Colletotrichum Gloeosporioides Species Complex Research Articles

Characterization and Fungicide Sensitivity of Colletotrichum Species Causing Strawberry Anthracnose in Eastern China.

Strawberry anthracnose caused by Colletotrichum spp. is one of the most serious diseases in the strawberry fields of China. In total, 196 isolates of Colletotrichum were obtained from leaves, stolons, and crowns of strawberry plants with anthracnose symptoms in eastern China and were characterized based on morphology, internal transcribed spacer (ITS), and β-tubulin (TUB2) gene sequences. All 196 isolates were identified as the Colletotrichum gloeosporioides species complex. In total, 62 strains were further identified at the species level by phylogenetic analyses of multilocus sequences of ITS, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat), calmodulin (CAL), and TUB2. Three species from the C. gloeosporioides species complex were identified: Colletotrichum siamense, C. fructicola, and C. aenigma. Isolates of C. siamense were tolerant to high temperatures, with a significantly larger colony diameter than the other two species when grown above 36°C. The inoculation of strawberry plants confirmed the pathogenicity of all three species. C. siamense isolates resulted in the highest disease severity. The in vitro sensitivities of C. siamense and C. fructicola isolates to azoxystrobin and three demethylation-inhibitor (DMI) fungicides (difenoconazole, tebuconazole, and prochloraz) were determined. Both species were sensitive to DMI fungicides but not to azoxystrobin. C. siamense isolates were more sensitive to prochloraz, while C. fructicola isolates were more sensitive to difenoconazole and tebuconazole. The present study provides valuable information for the effective management of strawberry anthracnose.

First Report of Colletotrichum aeschynomenes and C. tropicale Causing Anthracnose on Myrciaria dubia in Brazil

Camu-camu (Myrciaria dubia [Kunth] McVaugh) is a native fruit tree of Amazon region that produces fruit with high content of vitamin C. The enormous economic potential has encouraged its domestication process in the northern region of Brazil. Since 2016, brown, necrotic, anthracnose-like lesions were observed on 30 to 40% of the camu-camu plants in an experimental field located in the city of Boa Vista, Roraima, Brazil (2°52′20.7″ N; 60°42′44.2″ W). Small pieces of tissue taken from the lesions were surface disinfested previously (1 min in 70% alcohol, 1 min in 1% NaOCl, and rinsed with sterile water). Leaf sections were plated on water agar medium, incubated at 25°C for 4 days, and afterward were transferred to potato dextrose agar (PDA) medium. After 7 days on PDA at 25°C, the isolate CA3B formed a grayish-white colony. Conidia were hyaline, aseptate, subcylindrical with rounded ends, measuring (n = 20) 12.1 to 15.9 × 4.9 to 7.0 μm. Appressoria were brown, subglobose, clavate, measuring 7.4 to 16.7 × 4.6 to 9.9 μm. The isolate COL02 formed light gray colonies. Conidia were hyaline, aseptate, cylindrical, tapering slightly near both ends, measuring (n = 20) 12.3 to 16.1 × 4.7 to 6.5 μm. Appressoria were brown, mostly elliptic to subfusoid, deeply lobed, measuring 8.7 to 15.9 × 5.8 to 9.9 μm. Single-spore cultures were deposited in the Microorganisms Culture Collection of EMBRAPA Roraima, Roraima, Brazil. Total DNA was extracted, and fragments of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin 2 (TUB2), glutamine synthetase (GS), and manganese-superoxide dismutase (SOD2) genes were amplified (Weir et al. 2012) and sequenced. The obtained sequences showed 94 to 100% identity with sequences published in GenBank of the Colletotrichum gloeosporioides species complex, with a predominance of C. tropicale, C. aeschynomenes, and C. siamense. Sequences from the two isolates were deposited in GenBank (accession nos.: GAPDH, MK792453, MK792457; GS, MK792454, MK792458; SOD2, MK792455, MK792459; and TUB2, MK792456, MK792460). Phylogenetic Bayesian inference analysis based in a combined data set (concatenated sequences of GAPDH, TUB2, GS, and SOD2) showed that the isolate CA3B clustered with the ex-holotype specimen of C. tropicale (CBS 124949), and the isolate COL02 clustered with the ex-type specimen of C. aeschynomenes (ICMP 17673) with high support (posterior probability = 1). To confirm the pathogenicity, five seedlings of the camu-camu were sprayed with 10⁶ conidia/ml spore suspension for each isolate. Five seedlings were sprayed with sterile water as a negative control. All seedlings were covered with plastic bags 24 h after inoculation and kept at 27°C in a greenhouse with a 12-h photoperiod. After 7 days, depressed and necrotic lesions were observed in the inoculated leaves. In the third treatment, both isolates were coinoculated. Negative control plants showed no symptoms. To fulfill Koch’s postulates, both species were successfully reisolated from the inoculated and coinoculated leaves by cultural morphology. In Brazil, C. tropicale causes anthracnose on different hosts, such as mango and soursop (Costa et al. 2019; Lima et al. 2013). C. aeschynomenes was associated with Aeschynomene virginica in the United States (Weir et al. 2012), cassava in Thailand (Sangpueak et al. 2018), and cacao in Brazil (Nascimento et al. 2019). To our knowledge, this is the first report of C. aeschynomenes and C. tropicale causing anthracnose on M. dubia worldwide.

Characterization of the Colletotrichum gloeosporioides species complex causing grape ripe rot in Nagano Prefecture, Japan

Isolates of the Colletotrichum gloeosporioides species complex from grape ripe rot in Nagano Prefecture, Japan in 2016–2017 were identified and their morphology, virulence, and fungicide sensitivity clarified. Based on phylogenetic analysis of the ApMat locus, 80 isolates were identified as belonging to four species, C. perseae (17.5%), C. viniferum (8.8%), C. fructicola (7.5%), and C. gloeosporioides sensu stricto (2.5%) and another species designated Clade V (63.8%). Clade V was the most important pathogen in Nagano Prefecture because of its predominance and high virulence. Colletotrichum perseae and C. fructicola both had a sexual stage and were often found to be resistance to QoI fungicides; they were dominant in the northern region of Nagano Prefecture in 2016. These four species and Clade V can be distinguished by the growth rate of their colonies and morphology of their conidia and ascospores. The results of this study suggest that identification of dominant species in each field is necessary to determine the most suitable fungicide application for disease control.

First Report of Anthracnose on Dendrobium officinale Caused by Colletotrichum fructicola in Anhui Province, China

Dendrobium (Dendrobium officinale Kimura et Migo) is an epiphytic herbaceous plant mainly found in Anhui, Zhejiang, Yunnan, and Fujian provinces in China. The raw stems or processed products are commonly used as Chinese traditional medicine for treatment of cancer, stomach problems, and lowering blood sugar. In May 2019, approximately 60 to 85% of D. officinale plants were found with disease lesions on leaves in a greenhouse in Ningguo city, Anhui Province, China. The symptoms initially appeared as small, circular, brown spots. As disease developed, the center of the lesions was sunken with a dark brown border. To identify the pathogen, 16 symptomatic leaves were cut into pieces (3 × 3 mm), surface sterilized in 0.1% HgCl for 1 min followed by 70% ethanol for 15 s, and rinsed three times with deionized water. The dried tissues were placed on potato dextrose agar (PDA) medium supplemented with cefotaxime sodium salt (150 μg/ml) and then incubated at 28 ± 0.5°C in the dark. Nine fungal colonies with similar morphology consistently emerged from the diseased tissues after 3 to 4 days. Four single-spore isolates were randomly selected for morphological characterization and identification. The isolates showed whitish to pale gray cottony mycelial growth after 7 days of incubation on PDA at 28 ± 0.5°C in dark. Conidia were produced in small orange masses and were mainly cylindrical, colorless, rounded at both ends, 11.3 to 19.1 × 4.2 to 5.8 μm (n = 50), and single celled with one or two oil globules. Appressoria formed on hydrophobic cover glass at 28 ± 0.5°C were medium to dark brown, obovoid to ellipsoid. The morphological characteristics of these isolates were similar to the species belonging to the Colletotrichum gloeosporioides species complex (Cannon et al. 2012; Prihastuti et al. 2009; Weir et al. 2012). To further confirm the fungus, the rDNA internal transcribed spacer (ITS) region and partial sequences of two genes (ACT, TUB2) were amplified using primers ITS1/ITS4 (White et al. 1990), ACT512F/ACT783R (Carbone and Kohn 1999), and T1/T2 (O’Donnell and Cigelnik 1997), respectively. The resulting sequences for ITS, TUB2, and ACT were identical to each other in different strains, and representative sequences from strain SHCF-3 were submitted to GenBank with accession numbers MN173821, MN175549, and MN175550, respectively. A comparison using the ITS, TUB2, and ACT sequences revealed 99.62, 99.42, and 99.14% homology with the corresponding sequences (accession nos. JX010165, JX010405, and KJ954435) from the ex-epitype culture of C. fructicola (Hyde et al. 2014; Lu et al. 2018). To test pathogenicity, five 6-month-old D. officinale seedlings were sprayed with conidia suspensions (1 × 10⁶ spores/ml) from each of the isolates, respectively. On control plants, only sterilized water was used. Inoculated seedlings were incubated in a moist chamber (relative humidity > 90%) at 28°C. After 14 days, all the leaves from inoculated plants showed anthracnose lesions similar to those observed in greenhouse, whereas control plant leaves were asymptomatic. C. fructicola was reisolated from all the lesions and showed similar morphology to the original isolates, fulfilling Koch’s postulates. C. fructicola is known to cause anthracnose on Citrus sinensis (Hu et al. 2019) and Camellia sinensis (Shi et al. 2018) in China. To our knowledge, this is the first report of C. fructicola causing anthracnose on D. officinale in Anhui Province, China. This new disease may pose a serious threat to D. officinale quality and yield.

Diversity in species composition and fungicide resistance profiles in Colletotrichum isolates from apples

Outbreaks of bitter rot were observed in three commercial apple orchards in Illinois despite best management efforts during the 2018 production season. Three isolates from symptomatic fruit from these orchards and two isolates from an orchard in South Carolina were identified to the species level using morphological tools and calmodulin, glyceraldehyde-3-phosphate dehydrogenase, and beta-tubulin gene sequences. The isolates from Illinois were identified as Colletotrichum siamense of the Colletotrichum gloeosporioides species complex and the ones from South Carolina as Colletotrichum fioriniae and Colletotrichum fructicola of the Colletotrichum acutatum and the C. gloeosporioides species complex, respectively. Two of the three C. siamense isolates from Illinois were resistant to azoxystrobin and thiophanate-methyl as determined in mycelial growth tests in vitro. EC50 values were >100 μg/ml for both fungicides. One isolate was only resistant to azoxystrobin. None of the isolates from South Carolina was resistant to either of the two compounds. All five isolates were sensitive to fludioxonil (EC50 values <0.1 μg/ml), propiconazole (EC50 values ranged from 0.15 to 0.36 μg/ml), and benzovindiflupyr (EC50 values ranged from <0.1 to 0.33 μg/ml). Resistance in C. siamense to azoxystrobin and thiophanate-methyl was confirmed in detached fruit studies using apples treated with label rates of registered product. Resistance to thiophanate-methyl in C. siamense was based on E198A mutation in b-tubulin gene, whereas resistance to azoxystrobin was based on G143A in cytochrome b (CYTB). One isolate resistant to azoxystrobin possessed no amino acid variation in CYTB. This study shows that quinone outside inhibitor fungicide resistance in Colletotrichum from apple has emerged and is being selected for in Illinois apple orchards by current spray strategies. Resistance monitoring may alert growers to potential threats, but the employment of molecular tools based on current knowledge of resistance mechanisms will provide incomplete results.

Differentiation of lineages within “Colletotrichum gloeosporioides s.l.” associated with cassava anthracnose disease by BOX‐ and ERIC‐PCRs

AbstractSeveral molecular techniques have been used to differentiate species or genetic lineages of microorganisms prior to sequencing. Among them, BOX‐ and ERIC‐PCRs may provide specific banding patterns for different species, allowing its differentiation. Therefore, the objective of this study was to evaluate these techniques as a tool for differentiation of phylogenetic lineages belonging to the Colletotrichum gloeosporioides species complex associated with cassava anthracnose disease. Sets of BOX‐ and ERIC‐PCR primers were used to assess the differentiation of lineages belonging to the complex with 81 C. gloeosporioides sensu lato (s.l.) isolates from different cassava producing regions. Some were identified by sequencing, such as Colletotrichum fructicola, Colletotrichum tropicale, C. gloeosporioides s.s, Colletotrichum theobromicola, Colletotrichum siamense, Colletotrichum brevisporum and Colletotrichum sichuanensis. The primers were able to amplify DNA fragments from all isolates. The ERIC‐PCR presented a wider range of banding patterns in comparison to BOX‐PCR, providing better differentiation of the individuals, as well as a higher correlation with the phylogenetic data was obtained by ERIC‐PCR and the combined data set for “BOX‐/ERIC‐PCRs,” inferred by Mantel test. However, the use of concatenated data (BOX‐/ERIC‐PCRs) reduced the discriminatory capacity presented by ERIC‐PCR alone, probably due to the lowest resolution of BOX‐PCR. Therefore, ERIC‐PCR technique enabled efficient differentiation of isolates belonging to the C. gloeosporioides complex and can be used to analyse multiple isolates in a collection and also being an important tool as a guide in the decision‐making process prior to sequencing. Based on this methodology, it was possible to identify two new species associated with cassava anthracnose disease, C. brevisporum and C. sichuanensis, being the first report of these two species associated with cassava anthracnose disease in Brazil.

First Report of Anthracnose Caused by Colletotrichum aenigma on Actinidia arguta in China

HomePlant DiseaseVol. 103, No. 2First Report of Anthracnose Caused by Colletotrichum aenigma on Actinidia arguta in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Anthracnose Caused by Colletotrichum aenigma on Actinidia arguta in ChinaY. Wang, H. Y. Qin, Y. X. Liu, S. T. Fan, D. Sun, Y. M. Yang, C. Y. Li, and J. AiY. WangSearch for more papers by this author, H. Y. QinSearch for more papers by this author, Y. X. LiuSearch for more papers by this author, S. T. FanSearch for more papers by this author, D. SunSearch for more papers by this author, Y. M. YangSearch for more papers by this author, C. Y. LiSearch for more papers by this author, and J. Ai†Corresponding author: J. Ai; E-mail: E-mail Address: [email protected]http://orcid.org/0000-0002-7977-1574Search for more papers by this authorAffiliationsAuthors and Affiliations Y. Wang H. Y. Qin Y. X. Liu S. T. Fan D. Sun Y. M. Yang C. Y. Li J. Ai † , Institute of Special Animal and Plant Science, Chinese Academy of Agricultural Sciences, Changchun, 130112, Jilin, China. Published Online:17 Dec 2018https://doi.org/10.1094/PDIS-07-18-1137-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Actinidia arguta (Sieb. & Zucc.) Planch. ex Miq. is a fruit tree being newly cultivated worldwide and is a widely distributed wild fruit tree in China (Zhao 2002). Owing to their fruits rich in vitamin C, A. arguta enjoys the labels “world treasure” and “fruit king.” In September 2017, anthracnose symptoms were observed on the fruit of a 6-year-old A. arguta plantation in Dandong, Liaoning Province, China. Symptoms began with light brown round spots that developed into slightly sunken brown or black-brown lesions on infected pericarp; 15 to 20% of the trees exhibited typical anthracnose symptoms. For characterization, 5-mm2 pieces of symptomatic tissue were surface sterilized with 1% NaOCl for 2 min, triple rinsed with sterile water, dried on sterilized filter paper, and cultivated on acidified potato dextrose agar (PDA) medium at 25°C to obtain the pathogenic fungus. Three isolates were cultured on PDA at 25°C under a 12-h/12-h light/dark cycle for 7 days. The average resulting colony diameter was 6.80 ± 0.80 cm. Colonies were round with smooth edges and white aerial mycelia. The surfaces were fluffy and white, and the colonies’ undersides were pale yellow to white. Perithecia and ascospores were produced on PDA. The ascospores were cylindrical, both ends blunt, with slight depressions in the middle, and no setae or conidial discs. Conidial spores were smooth and transparent, cylindrical or stick type, both ends blunt, with small particles inside, evenly distributed, 16.1 to 19.7 × 5.0 to 8.1 µm (average = 17.8 ± 1.03 × 6.01 ± 0.72, n = 50). Appressoria were brown to dark brown, usually scattered or irregular, 7.2 to 11.4 × 4.3 to 8.2 µm (average = 9.29 ± 1.08 × 6.33 ± 0.86, n = 20). Based on morphological and cultural characteristics, the isolates were identified as part of the Colletotrichum gloeosporioides species complex (Deng et al. 2017; Phoulivong et al. 2010; Weir et al. 2012). To confirm the species identification, six DNA regions of the randomly selected strain AACAWY203DRT5 were amplified by polymerase chain reaction and sequenced: rDNA internal transcribed spacers regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), chitin synthase (CHS) (Gan et al. 2017), actin (ACT) (Liang et al. 2017), and β-tubulin (TUB2). A BLASTn algorithm-based search revealed that all sequences (GenBank accession nos. MH476561 for ITS, MH476562 for GAPDH, MH476563 for ACT, MH476564 for CAL, MH476565 for CHS, and MH476566 for TUB2) had 99 to 100% sequence identities with Colletotrichum aenigma (NR_120140 for ITS, JX009913 for GAPDH, KX885151 for ACT, KU251793 for CAL, KX513884 for CHS, and KY820891 for TUB2). Based on these results, the isolate was identified as C. aenigma. To confirm pathogenicity, 18 A. arguta fruits were surface disinfested by immersion in 1% NaOCl for 1 min, triple rinsed with sterile water, and then dried on sterilized filter paper. Inoculations were performed by inoculating 10 μl of a conidial suspension (106 spore/ml) on the fruit surface. As controls, nine fruits were mock inoculated with sterile distilled water. All fruits were maintained at 25°C, about 90% relative humidity and with natural light. After 1 week, symptoms started to develop only on inoculated fruit, whereas control fruit did not develop symptoms. The fungus was only consistently reisolated from the diseased fruits, thus fulfilling Koch’s postulates. To our knowledge, this is the first report C. aenigma causing anthracnose of A. arguta in China. The disease leads to lower fruit quality and decreased yield, and research is needed on management options to minimize losses.

Keragaman Cendawan Pascapanen pada Umbi Bawang Merah Varietas Bima Brebes

Diversity of Postharvest Fungi on Shallot Bulbs Variety Bima BrebesIn Indonesia, shallot (Allium ascalonicum) is horticultural main commodity after hot pepper. Significant yield losses can be caused by postharvest fungi infection. Research on the diversity of postharvest fungi on shallot bulbs has been conducted in some countries, unfortunately little is done in Indonesia. The study was aimed to obtain information on the diversity of postharvest fungi infecting shallot bulbs variety Bima Brebes from several traditional markets in Bogor City. Shallot bulbs were collected in January and February 2016. The study consisted of fungal isolation from shallot bulbs, fungal pathogenicity test, and identification of pathogenic fungi based on morphological and molecular characteristics. Morphology identification was based on the color of fungal colony, growth pattern, as well as somatic and reproduction structures. Several species of pathogenic fungi were successfully identified from shallot bulbs i.e. Alternaria alternata, Aspergillus niger, Colletotrichum gloeosporioides species complex, Fusarium fujikuroi species complex, F. oxysporum, F. solani, Penicillium citrinum and P. pinophilum. Among these fungi, the highest pathogenicity was shown by C. gloeosporioides species complex.

First Report of Colletotrichum gloeosporioides Causing Anthracnose of Tejocote (Crataegus gracilior) Fruits in Mexico

Crataegus is a genus classified in family Rosaceae and includes several tree species commonly called tejocote that are widely cultivated for their pome fruits in Mexico. During fall of 2014, 2015, and 2016, severe symptoms of anthracnose were observed on approximately 60% of tejocote (Crataegus gracilior) fruits in an orchard located in Tulancingo, Oaxaca, Mexico. Affected fruits showed sunken, prominent, dark brown to black necrotic lesions and were exuding salmon spore masses. To isolate the fungus, small pieces from tissue adjacent to the lesions of 10 symptomatic fruits were excised and surface disinfested by immersion in a 1% sodium hypochlorite solution for 2 min, rinsed three times in sterile distilled water, placed in Petri plates containing potato dextrose agar (PDA), and incubated at 25°C for 5 to 7 days in darkness. Mycelial plugs were excised from the edge of the actively growing fungal colony and aseptically transferred to fresh PDA medium and incubated at 25°C for 6 days. Five monoconidial cultures were obtained by transferring germinated spores to Petri plates with fresh PDA. One isolate was selected as representative for morphological and molecular identification. Colonies of pure cultures exhibited greyish-white aerial mycelium and abundant salmon-pink conidial masses. Conidia (n = 100) were subcylindrical, hyaline, straight, one-celled, with rounded ends, measuring 13.6 to 17.7 × 4.4 to 5.9 μm. Conidial appressoria were ovoid and brown to dark brown. Based on morphological characteristics, the fungus was identified within the Colletotrichum gloeosporioides species complex (Weir et al. 2012). The isolate was designated UACH-177 and deposited in the Culture Collection of Phytopathogenic Fungi at the Chapingo Autonomous University. For molecular identification, the internal transcribed spacer (ITS) region (White et al. 1990) and fragments of Apn2 (Rojas et al. 2010), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin 2 (TUB2) genes (Weir et al. 2012) were amplified by polymerase chain reaction and sequenced. The sequences were deposited in GenBank (accession nos.: ITS, MG821312; Apn2, MG821310; GAPDH, MG821311; and TUB2, MG821313). A phylogenetic analysis using Bayesian inference and including published ITS, Apn2, GAPDH, and TUB2 data for C. gloeosporioides and other Colletotrichum species was performed. The phylogenetic analysis showed the sequences were grouped into the clade of C. gloeosporioides. To confirm the pathogenicity of the fungus, 20 tejocote fruits were surface disinfested by immersion in a 1% sodium hypochlorite solution for 1 min, washed three times with sterile distilled water, and dried on sterilized filter paper. Inoculations were performed by deposition of 10 μl of a conidial suspension (10⁶ spores/ml) on the fruit surface. Ten fruit were mock inoculated with distilled water as a control. All fruits were kept in a moist chamber at 25°C for 10 days. The pathogenicity test was repeated twice. Disease symptoms were observed on all inoculated fruit after 7 days, whereas control fruit did not develop symptoms. Fungal colonies were reisolated from all symptomatic fruits and were found to be morphologically identical to the original isolate inoculated on tejocote fruits, thus fulfilling Koch’s postulates. In Mexico, Garcia-Alvarez (1976) reported Colletotrichum sp. on fruits of Crataegus mexicana; however, that report was not supported by morphological characterization nor pathogenicity tests. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose of C. gracilior in Mexico and worldwide.

Differentiation of phylogenetic lineages within the ‘Colletotrichum gloeosporioides species complex’ associated with cassava anthracnose disease by PCR-RFLP

Different species were found associated with cassava anthracnose, considered a major disease of this crop. The correct identification of the causal agent is a first step for defining appropriate control strategies, such as resistant varieties. In silico analyses used sequences of six genomic regions of ex-type specimens from the ‘Colletotrichum gloeosporioides species complex’ (C.g.SC) and 21 restriction enzymes to identify phylogenetic lineages. The three best combinations of region/enzymes were validated in 18 Colletotrichum spp. isolates from cassava. Dendrograms for in silico PCR-RFLP for CAL, ITS and TUB2 showed considerable agreement with the phylogenetic analysis of each genomic region; however, the CAL gene presented greater discriminatory power. Since the band patterns from in gel analysis were almost the same as expected for the in silico analysis for the CAL region, a new approach was proposed based on the combined data from these two methodologies, allowing the differentiation of five phylogenetic lineages within the C.g.SC (C. tropicale; C. fructicola; C. siamense; C. gloeosporioides sensu stricto and C. theobromicola), and one outside of this complex (C. cliviae). This evaluation showed to be a reliable technique for preliminary identification of species prior to sequencing.

First Report of Colletotrichum aenigma Causing Anthracnose on Sedum kamtschaticum in Korea

HomePlant DiseaseVol. 101, No. 12First Report of Colletotrichum aenigma Causing Anthracnose on Sedum kamtschaticum in Korea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Colletotrichum aenigma Causing Anthracnose on Sedum kamtschaticum in KoreaH.-W. Choi, Y. K. Lee, and S. K. HongH.-W. ChoiSearch for more papers by this author, Y. K. LeeSearch for more papers by this author, and S. K. Hong†Corresponding author: S. K. Hong; E-mail: E-mail Address: [email protected]Search for more papers by this authorAffiliationsAuthors and Affiliations H.-W. Choi Y. K. Lee S. K. Hong † , National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365, Republic of Korea. Published Online:10 Oct 2017https://doi.org/10.1094/PDIS-07-17-0938-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Orange stonecrop (Sedum kamtschaticum Fisch. and C.A. Mey.) is a perennial herbaceous plant belonging to the family Crassulaceae. It has been used as an ornamental vegetable and for folk medicine for treating inflammatory disorders in Korea. In October 2010, anthracnose occurred on leaves of orange stonecrop grown in a field in Bonghwa, Korea (36.92°N, 128.77°E). Diseased plants were about 5 to 10% of 1,000 total orange stonecrops in the field. Symptoms appeared as circular to irregular lesions with blackish brown discoloration on margins of leaves. Fragments of diseased leaves were surface sterilized with 1% NaOCl for 30 s, then rinsed in sterile water. Tissue pieces were placed on water agar (WA) and incubated at 25°C for 5 days. Three isolates were cultured on potato dextrose agar (PDA) at 25°C in darkness for 7 days. Colonies were at first cottony, floccose, grayish white, becoming gray with age, dark brown at the center in reverse, with mycelial growth of 58.3 mm after 7 days at 25°C. Conidiomata on PDA were slimy, dark brown, forming setae in abundance. Setae were brown, 1 to 3 septate, with tapering acute apices, and 41.5 to 103.2 × 4.6 to 5.9 μm (mean 78.1 × 5.2). There were some differences between shape and size of conidia from natural lesions and those from cultures. The former was cylindrical, tapering toward one or both ends, rarely fusiform, sometimes slightly curved, and 14.1 to 19.1 × 6.7 to 8.8 μm (mean 16.6 × 7.5, l/b 2.2) in size, while the latter was usually cylindrical, straight, with both ends rounded, rarely tapering toward one end, and 16.5 to 22.7 × 6.0 to 7.0 μm (mean 18.7 × 6.4, l/b 2.9) in size. Appressoria were brown to dark brown, usually clavate to ovate, crenate or irregular, and 8.7 to 15.2 × 6.1 to 10.2 μm (mean 11.1 × 8.6) in size. Based on morphological and cultural characteristics, the three isolates were identified as Colletotrichum gloeosporioides species complex (Phoulivong et al. 2010; Weir et al. 2012). To clarify taxonomic placement of the present isolate (IY1059) within the C. gloeosporioides complex, chitin synthase 1 (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), β-tubulin (TUB2), actin (ACT), rDNA internal transcribed spacers regions (ITS), and glutamine synthetase (GS) from IY1059 were amplified by PCR as previously described (Prihastuti et al. 2009). The PCR products were sequenced and deposited in GenBank with accession numbers KY820888–93, and MF401417, respectively. Six sequences of IY1059, except the GS gene, were 99 or 100% identical with the reference strains, C. aenigma C1253.4 or ICMP 18608. The GS sequence of IY1059 showed 98% identity with the reference strain. Based on morphological and molecular characteristics, the isolate was identified as C. aenigma. A pathogenicity test of the three isolates was made on wounded and unwounded leaves of orange stonecrop by dropping 30 µl of conidial suspensions (5 × 105 conidia ml−1). The same quantity of sterile distilled water placed on unwounded or wounded leaves was used as control. Both inoculated and control leaves were placed in plastic boxes with 100% relative humidity at 25°C. After 3 days, symptoms started to develop only on inoculated and wounded leaves. No symptoms developed on the unwounded and control leaves for 7 days after inoculation. The fungus was consistently reisolated from the only inoculated leaves, thus fulfilling Koch’s postulates. To our knowledge, this is the first report that C. aenigma causes anthracnose on leaves of orange stonecrop. Further studies are required to better define the host range of C. aenigma in Korea.

The impact of phenotypic and molecular data on the inference of Colletotrichum diversity associated with Musa

ABSTRACTDeveloping a comprehensive and reliable taxonomy for the Colletotrichum gloeosporioides species complex will require adopting data standards on the basis of an understanding of how methodological choices impact morphological evaluations and phylogenetic inference. We explored the impact of methodological choices in a morphological and molecular evaluation of Colletotrichum species associated with banana in Brazil. The choice of alignment filtering algorithm has a significant impact on topological inference and the retention of phylogenetically informative sites. Similarly, the choice of phylogenetic marker affects the delimitation of species boundaries, particularly if low phylogenetic signal is confounded with strong discordance, and inference of the species tree from multiple-gene trees. According to both phylogenetic informativeness profiling and Bayesian concordance analyses, the most informative loci are DNA lyase (APN2), intergenic spacer (IGS) between DNA lyase and the mating-type locus MAT1-2-1 (APN2/MAT-IGS), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), β-tubulin (TUB2), and a new marker, the intergenic spacer between GAPDH and an hypothetical protein (GAP2-IGS). Cornmeal agar minimizes the variance in conidial dimensions compared with potato dextrose agar and synthetic nutrient-poor agar, such that species are more readily distinguishable based on phenotypic differences. We apply these insights to investigate the diversity of Colletotrichum species associated with banana anthracnose in Brazil and report C. musae, C. tropicale, C. theobromicola, and C. siamense in association with banana anthracnose. One lineage did not cluster with any previously described species and is described here as C. chrysophilum.

Phylogenetic relationship and fungicide sensitivity of members of the Colletotrichum gloeosporioides 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.

Characterization, Pathogenicity, and Phylogenetic Analyses ofColletotrichumSpecies Associated with Brown Blight Disease onCamellia sinensisin China

Brown blight disease caused by Colletotrichum species is a common and serious foliar disease of tea (Camellia sinensis). Fungal isolates from several tea plantations causing typical brown blight symptoms were identified as belonging to the Colletotrichum acutatum species complex and the Colletotrichum gloeosporioides species complex based on morphological characteristics as well as DNA analysis of the internal transcribed spacer (ITS) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Colletotrichum acutatum, a new causal agent associated with C. sinensis, showed high phenotypic and genotypic diversity compared with the more commonly reported C. gloeosporioides. Phylogenetic analysis derived from individual and combined ITS and GAPDH sequences clearly clustered C. acutatum and C. gloeosporioides into separate species. Pathogenicity tests validated that both species were causal agents of tea brown blight disease and were highly pathogenic to tea leaves. However, the two groups of C. gloeosporioides with low levels of variability within their ITS and GAPDH regions differed in their virulence. This study reports for the first time the characterization of C. acutatum and C. gloeosporioides causing brown blight disease on tea (Camellia sinensis (L.) O. Kuntze) in China.

Markers to differentiate species of anthracnose fungi identify Colletotrichum fructicola 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.

Distribution and Characteristics of Colletotrichum spp. Associated with Anthracnose of Strawberry in Hubei, China.

Anthracnose caused by Colletotrichum spp. is a serious disease of strawberry. The etiology of anthracnose of strawberry is complex, and several Colletotrichum spp. have been regarded as causal agents. In the present study, multilocus (actin, β-tubulin, calmodulin, glyceraldehyde-3-phosphate dehydrogenase, and chitin synthase) phylogenetic analysis revealed that 100 isolates of Colletotrichum associated with anthracnose of strawberry in central China belong to five species. In total, 97 isolates were identified belonging to the Colletotrichum gloeosporioides species complex, with C. murrayae, C. gloeosporioides, C. fructicola, and C. aenigma accounting for 81, 8, 4, and 4% of the total isolates, respectively. Three isolates belonging to the C. acutatum complex were identified as C. nymphaeae. On inoculated strawberry plants, isolates of C. fructicola and C. murrayae species showed strong pathogenicity to both leaves and petioles of strawberry, with plant mortality 30 days after inoculation of 77.8 and 55.6%, respectively. C. gloeosporioides, C. aenigma, and C. nymphaeae showed strong pathogenicity to leaves but weak pathogenicity to petioles, with plant mortality 30 days after inoculation of 5.6, 16.7, and 11.1%, respectively. The five species were divided into four classes based on their maximum growth temperatures. Isolates of C. murrayae and C. gloeosporioides were more tolerant to high temperature (>34°C) than isolates of other species, followed by C. fructicola and C. aenigma. Isolates of C. nymphaeae, which are only distributed in areas of higher altitude (1,100 m), were highly sensitive to higher temperature. These results indicate that pathogenicity and adaptation to temperature are important factors in the distribution of Colletotrichum spp. on strawberry plants. This research may increase our understanding of how Colletotrichum spp. emerge and spread to geographical regions with different latitudes or elevations.

When Do We Call Genetically Distinct Strains Different Species? - A Cautionary Case Study of the Colletotrichum gloesporioides Species Complex

Different criteria have been used to define fungal species. Due to the increasing availability of DNA sequence information, the phylogenetic species concept (PSC) has become popular among fungal taxonomists. However, controversies remain about the criteria used to define the “phylogenetic distinctiveness” between closely related taxa and what other criteria are needed in order to derive more robust species. In a recent study, we reported the DNA sequences of four gene loci for each of 199 strains of the fungal pathogen Colletotrichum gloeosporioides species complex (CGSC) causing leaf anthracnose on the tea-oil tree Camellia oleifera in southern China. Our combined sequence analyses clustered 194 of the 199 isolates into four of the 22 previously described species within CGSC: Colletotrichum fructicola (167 isolates), Colletotrichum siamense (19 isolates), Colletotrichum gloeosporioides (6 isolates) and Colletotrichum camelliae (2 isolates). The remaining five isolates clustered distinctly from all 22 known species. Using the data, here we further investigate the extent of reproductive isolation among these species in our samples. Our analyses revealed extensive allele - sharing among three of the “phylogenetic species” and between them and the five unassigned isolates. Furthermore, there was extensive evidence for prevalent recombination among three of the four species. Our results suggest that caution should be taken in naming new species and highlight the importance of large-scale population genetic studies in helping define reproductive isolation and species boundaries.

Identificación genética de aislamientos de Colletotrichum spp. causantes de antracnosis en frutos de aguacate, banano, mango y tomate de árbol

Anthracnose is a major disease affecting the production of tropical fruit trees on a global level. Our objective was to identify at the species level Colletotrichum isolates causing anthracnose in fruits of avocado, banana, mango, and tamarillo by multilocus phylogenetic analysis based on sequencing data from the ITS region and from the βtubulin-2 and glyceraldehyde-3-phosphate dehydrogenase genes. We evaluated the morphology of colonies, conidia, and appressoria for each species. We obtained 16 isolates from fruits exhibiting symptoms of anthracnose, which were confirmed using the wound and drip inoculation method as causing anthracnose in fruits of their respective hosts. The isolates were identified as Colletotrichum asianum, Colletotrichum fructicola, or Colletotrichum musae within the Colletotrichum gloeosporioides species complex, or as Colletotrichum tamarilloi within the Colletotrichum acutatum species complex. This study contributes to the knowledge of species causing anthracnose in tropical fruits in Colombia, which will facilitate the design of diagnosis techniques and the selection of better disease management strategies. © Acad. Colomb. Cienc. Ex. Fis. Nat. 2015.

チャの葉に発生した大型ロゼット紋様褐色斑点

Big rosette type brown-spot was found on tea (Camellia sinensis (L)O.Kuntze) leaves in 2013 and 2014. Fungi isolated from the diseased plants were identified as the Colletotrichum gloeosporioides species complex. Inoculation tests confirmed that this fungus was the causal agent of the disease. Big rosette brown-spot symptom is a special symptom of brown-blight disease on tea.

ITS1, 5.8S and ITS2 secondary structure modelling for intra-specific differentiation among species of the Colletotrichum gloeosporioides sensu lato species complex.

The Colletotrichum gloeosporioides species complex is among the most destructive fungal plant pathogens in the world, however, identification of member species which are of quarantine importance is impacted by a number of factors that negatively affect species identification. Structural information of the rRNA marker may be considered to be a conserved marker which can be used as supplementary information for possible species identification. The difficulty in using ITS rDNA sequences for identification lies in the low level of sequence variation at the intra-specific level and the generation of artificially-induced sequence variation due to errors in polymerization of the ITS array during DNA replication. Type and query ITS sequences were subjected to sequence analyses prior to generation of predicted consensus secondary structures, including the pattern of nucleotide polymorphisms and number of indel haplotypes, GC content, and detection of artificially-induced sequence variation. Data pertaining to structure stability, the presence of conserved motifs in secondary structures and mapping of all sequences onto the consensus C. gloeosporioides sensu stricto secondary structure for ITS1, 5.8S and ITS2 markers was then carried out. Motifs that are evolutionarily conserved among eukaryotes were found for all ITS1, 5.8S and ITS2 sequences. The sequences exhibited conserved features typical of functional rRNAs. Generally, polymorphisms occurred within less conserved regions and were seen as bulges, internal and terminal loops or non-canonical G–U base-pairs within regions of the double stranded helices. Importantly, there were also taxonomic motifs and base changes that were unique to specific taxa and which may be used to support intra-specific identification of members of the C. gloeosporioides sensu lato species complex.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-684) contains supplementary material, which is available to authorized users.