Markers to differentiate species of anthracnose fungi identify <Emphasis Type="Italic">Colletotrichum fructicola</Emphasis> as the predominant virulent species in strawberry plants in Chiba Prefecture of Japan

Colletotrichum fungi belonging to the Colletotrichum gloeosporioides species complex include a number of economically important postharvest pathogens that often cause anthracnose. Until now, different species within this group could only be distinguished from one another reliably using multigenic phylogenetic analyses. Using a comparative genomics approach, we developed a marker that can differentiate Colletotrichum fructicola, Colletotrichum aenigma and Colletotrichum siamense within the C. gloeosporioides species complex based on PCR amplicon size differences. When we used this marker to classify 115 isolates collected over 20 years from strawberries in Chiba Prefecture, Japan, the isolates were predominantly C. fructicola. To our knowledge, this is the first report characterizing different species of Colletotrichum infecting strawberries in Japan and contributes to our understanding on the diversity of anthracnose pathogens in Japan.     

Phylogenetic relationship and fungicide sensitivity of members of the <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis> species complex from apple

The members of the Colletotrichum gloeosporioides species complex (CGSC), the dominant pathogens of apple bitter rot in Nagano prefecture, Japan, were reidentified and the relationship between the species and fungicide sensitivity was revealed. Based on phylogenetic analysis of the ApMat locus with the neighbor-joining (NJ) method, isolates from apple contained three species of the CGSC; C. fructicola, C. aenigma, C. siamense, and three clades of the CGSC: Clade V, S and K. Colletotrichum fructicola and Clade S dominated in Nagano Prefecture. Isolates of C. siamense, C. aenigma and Clade V, S and K remained sensitive to benomyl and quinone outside inhibitor (QoI) fungicides, while C. fructicola often developed resistance to benomyl and QoI fungicides. These results suggest that the development of fungicide resistance differs among members of the CGSC.     

The potential of fludioxonil for anthracnose control on China chili fruit

Anthracnose, caused by Colletotrichum scovillei, is one of the most destructive disease which causes massive yield losses in chili. Our preliminary study has demonstrated that fludioxonil had high activity against mycelial growth and spore germination of C. scovillei but it has not been labeled on chili. The aim of this study was to investigate the impact of fludioxonil on C. scovillei infection. In greenhouse and field trials, fludioxonil was applied to chili plants to determine the duration of protection of fruit provided by this compound against chili anthracnose. Fludioxonil may have impact on the early stage of chili anthracnose infection by inhibiting the germ tube elongation and appressorium formation. Moreover, it inhibited the secondary infection of C. scovillei by inhibiting the sporulation, germination of spores and formation of appressoria. Fludioxonil provided high level of protective activity for up to 96 h, as well as excellent curative activity of within 24 h in vivo tests. In field trials, fludioxonil can reduce the incidence and severity of chili anthracnose while giving a higher chili yield. These results suggest that fludioxonil could be a promising fungicide for anthracnose control in chili production.     

Molecular and phenotypic characterization revealed six <Emphasis Type="Italic">Colletotrichum</Emphasis> species responsible for anthracnose disease of small cardamom in South India

Small cardamom (Elettaria cardamomum Maton) is extensively cultivated in the Western Ghats of South India either as a monocrop under the forest trees or as an intercrop along with arecanut and coffee plantations. Colletotrichum species responsible for severe outbreaks of anthracnose on small cardamom in South India are reported. Small cardamom anthracnose, popularly known as “Chenthal”, manifests itself on the foliage as yellowish lesions, which later coalesce to form large blighted areas. In advanced stages, the affected leaves dry up giving a burnt appearance to the plant. Twenty-five isolates of Colletotrichum were isolated from leaves of small cardamom in Karnataka, Kerala and Tamil Nadu states of India. The isolates were characterized through morphological studies and multilocus phylogenetic analysis (ITS, ACT, CHS-1, GAPDH, TUB2, CYLH3, GS and ApMat gene regions) to test whether different species are present and identified: C. karstii (2 isolates), C. gloeosporioides (1), C. siamense (7), C. syzygicola (6), Colletotrichum sp (5), and C. guajavae (4), as the cause of anthracnose on small cardamom for the first time. Pathogenicity of the six species was confirmed. To our knowledge, this is the first detailed study of Colletotrichum species which cause anthracnose diseases on small cardamom.     

Diversity of <Emphasis Type="Italic">Colletotrichum</Emphasis> species in coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis>) plantations in Mexico

The aim of this study was to identify the Colletotrichum species associated with anthracnose symptoms in coffee (Coffea arabica L.) plantations in northern Puebla, Mexico. In 2013, five surveys were conducted in different production areas and at different altitudes. Symptomatic leaves, shoots, and ripe and unripe fruits of the coffee variety Red Caturra were collected. Isolates were obtained and the Colletotrichum species were identified morphologically and characterized by multilocus sequence analyses of the ACT, CAL, GAPDH, and TUB2 genes and the rDNA region. Additionally, pathogenicity tests were conducted using six isolates. We identified C. gigasporum, C. gloeosporioides, C. karstii (two isolates), C. siamense, and C. theobromicola. This is the first report of these five species infecting leaves of coffee. The symptoms caused by these species were characterized, but the species causing Coffee Berry Disease was not found. This is the first report of a complex of species affecting coffee plants in the same geographical area in Mexico, and suggests that other complexes of species may be important pathogens in coffee-producing areas elsewhere.     

Weeds as the potential inoculum source of <Emphasis Type="Italic">Colletotrichum fructicola</Emphasis> responsible for strawberry anthracnose in Nara,Japan

Colletotrichum fructicola is a major causal agent among anthracnose pathogens of strawberry in Nara, Japan. We hypothesized that a wide range of weeds growing in and around strawberry fields are inoculum sources of the disease and investigated their potential as hosts of C. fructicola. We also examined the influence of herbicide treatment on C. fructicola sporulation on weeds. The fungus was detected on 31 of 541 (5.7%) leaves sampled from 13 weed species from 2005 to 2008. The fungus was most frequently isolated from leaves of Amaranthus blitum with an isolation frequency of 17.9%; inoculation of A. blitum with the pathogen caused brown leaf spots. Other weeds such as Digitaria ciliaris, Galinsoga ciliata, Solidago altissima, Erigeron annuus, and Sonchus oleraceus were found to harbor the fungus at lower rates (4.3–8.1%) without symptoms. C. fructicola formed acervuli on leaves of A. blitum, D. ciliaris, and S. oleraceus after plants were killed by a herbicide (glyphosate). These results demonstrated that infected weeds associated with strawberry cultivation are potential inoculum sources of C. fructicola, especially after herbicide treatment.     

Fungicide sensitivity,growth rate,aggressiveness and frost hardiness of <Emphasis Type="Italic">Monilinia fructicola</Emphasis> and <Emphasis Type="Italic">Monilinia laxa</Emphasis> isolates

Monilinia fructicola, the most destructive pathogen of the genus Monilinia, has recently been introduced into Serbia and many other European countries. Since then, many studies have been conducted to evaluate the characteristics of Monilinia species that have a role in the establishment and survival of the pathogen in new areas. The present study assessed the capacity of M. fructicola to repress and replace Monilinia laxa in Serbia based on: fungicide sensitivity, growth rate and aggressiveness at different temperatures, as well as frost hardiness of the isolates of both species. The results showed that the isolates of M. fructicola, compared to M. laxa, were significantly less sensitive to the following fungicides: iprodione, tebucanozole, chlorothalonil, azoxystrobin, fluopyram, and boscalid. In addition, M. laxa isolates exhibited little variation in sensitivity to all of the tested fungicides, whereas M. fructicola isolates displayed a wide range of sensitivity. The temperature of 5°C favored M. laxa growth and aggressiveness, while at 30°C M. fructicola grew faster and had higher lesion expansion rate. These results support an assumption that M. fructicola will continue to spread in Serbian orchards in coming years, particularly on stone fruits harvested during hot summer weather.     

Application of the consolidated species concept to identify the causal agent of strawberry anthracnose in Iran and initial molecular dating of the <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> species complex

Anthracnose caused by Colletotrichum spp. is one of main strawberry diseases worldwide. The disease recently arrived in Iran and become epidemic in the Kurdistan province. A combination of morphological, molecular and pathogen-host interaction approaches (referred to the consolidated species concept) were applied to isolates from symptomatic strawberry tissues collected in Iran. Multi-gene phylogenetic analysis based on ITS-rDNA, beta-tubulin (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) identified the species as the S5 clade of Colletotrichum nymphaeae. No significant intraspecies variation was observed in terms of morphology and pathogenicity. No significant genetic variation was observed among the isolates using inter simple sequence repeat analysis (ISSR) and primer pair combination of ERIC1/BOX and ERIC2/BOX fingerprinting markers. Based on the results of this study, it can be postulated that the C. nymphaeae population in Iran established itself from a single origin due to a founder effect. More generally, molecular dating based on relaxed clocks indicates co-radiation of C. nymphaeae S5 and strawberry plants and suggests high reciprocal specificity between the host and pathogen.     

Rapid diagnosis of soybean anthracnose caused by <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis> using a loop-mediated isothermal amplification (LAMP) assay

A loop-mediated isothermal amplification (LAMP) assay that directly detects Colletotrichum truncatum in diseased soybean tissues is described, thus allowing rapid diagnosis of soybean anthracnose. Using the target gene Rpb1 (that codes for the large subunit of RNA polymerase II), we designed and screened a set of species-specific primers allowing amplification at 62 °C over 70 min. After addition of SYBR Green I to the LAMP reaction products, a yellow-green color (visible to the unaided eye) developed only in the presence of C. truncatum. The detection limit of the LAMP assay was 100 pg (per μL genomic DNA). The Rpb1-Ct-LAMP assay has been successfully used to diagnose soybean anthracnose in field samples collected from Jiangsu, Anhui and Hubei provinces of China, and to detect C. truncatum in soybean seeds from farmers’ markets. Our results show that the Rpb1-Ct-LAMP assay is a useful and convenient method for detecting C. truncatum, and thus for diagnosis of soybean anthracnose.     

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.     

Chili anthracnose: Colletotrichum taxonomy and pathogenicity

Chili anthracnose is caused by Colletotrichum species mostly associated with the acutatum, truncatum and gloeosporioides complexes. Since 2009 the Colletotrichum taxonomy has been extensively revised based on multigene phylogenetics, which has had a large impact on the number of species known to cause anthracnose disease of chili. This review discusses (i) the taxonomy of Colletotrichum spp. infecting chili, and (ii) the impact of Colletotrichum pathotypes on breeding for resistance to anthracnose. To date, 24 Colletotrichum species have been identified as pathogens of chili anthracnose, with the three main pathogens being C. scovillei, C. truncatum and C. siamense. Identification of several pathotypes within these three Colletotrichum species, particularly pathotypes that can overcome resistance in the related Capsicum species, Ca. chinense and Ca. baccatum, will be of major concern to plant breeders as they develop resistant chili genotypes from the transfer of resistance genes from these Capsicum species into Ca. annuum. Accurate identification of the Colletotrichum species causing anthracnose and improved understanding of the biology of the Colletotrichum species and their interaction with the host will enable the application of improved integrated disease management techniques.     

Pathogenicity and characterization of <Emphasis Type="Italic">Colletotrichum lentis</Emphasis>: A causal agent of anthracnose in common vetch (<Emphasis Type="Italic">Vicia sativa</Emphasis>)

A series of studies were carried out on Colletotrichum lentis which had been been identified in 2015 based largely on the distinctive shape of conidia and ITS sequences, and which has been causing severe anthracnose disease symptoms on common vetch plants (Vicia sativa) in Gansu Province in the northwest region of China. A key focus of the present studies was to determine how vetch crops become infected. The addition of residues from harvested common vetch crops to land being prepared as a seedbed was shown to result in the highest levels of disease severity indicating that this management practice was the most likely way for crops to become severely infected. Seed transmission was unlikely to be the cause of severe outbreaks as less than 5% of seeds harvested from severely infected plants carried C. lentis. To verify that the species causing the severe outbreaks of anthracnose disease of vetch crops was C. lentis, sequence analysis of the ITS, TUB2, ACT, HIS3 and GAPDH genes was conducted. C. lentis isolates from common vetch and lentil (Lens culinaris) formed a distinctive group among Colletotrichum species, including those species that infect other forage and field crops. The unique shape of conidia of C. lentis, straight with only one end curved, was confirmed as being reliable for rapid identification of disease outbreaks caused by this damaging fungal pathogen.     

Nonhost resistance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> against <Emphasis Type="Italic">Colletotrichum</Emphasis> species

Arabidopsis thaliana exhibits a durable resistance called nonhost resistance against nonadapted fungal pathogens. A. thaliana activates preinvasive resistance and terminates entry attempts by nonadapted fungi belonging to the genus Colletotrichum, which cause anthracnose disease in many plants. In the interaction between A. thaliana and nonadapted C. tropicale, the preinvasive resistance involves the PENETRATION 2-related antifungal secondary metabolite pathway and the ENHANCED DISEASE RESISTANCE 1-dependent antifungal peptide pathway. The development of invasive hyphae by C. tropicale owing to the reduction of preinvasive resistance then triggers the blockage of further hyphal expansion via the activation of the second layer of resistance, i.e., postinvasive resistance, which guarantees the robustness of the nonhost resistance of A. thaliana against Colletotrichum pathogens. Both the tryptophan-derived metabolic pathway and glutathione synthesis play critical roles in the postinvasive resistance against C. tropicale, although the molecular mechanism of postinvasive resistance remains to be elucidated. In this review, we describe the current understanding of the molecular background of the Arabidopsis nonhost resistance against Colletotrichum fungi and discuss perspectives for future research on this durable resistance.     

Colletotrichum species associated with chili anthracnose in Australia

Phylogenetic relationships were determined for 45 Colletotrichum isolates causing anthracnose disease of chili in Queensland, Australia. Initial screening based on morphology, ITS and TUB2 genes resulted in a subset of 21 isolates being chosen for further taxonomic study. Isolates in the C. acutatum complex were analysed using partial sequences of six gene regions (ITS, GAPDH, ACT, CHS‐1, TUB2 and HIS3), and in the C. gloeosporioides complex were analysed using four gene regions (ITS, TUB2, ApMat and GS). Phylogenetic analysis delineated four Colletotrichum species including C. siamense, C. simmondsii, C. queenslandicum, C. truncatum and a new Colletotrichum species, described here as C. cairnsense sp. nov. This is the first reported association of C. queenslandicum, C. simmondsii and C. siamense with chili anthracnose in Australia; these species were previously associated with anthracnose on papaya and avocado. Furthermore, the dominant species causing anthracnose of chili in Southeast Asia, C. scovillei, was not detected in Australia. Inoculations on chili fruit confirmed the pathogenicity of C. cairnsense and the other four species in the development of chili anthracnose in Australia.     

Disease suppressive activity of extracts from entomopathogenic fungus <Emphasis Type="Italic">Ophiocordyceps sobolifera</Emphasis> against chili anthracnose fungi <Emphasis Type="Italic">Colletotrichum</Emphasis> spp. in a pot experiment

This study evaluated the efficacy of the extracts of Ophiocordyceps sobolifera isolate Cod-NB1302 for the biological control of chili anthracnose disease caused by Colletotrichum capsici and C. gloeosporioides under pot conditions. Among the extracts, mycelial extract treatments provide the best reduction in disease severity. Interestingly, two bioactive constituents, adenosine and cordytropolone, from the mycelial extract, inhibited growth of the fungal pathogens. Moreover, these bioactive compounds had a synergistic effect against the fungal pathogens in a pot experiment. These results confirmed the disease suppressive activity of the mycelial extract.     

Biochemical,physiological, and molecular characterization of <Emphasis Type="Italic">Dickeya dianthicola</Emphasis> (formerly named <Emphasis Type="Italic"> Erwinia chrysanthemi</Emphasis>) causing potato blackleg disease in Japan

The taxonomic assignment of Japanese potato blackleg isolates of Dickeya spp. has not been confirmed after the changes in their former name, Erwinia chrysanthemi. Therefore, we investigated and identified 23 representative isolates of Dickeya spp. from symptomatic stems of potatoes in Japan, with biochemical tests and phylogenetic sequence analysis using recA, dnaX, rpoD, gyrB, and 16S rDNA sequences. Results of our biochemical tests showed that all isolates can be assigned to phenon 5 and biovar 1, which are associated with D. dianthicola. Based on the recA, dnaX, rpoD, gyrB, and 16S rDNA sequences, all isolates are in the same clade with D. dianthicola and were clearly distinguished from D. chrysanthemi, D. dadantii, D. dadantii subsp. dieffenbachiae, D. solani, D. zeae, and D. paradisiaca. Therefore, we conclude that Dickeya spp. isolated from potatoes with blackleg symptoms in Japan are D. dianthicola.     

Identification and species status of the mango biotype of Colletotrichum gloeosporioides in Ghana

Research work was carried out to identify and ascertain the species status of the mango biotype of Colletotrichum gloeosporioides infecting mangoes in Ghana. Forty five isolates of Colletotrichum species were collected from 12 districts in Ghana while five each were obtained from mango fruits from Florida, Mexico and Puerto Rico. The entire internal transcribed spacer region, partial beta-tubulin gene and partial glyceraldehyde-3-phosphate dehydrogenase gene of isolates were sequenced and used in phylogenetic studies. The results of the sequence analysis of the first ribosomal transcribed spacer (ITS 1) region showed that 35 % of the isolates from Ghana and all the five isolates from Mexico were the mango biotype of C. gloeosporioides, while the others were not. Phylogenetic studies showed that the mango biotype of the pathogen was Colletotrichum asianum but not C. gloeosporioides as previously thought. However, the other isolates that were not the mango biotype were identified as Colletotrichum siamense and Colletotrichum species which had probably cross-infected mango from other fruit crops in the field.     

Relative potency of culture supernatants of <Emphasis Type="Italic">Xenorhabdus</Emphasis> and <Emphasis Type="Italic">Photorhabdus</Emphasis> spp. on growth of some fungal phytopathogens

In previous research, concentrated metabolites produced by bacteria of the genera Xenorhabdus and Photorhabdus (which are symbionts of entomopathogenic nematodes) were reported to be highly suppressive to fungal and oomycete plant pathogens. Conceivably, application of non-concentrated bacterial filtrates would be more economically feasible compared to using concentrated metabolites. We evaluated the potency of 10 % v/v cell-free supernatants of the bacteria X. bovienii, X. nematophila, X. cabanillasii, X. szentirmaii, P. temperata, P. luminescens (VS) and P. luminescens (K22) against Fusicladium carpophilum (peach scab), F. effusum (pecan scab), Monilinia fructicola (brown rot), Glomerella cingulata (anthracnose) and Armillaria tabescens (root rot). A bioactive compound derived from Photorhabdus bacteria, trans-cinnamic acid (TCA), was also compared with the bacterial filtrates. Fungal colony size based on manual measurements was compared for accuracy to measurements taken by image analysis. Supernatants of Xenorhabdus spp. exhibited stronger suppressive effects on spore germination and vegetative growth when compared with Photorhabdus spp. Overall, TCA was the most effective treatment; vegetative growth was completely inhibited by TCA (1.27 mg/ml). TCA treatments also suppressed spore germination of F. carpophylium and F. effussum by approximately 90 %. The efficacy of supernatants varied among Xenorhabdus species depending on the species tested, but X. szentirmaii filtrates tended to cause greater inhibition relative to the other bacteria supernatants. Manual measurement of colony diameter required at least two replicate estimates of the colony to avoid a type II error. Area measurements were slightly overestimated based on ruler measurements, but did not affect the outcome of the analysis. Supernatants of Xenorhabdus spp., Photorhabdus spp., or TCA, did not cause any phytotoxic effects when applied to various plant species in the greenhouse. Our results indicate the potential of using TCA or Xenorhabdus cell free supernatants as bio-fungicides. Such a product, based on bacterial culture supernatants, would be economically viable, marketable and easily applicable by the end-users in many situations.