Morphological and molecular diversity of Colletotrichum spp. causing pepper spot and anthracnose of lychee (Litchi chinensis) in Australia

Since the 1980s a new disease has been affecting Australian lychee. Pepper spot appears as small, black superficial lesions on fruit, leaves, petioles and pedicels and is caused by Colletotrichum gloeosporioides, the same fungus that causes postharvest anthracnose of lychee fruit. The aim of this study was to determine if a new genotype of C. gloeosporioides is responsible for the pepper spot symptom. Morphological assessments, arbitrarily‐primed PCR (ap‐PCR) and DNA sequencing studies did not differentiate isolates of C. gloeosporioides from anthracnose and pepper spot lesions. The ap‐PCR identified 21 different genotypes of C. gloeosporioides, three of which were predominant. A specific genotype identified using ap‐PCR was associated with the production of the teleomorph in culture. Analysis of sequence data of ITS and β‐tubulin regions of representative isolates did not group the lychee isolates into a monophyletic clade; however, given the majority of the isolates were from one of three genotypes found using ap‐PCR, the possibility of a lychee specific group of C. gloeosporioides is discussed.     

Quantification of the aggressiveness of a foliar pathogen, Colletotrichum gloeosporioides, responsible for water yam (Dioscorea alata) anthracnose

In the absence of a gene-for-gene relationship between a pathogen and its host, knowledge about aggressiveness is crucial to characterize novel pathogen populations that potentially emerge in agricultural pathosystems. Information about pathogen aggressiveness is also critical when establishing representative panels of pathogen isolates to test host resistance and in mapping quantitative trait loci involved in the host resistance. In this study, we focused on the fungus C. gloeosporioides that causes necrosis on the aerial part of one of its host plants, Dioscorea alata, and identified the in vitro conditions required to assess fungal aggressiveness on this host. Our main purpose was to convert the necrosis area development into a unique index for quantifying pathogen aggressiveness. The ??Ag?? index described here has two advantages. First, it integrates the variance of symptom evolution curves to estimate the lesion development rates (initial and secondary) and the maximal necrosis area. Secondly, the new index takes two different symptoms commonly observed when inoculating D. alata leaves with C. gloeosporioides into account, one correlated with high leaf colonisation efficiency and the other with low colonisation efficiency. The weights accorded to each symptom in the index were proportional to leaf colonisation efficiency. We propose a framework for the acquisition of this index that has been designed to be conveniently combined with the routine bioassays required to establish representative panels of pathogen isolates. The general framework for the construction of this index can be broadly applied to diseases with necrotic symptoms.     

Olive anthracnose caused by Colletotrichum in Uruguay: symptoms,species diversity and pathogenicity on flowers and fruits

European Journal of Plant Pathology - Olive anthracnose caused by C. acutatum, C. gloeosporioides and C. boninense species complexes, is the most widespread and economically important olive disease...     

Infection of Nicotiana species by the anthracnose fungus, Colletotrichum orbiculare

Colletotrichum gloeosporioides f. sp. malvae, isolate Biomal®, ATCC 20767, was originally isolated from round-leaved mallow (Malva pusilla) and developed as a weed biocontrol agent. Ribosomal DNA sequence analysis was recently used to re-classify this fungus as C. orbiculare, which is an aggregate species with a number of formae speciales. Several morphological features of ATCC 20767 were examined that were consistent with those described for C. orbiculare, and inoculation of a number of Nicotiana species and several cultivars of N. tabacum showed that this fungus was pathogenic to many of these previously undescribed hosts. Spore germination and appressorium formation were higher on tobacco than previously observed on round-leaved mallow. The pathogen produced melanized appressoria on N. tabacum leaves that formed preferentially at the anticlinal epidermal cell wall. A symptomless phase of infection persisted for 72–96 h postinoculation, during which time the fungus first produced a spherical infection vesicle from an infection peg, and then large primary hyphae which grew through the epidermal cells. The large primary hyphae were highly constricted at the points of penetration of the host cell walls. Thin secondary hyphae appeared at 96–120 h postinoculation coinciding with the appearance of light green, water-soaked spots and the formation of acervuli. The infection of tobacco by C. orbiculare ATCC 20767 is not a non-specific interaction but appears to follow an intracellular hemibiotrophic infection process that is very similar to that established for the C. orbiculare infection of round-leaved mallow, cucurbits and beans.     

Pathogenicity of Colletotrichum species causing anthracnose of Capsicum in Asia

Anthracnose of chili is caused by a complex of Colletotrichum species, with recent surveys reporting at least 28 different species implicated. However, there have been very few studies to identify the relative pathogenicity of the various species or to optimize a bioassay to assess pathogenicity. A detached Capsicum fruit bioassay to determine the pathogenicity of a diverse geographical range of isolates of Colletotrichum scovillei showed fruit maturity, host genotype, and inoculation method all interact to affect infection and rate of lesion development. On Capsicum annuum ‘Bangchang’ fruit wounded prior to inoculation, pathogenicity was consistent regardless of fruit maturity. In contrast, without wounding there was variability in pathogenicity. On the relatively resistant host Capsicum chinense PBC932, pathogenicity was dependent on both the inoculation method and the maturity stage of the fruit. In addition, lack of correlation in pathogenicity of isolates between the two Capsicum lines indicated that there was host–isolate specialization that would make prediction of pathogenicity of isolates on host difficult. In a further study, 10 species of Colletotrichum isolated from diseased chili fruits in Asia caused anthracnose symptoms on C. annuum ‘Bangchang’ under all testing conditions, with large differences in aggressiveness. C. chinense PBC932 was generally more resistant to all the species, with smaller lesions produced in different host conditions. Colletotrichum javanense and C. scovillei were highly aggressive relative to other species, especially when inoculated on nonwounded fruit. Pathotype differences were identified within multiple isolates of C. scovillei and C. siamense, the two most frequently identified pathogenic species on chili.     

Characterization of Colletotrichum gloeosporioides responsible for anthracnose disease of Trichosanthes kirilowii Maxim in central China

Anthracnose disease is an important fungal disease on Trichosanthes kirilowii Maxim. (T. kirilowii), which has recently become a major problem in production areas of T. kirilowii in Anhui Province, central China. A total of 78 putative Colletotrichum spp. isolates were obtained from infected leaves and fruits of T. kirilowii showing anthracnose symptoms during the years 2009-12. All the fungal isolates were consistently identified as Colletotrichum gloeosporioides by a combination of morphological characteristics, species-specific PCR assays and sequence analysis of the ITS region. The results of pathogenicity tests showed difference in virulence among 24 isolates of C. gloeosporioides representatively selected from different regions, when disease symptoms were evaluated after artificially inoculation on the detached T. kirilowii fruits for 2 weeks. The mycelial growth and germination rate of C. gloeosporioides isolates in vitro were determined under temperatures of 10, 15, 20, 25, 28, 30, 33 and 36°C. The optimum growth temperatures of these isolates were observed at 25-30°C, and the germination rates were rather high in the range of 20-33°C. Host range study revealed that the pathogen, under experimental conditions, can infect nine cultivated and weed species belonging to six families (Cucurbitaceae, Rosaceae, Solanaceae, Convolvulaceae, Celastraceae and Cannabinaceae). Moreover, C. gloeosporioides can overwinter safely in infected tissues in field soil, suggesting the potential contribution of plant debris to disease epidemics in the region. The study presents useful information for epidemiological surveillance of T. kirilowii anthracnose caused by C. gloeosporioides in the central region of China.     

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.     

Comparison of disease progress curves for yam anthracnose (Colletotrichum gloeosporioides)

The incidence and severity of foliar anthracnose, caused by Colletotrichum gloeosporioides , were recorded for seven fields of yams ( Dioscorea alata var. White Lisbon) in Barbados over a complete growing season. A scoring system was developed to estimate severity, placing the emphasis on the earliest, least visible stages of the disease. Disease progress curves were described in terms of the logistic model and the effects of site and leaf age on the parameters as well as on disease at tuber bulking were analysed. Differences between sites indicated that disease was more severe in areas of higher rainfall. In severe epidemics, disease predominated on young leaves. Fungicides applied sufficiently early can delay the onset of disease and slow the rate of disease progress.     

Stomatal penetration of cowpea (Vigna unguiculata) leaves by a Colletotrichum species causing latent anthracnose

The infection process of a Colletotrichum species causing latent infection and anthracnose in cowpea ( Vigna unguiculata ) was studied in seedlings by light and confocal microscopy. Leaf surfaces were extensively colonized by an anastomosing network of germ-tubes and conidia. This epiphytic mycelium produced abundant secondary conidia on short conidiophores. Although melanized appressoria were developed, the host surface was not penetrated directly. The fungus only gained ingress into leaves through stomatal openings, by means of undifferentiated germ-tubes, and slowly colonized the mesophyll by intercellular hyphae, without initially producing visible symptoms. Anthracnose lesions with multisetate acervuli appeared on senescent leaves after a prolonged symptomless period of host colonization lasting > 2 weeks. Analysis of the nucleotide sequences of the amplified D2 and ITS-2 regions of rDNA revealed close similarities (95–96%) between this cowpea pathogen and isolates of C . gloeosporioides from Aeschynomene virginica, Stylosanthes scabra and Mangifera indica. These results, in addition to other morphological and growth attributes, identify this endophytic anthracnose pathogen of cowpea as a Colletotrichum species distinct from C. capsici and C. destructivum .     

Genetic diversity and pathotype determination of Colletotrichum sublineolum isolates causing anthracnose in sorghum

Amplified fragment length polymorphism (AFLP) based genetic diversity was analyzed for 232 Colletotrichum sublineolum isolates collected between 2002 and 2004 from three geographically distinct regions of Texas, and from Arkansas, Georgia, and Puerto Rico. Results revealed significant levels of polymorphism (59%) among the isolates. Even so, genetic similarity between isolates was high, ranging from 0.78 to 1.00. Clustering of similar isolates did not correlate with either geographic origin or year of collection. Pathotypes of 20 of the isolates were determined using 14 sorghum lines previously used in Brazil and the United States and 4 from Sudan. Seventeen new pathotypes were established from the 18 isolates that gave uniform and consistent reactions on all host differentials over 2?years of greenhouse testing. Differentials BTx378 and QL3 were resistant to all isolates while BTx623 and TAM428 were universally susceptible both years. Each of these lines had shown differential responses in prior studies indicating that the pathogen population has sufficient diversity to adapt rapidly to changes in resistant host lines deployed. When the 2-step pathotype classification scheme was used, the 18 isolates examined in this study were placed in four pathotype groups (A, C, D and G), which would further then be separated into ten distinct pathotypes. Common sets of differentials and a standardized nomenclature will allow for comparison to be made among pathotypes of C. sublineolum detected from different regions and also could help direct planting of appropriate sorghum lines and aid in the development of more durable forms of resistance.     

Distribution and phylodynamics of papaya ringspot virus on Carica papaya in Cuba

Orchard and garden papaya crops grown in 47 Cuban municipalities were surveyed from 2008 to 2013, revealing the widespread distribution of papaya ringspot virus (PRSV) in Cuba. Phylodynamic analyses performed with the partial coat protein gene of all Cuban PRSV-P isolates (34 sequences) and 107 sequences of isolates from the American continent and the Caribbean islands showed a most recent common ancestor in 1942 (95% highest posterior density, HPD 95% = 1911–1967). The substitution rate was estimated to be 7.7 × 10⁻⁴ substitutions per site per year (HPD 95% = 4.6 × 10⁻⁴ to 1.1 × 10⁻³), which is equivalent to those detected in other RNA viruses. Demographic reconstruction of PRSV showed that viral diversity increased in the 1985–1990 period, which coincides with the implementation of extensive production practices. Moreover in Cuba, viral dispersion occurred from Mexico and other unknown ancestral locations. The spatiotemporal diffusion analysis proposed Mexico as an ancestral area for the origin of diversification in the American continent and suggests new dispersion events between American and Caribbean isolates. The observed widespread distribution, clear geographic grouping of Cuban isolates, virus growth and genetic diversity provide strong evidence of the PRSV dispersion patterns, which has implications for the control strategies of PRSV.     

Identification and characterization of Colletotrichum species associated with anthracnose disease of banana

Banana (Musa spp.) is one of the five most abundantly produced fruits in the world and is widely planted in tropical and subtropical areas. Banana anthracnose is one of the main diseases during the growth and postharvest storage period of banana, seriously affecting quality and production. In this study, 24 samples of banana anthracnose were collected near the cities Nanning, Qinzhou, Baise, and Chongzuo in Guangxi Province, China. Based on colony features, conidial and appressorial morphology, and sequence analysis of several genomic regions (internal transcribed spacer [ITS] region, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], actin [ACT], β-tubulin [TUB2], chitin synthase [CHS-1], calmodulin [CAL], and the intergenic region of apn2 and MAT1-2-1 [ApMAT]), the 32 Colletotrichum isolates obtained were identified as five species: C. fructicola (41%), C. cliviicola (28%), C. siamense (16%), C. karstii (9%), and C. musae (6%). A conidial suspension (10⁶ spores/ml) was used to inoculate banana seedlings for pathogenicity tests by applying 20 μl to wound sites. Lesions caused by C. musae developed most rapidly while those of C. karstii took the longest time to develop. This is the first report of C. siamense, and C. karstii associated with banana anthracnose in China, and the first report of C. fructicola and C. cliviicola associated with banana anthracnose worldwide.     

Characterization of Erwinia sp. causing black rot of papaya (Carica papaya) first recorded in Okinawa Main Island,Japan

Since 2002, papaya black rot has been spreading over several islands of Okinawa Prefecture, Japan. To devise a prevention strategy for the disease, microbiological research on the pathogen was conducted. Twelve strains were isolated from papaya infected with black rot showing symptoms such as water-soaked lesions on stems and petioles, black spots on fruits, and rotted leaves turning yellow with necrotic spots. Through Koch's postulates, we confirmed that the isolated strains caused papaya black rot. Bacteriological assays showed that the strains have characteristics different from the type strains of Erwinia mallotivora, E. papayae, and E. psidii. Moreover, 16S rDNA sequence similarity searches showed that the isolated strains had less than 98.6% similarity with type strains. Additionally, phylogenetic analysis of 16S rDNA sequences suggested that the isolated strains were possibly a novel species belonging to the genus Erwinia, as the strains formed an independent cluster and had low sequence similarity with the type strains. Earlier studies indicated that papaya black rot is caused by E. cypripedii. Therefore, we propose to add the Erwinia sp. isolated in this study to the list of papaya black rot pathogens.     

Influence of native endophytic bacteria on the growth and bacterial crown rot tolerance of papaya (Carica papaya)

The native plant microbiome is composed of diverse microbial communities that influence overall plant health, with some species known to promote plant growth and pathogen resistance. Here, we show the antibacterial and growth promoting activities of autoclaved culture metabolites (ACM) from native endophytic bacteria (NEB). These NEB were isolated from a papaya cultivar (var. Cariflora) that is tolerant to bacterial crown rot (BCR) caused by Erwinia mallotivora. In this cultivar, bacterial colonization in tissues recovering from the disease was observed before onset of tissue regeneration or ‘regrowth’. We further isolated and characterized these bacteria and were able to identify two culturable stem NEB related to plant endophytic genera Kosakonia sp. (ex. Enterobacter sp., isolate EBW), and to Sphingomonas sp. (isolate EBY). We also identified root NEB under genus Bacillus (isolates BN, BS, and BT). Inhibition assays indicated that ACM from these NEB promptly (within 18-30 h) and efficiently inhibited (60–65% reduction) E. mallotivora proliferation in vitro. When surface-sterilized papaya seeds were soaked in ACM from isolates EBY and EBW, germination was variably retarded (20–60% reduction) depending on plant genotype, but plant biomass accumulation was significantly stimulated, at around two-fold increase. Moreover, greenhouse experiments show that ACM from all isolates, especially isolate EBW, significantly reduced BCR incidence and severity in a susceptible genotype (var. Solo), at around two-fold. In general, our observations of pathogen antagonism and plant growth promotion leading to disease reduction, suggested the influence of native endophytic bacteria to increased fitness in plants, and tolerance against the re-emerging crown rot disease of papaya.

     

Identification of colletotrichum species responsible for anthracnose and root necrosis of strawberry in Israel

ABSTRACT Strawberry anthracnose was observed for the first time in Israel in 1995. The disease reached epidemic proportions in Israeli nurseries and production fields in 1995 and 1996. Using morphological and cultural characteristics, the species responsible for anthracnose was identified as Colletotrichum acutatum. A reliable semi-selective medium, amended with iprodione and lactic acid, was used to isolate the fungus from infected tissues. In addition, C. acutatum was subsequently isolated from necrotic roots of stunted, chlorotic plants that exhibited no symptoms of anthracnose. High levels of the pathogen from naturally infested field soil and perlite growth substrate were quantified from the rhizosphere of diseased plants on the iprodione-amended medium. Both foliar- and rootinfecting isolates were equally pathogenic to strawberry, causing 95 to 100% plant mortality, when inoculated on roots and foliage. In complementation (heterokaryon) tests using nitrate nonutilizing mutants, 113 out of 115 isolates from different plant parts and locations belonged to a single vegetative compatibility group. Arbitrarily primed polymerase chain reaction of genomic DNA using four repetitive-motif primers produced nearly uniform amplified DNA banding patterns for 141 of the Israeli strawberry isolates from different sites, plots, plant tissues, and cultivars. When compared to reference isolates from the US, these band patterns suggested that a single introduction of C. acutatum was responsible for strawberry anthracnose on foliage and necrosis of roots in Israel.     

Quantitatively expressed resistance to anthracnose (Colletotrichum gloeosporioides) in Stylosanthes scabra

Nineteen accessions of Stylosanthes scabra that had low terminal severities of anthracnose in the field were evaluated for their response in the glasshouse using four isolates representing three different races of Colletotrichum gloeosporioides. Accessions could be allocated to one of three groups: one showed a higher disease severity after inoculation with isolate UQ14 compared to the other isolates; the second showed a higher severity after inoculation with isolates WRS32 and/or WRS20 compared to isolates SR4 and UQ14, and the third group showed moderate to low disease severities following inoculation with all four isolates. Based on reduced disease severity and compatible infection types, a set of six accessions was selected from the three groups for further testing, two of which showed no significant difference in their response to the four isolates. Two components of resistance, incubation period and infection efficiency were studied in the selected accessions. Of these, incubation period appeared unrelated to the expression of resistance. Criteria for identifying this form of quantitatively expressed resistance are discussed.     

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.