase (GS) gene was evaluated utilizing representative isolates from a world-wide collection. Unique band patterns of the 1-kb GS intron were obtained for C. acutatum (two fragments with 600 and 350 bp) and C. gloeosporioides (four fragments with 238–340, 252–254, 204, and 108–116 bp) based on PstI enzyme digestion of the amplified PCR product. These data were also confirmed by PstI digestion of the intron DNA sequences using BioEdit software. The identification based on RFLPs of the 1-kb GS intron was consistent with the identification based on previously evaluated species-specific primers (CaInt2 and CgInt). In addition, both species can be differentiated by multiplex PCR. CaInt2, CgInt and ITS4 in one PCR will distinguish between C. acutatum and C. gloeosporioides by differences in PCR product fragment size: 490 bp and 470 bp, respectively. Also, a rapid DNA extraction method was developed, which reduced the time for DNA extraction from two hours to five minutes. In summary, RFLP of the 1-kb GS intron is a reliable technique for identification and differentiation between both species, does not require a sequencing step, and may be useful to diagnostic clinics in helping to make disease management recommendations.     

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.     

Molecular characterization of <Emphasis Type="Italic">Pythium</Emphasis> group F isolates by ribosomal-and intermicrosatellite-DNA regions analysis

Pythium group F is a ubiquitous, though minor, pathogen in several soilless and soil cultures; investigations were carried out to analyze different regions of the DNA and better understand the nature of this group. Fourty-two isolates were obtained from a variety of plants (cucumber, lettuce, tomato) grown in soil or soilless cultures collected in various countries (Canada, Denmark, France, Norway, Sweden and United Kingdom). All Pythium group F isolates displayed amplified ITS1-5,8S-ITS2 ribosomal DNA region (rDNA) of similar length, whereas polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) revealed that, among the seven enzymes used, polymorphism was only identified with Hin6I. After cloning of ITS1-5,8S-ITS2 rDNA region from Pythium group F isolates that displayed restriction polymorphism patterns with Hin6I, comparisons of sequence and restriction mapping data showed a slight variation consisting in a single base change. Inter Simple Sequence Repeat (ISSR)-PCR method was also used to obtain data related to the entire genome and not only to a single DNA region. It identified repeated motifs in the genome of Pythium group F isolates. Two primers (CAC)₅ and (CCA)₅ detected polymorphism, and isolates were classified among 11 molecular clusters. The genetic diversity of this group was not correlated with the geographical locations or the host plants from which the isolates originated. Polymorphism of Pythium group F isolates pointed out by ISSR is discussed     

Improved method to induce sporulation of <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>, causal fungus of grape ripe rot

Colletotrichum gloeosporioides and C. acutatum are causal agents of grape ripe rot, but with available methods, sporulation of C. gloeosporioides on plate media has been unstable and inferior to that of C. acutatum. To facilitate studies on C. gloeosporioides, I developed an improved method to induce conidiation of this fungus. Isolates of C. gloeosporioides were pre-cultured in potato dextrose broth for 1 week, then pulverized in whole broth. The homogenate was then spread on diluted oatmeal agar (15–20% commercial oatmeal agar medium, 1.5% agar) plates. After the plates were cultured at 25°C under continuous light for another week, the C. gloeosporioides isolates sporulated stably on the plate medium.