Identification and characterization of <Emphasis Type="Italic">Colletotrichum</Emphasis> spp. associated with chili anthracnose in peninsular Malaysia

Anthracnose fruit rot caused by Colletotrichum spp. is a serious post-harvest disease of chili fruits (Capsicum spp.). One hundred-thirty isolates of Colletotrichum spp. were isolated from anthracnose of green and red cayenne pepper (Capsicum annuum) and bird’s eye chili (Capsicum frutescens). The isolates were morphologically identified as Colletotrichum acutatum sensu lato (62 isolates), Colletotrichum truncatum (54 isolates), and Colletotrichum gloeosporioides sensu lato (14 isolates). Molecular identification and phylogenetic analyses were based on internal transcribed spacer regions, β-tubulin, actin, and glyceraldehyde-3-phosphate dehydrogenase genes, and the isolates were re-identified as C. scovillei (58 isolates), C. truncatum (54 isolates), C. siamense (11 isolates), C. fioriniae (four isolates), and C. fructicola (3 isolates). Maximum likelihood trees using combined sequences showed that isolates of the same species grouped in the same main clade with the isolates used for comparison. Pathogenicity testing showed that the tested isolates from each species were pathogenic towards green and red Capsicum annuum and Capsicum frutescens upon treatment of wounded fruit, using both the mycelial plug and conidial suspension methods. Only five isolates of C. truncatum and seven isolates of C. scovillei were found to be pathogenic upon treatment of unwounded fruit. The occurrence of five Colletotrichum spp. (C. siamense, C. fructicola, C. scovillei, C. fioriniae, and C. truncatum) associated with chili anthracnose in Peninsular Malaysia indicates that correct species identification is important to formulate not only effective disease management, but also effective quarantine policy.     

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.     

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.     

Assessment of the baseline sensitivity and resistance risk of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> to fludioxonil

Anthracnose, which is caused by Colletotrichum acutatum, is a destructive disease of pepper. A preliminary study demonstrated that fludioxonil (a phenylpyrrole fungicide) has good activity against C. acutatum and thus has potential to be used as an alternative fungicide for the management of pepper anthracnose. However, there is no information regarding the baseline sensitivity and resistance risk of C. acutatum to fludioxonil. Thus, the sensitivities of 205 isolates of C. acutatum to fludioxonil were determined. The results showed that the frequency distributions of the EC₅₀ values were unimodal, and the mean EC₅₀ values for the inhibition of mycelial growth and spore germination were 0.031 μg/mL and 0.035 μg/mL, respectively. Three stable mutants with high resistance to fludioxonil were obtained in the laboratory. Two parameters, namely in vitro sporulation and the in vitro and in vivo germination of spores, showed significant difference (P < 0.01) when the mutants were compared to the sensitive isolates. Moreover, the mutants were more sensitive to osmotic stress compared to the parents. No significant differences (P ≥ 0.05) were detected in colony diameter, mycelia weight, pathogenicity or sporulation in vivo between the fludioxonil-resistant mutants and their corresponding parents. Cross-resistance occurred between fludioxonil, iprodione and procymidone. Overall, resistance risk of C. acutatum to fludioxonil was low to medium, and thus resistance management should be considered.     

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

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

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

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

Colletotrichum gloeosporioides, a new causal agent of citrus post-bloom fruit drop

Citrus post-bloom fruit drop (caused by Colletotrichum acutatum) frequently occurs in the southwestern region of São Paulo State, Brazil. A survey of Colletotrichum isolates associated with symptoms of post-bloom fruit drop in São Paulo State showed C. gloeosporioides in addition to C. acutatum. The objectives of this study were to confirm the identification of C. gloeosporioides isolated from symptomatic citrus flowers, to test the pathogenicity of C. gloeosporioides isolates, to compare the development of disease caused by C. gloeosporioides and C. acutatum, and to determine the frequency of C. gloeosporioides in a sample of isolates obtained from symptomatic flowers in different regions of São Paulo State. Through the use of species-specific primers by PCR, 17.3% of 139 isolates were C. gloeosporioides, and the remaining 82.7% were C. acutatum. The pathogenicity tests, carried out in 3-year old potted plants of sweet oranges indicated that both species caused typical symptoms of the disease including blossom blight and persistent calyces. Incubation periods (3.5 and 3.9 days, respectively, for C. acutatum and C. gloeosporioides) and fruit sets (6.7 and 8.5%, respectively for C. acutatum and C. gloeosporioides) were similar for both species. The incidences of blossom blight and persistent calyces were higher on plants inoculated with C. acutatum than in those inoculated with C. gloeosporioides. Conidial germination was similar for both species under different temperatures and wetness periods. Under optimal conditions, appressorium formation and melanisation were higher for C. gloeosporioides than for C. acutatum. These results indicated that Colletotrichum gloeosporioides is a new causal agent of post-bloom fruit drop.     

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.     

Role of secondary metabolites in the biocontrol activity of <Emphasis Type="Italic">Pseudomonas corrugata</Emphasis> and <Emphasis Type="Italic">Pseudomonas mediterranea</Emphasis>

Colletotrichum gloeosporioides is the causal agent of Camellia oleifera anthracnose, mainly infecting fruits and leaves. The fungus secretes degrading enzymes to destroy the cuticle of aerial plant parts and help infect the host successfully. To validate whether a cutinase gene (CglCUT1) was required for cutinase activity and pathogenicity of C. gloeosporioides, the CglCUT1 gene was cloned and analyzed. The characterization of CglCUT1 predicted protein suggests that the cloned DNA encoded a cutinase in C. gloeosporioides affecting C. oleifera. The CglCUT1 showed a high homology to those from C. gloeosporioides causing papaya anthracnose and C. capsici causing pepper anthracnose, as well as those of other ascomycetes. The whole CglCUT1 gene was knocked-out and the knockout mutant (?CglCUT39) was subsequently complemented using Agrobacterium tumefaciens mediated transformation. The knockout transformants exhibited significant decreases in cutinase activity and virulence compared with the wild-type strain. The complemented transformants of the disrupted transformant ?CglCUT39 showed a significant increase in cutinase activity and virulence compared with the disrupted transformant ?CglCUT39. This study suggests that the CglCUT1 gene has a positive effect on fungal virulence of the hemibiotrophic C. gloeosporioides on C. oleifera.     

<Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> occurs asymptomatically on apple leaves

Fungi within the Colletotrichum acutatum species complex occur asymptomatically on plant parts of many different plant species. Leaves from apple orchards in southern Norway were sampled, frozen for five hours and incubated for six days to reveal presence of asymptomatic infections of C. acutatum. Number of leaves (incidence) and leaf area covered (severity) with conidial masses of C. acutatum were assessed biweekly on cv. Aroma from late May to late September during three growing seasons. The first finding of conidial masses occurred in the second half of July, and there was a higher incidence occurring in August and September. Sampling of leaves from fruit spurs and vegetative shoots of cvs. Aroma and Elstar showed that conidial masses of C. acutatum developed on leaves on both shoot types, and there was no difference in incidence between these two types. The fungus was detected on leaves from six of eight commercial orchards of cv. Aroma over three years, with a mean incidence of 5.5 %. After storage, bitter rot was found on apple fruit from all eight orchards. There was no correlation between incidence of conidial masses of C. acutatum on leaves and on fruit. In all orchards and seasons investigated, incidence and severity on leaves varied from 0 to 67 % and 0 to 85 %, respectively. The discovery of apple leaves containing conidial masses of C. acutatum clearly indicate for leaves as a potential source of inoculum for fruit infections.     

Functional characterization of CgPBS<Subscript>2</Subscript>, a MAP kinase kinase in <Emphasis Type="Italic">Colletotrichum gloeosporioides,</Emphasis> using osmotic stress sensitivity as a selection marker

Colletotrichum leaf disease of Hever brasiliensis (rubber tree) caused by C. gloeosporioides is one of the major causes of declining rubber tree yields. Little is known about the fungal molecular characters that are important for pathogenicity on rubber tree and fungicide resistance. In this study, we cloned the CgPBS₂ gene, the key component of the Hog1 pathway which controls various aspects of osmoregulation and fungicide resistance in various fungal pathogens, including the causal agent of Colletotrichum leaf disease of rubber tree. We characterized the function of the CgPBS₂ gene by reverse genetics. Because the Hog1 pathway plays an important role in stress responses, we obtained a CgPBS₂ gene deletion mutant by PEG-mediated transformation of protoplasts after reducing the concentration of sucrose in the screening medium from 1.0 M to 0.2 M. Then, the complemented transformants and GFP-labelled CgPBS₂ gene transformants were selected directly under highly hyperosmotic medium (PDA?+?1.5 M sorbitol) without using other selectable gene markers. Phenotypic observations showed that the CgPBS₂ protein was mainly localized in the conidial cytoplasm of the CgPBS₂-GFP transformants. In addition, disruption of CgPBS₂ led to sensitivity to hyperosmosis and high salt concentration as well as resistance to the fungicide fludioxonil. No obvious difference in virulence was observed between the null mutant and the wild-type strain. These results provide insights into the role of the CgPBS₂ gene in osmotic stress, salt stress and fludioxonil resistance and suggest that osmotic stress sensitivity can be used as a selection marker.     

Honeybees can spread Colletotrichum acutatum and C. gloeosporioides among citrus plants

Postbloom fruit drop (PFD) is an important citrus disease that causes up to 100% yield losses during years in which conditions are favourable for the occurrence of epidemics. The conidia of Colletotrichum acutatum and C. gloeosporioides, causal agents of PFD, are predominantly dispersed by rain splash. At the beginning of epidemics, the distribution of diseased plants is random and the disease progress rate is very high, which is unusual for pathogens spread by rain splash. As the pathogen produces abundant conidia on diseased petals, pollinating insects may contribute to disease dispersal. This study investigated honeybees (Apis mellifera) as dispersal agents of C. acutatum and C. gloeosporioides among citrus plants. Two experiments were carried out in a screenhouse in which citrus plants were protected (or not) in insect‐proof cages. The source of inoculum was placed on one side of the screenhouse, and a honeybee hive was placed on the opposite side. All uncaged plants showed symptoms of the disease, and none of the caged plants exhibited PFD symptoms. The monomolecular model showed a good fit to disease progress in both experiments. Conidium‐like structures of Colletotrichum spp. were identified attached to the bodies of the honeybees by scanning electron microscopy. These results have revealed that honeybees disperse Colletotrichum among citrus plants.     

Infection criteria,inoculum sources and splash dispersal pattern of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> causing bitter rot of apple in New Zealand

Infection of Malus x domestica cv. Royal Gala fruit by Colletotrichum acutatum causing bitter rot was studied in the temperate climate of New Zealand. Temperatures above 15 °C were required for lesions to develop on detached apple wound-inoculated or inoculated without wounding with C. acutatum spores, regardless of maturity. A wetness period of 72 h was required for infection of mature detached apple fruit without wounding. On wound-inoculated detached apple fruits, sporulation was related to temperature and followed a similar pattern. In the field, a mean temperature above 15 °C for 72 h after wound-inoculation was required for lesions to develop. Buds were a more important source of inoculum than twigs, and it was shown that C. acutatum could be isolated more frequently from outer bud scales than from inner scales. Asymptomatic infection of vegetative and reproductive buds was detected. C. acutatum was detected on asymptomic surface-sterilised petals and fruit, more commonly during summer than spring. Symptomless sterilised leaves generally yielded C. acutatum throughout the season, but isolations were more frequent in summer. Recovery of inoculum using a splash meter to detect vertical dispersal showed that in summer inoculum was primarily splashed up from the ground. In spring, inoculum was recovered in similar quantities from all heights up to a metre, suggesting that splash dispersal occurs from the canopy as well as from the ground. A disease cycle for C. acutatum infecting apples and causing bitter rot in New Zealand is suggested.     

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.     

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.     

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.     

Diversity and pathogenicity of Colletotrichum species isolated from soursop in Colombia

Anthracnose, caused by Colletotrichum species is a highly limiting disease for the production of the tropical fruit tree crop, soursop (Annona muricata L.). In this study, 83 single-spore isolates of Colletotrichum were obtained from diseased soursoup tissues and subjected to a species complex-specific PCR assay. The isolates were identified as C. gloeosporioides sensu lato (n?=?60), C. boninense s. lat. (n?=?22), or C. acutatum s. lat. (n?=?1). A subset of 21 selected isolates was identified to species level by means of a multi-locus phylogenetic analysis using sequences from the ITS region and partial sequences of the actin, β-tubulin-2, glyceraldehyde-3-phosphate dehydrogenase, and chitin synthase-1 genes. The multi-locus phylogenetic analysis resolved C. theobromicola, C. tropicale, C. siamense, and C. gloeosporioides sensu stricto in the C. gloeosporioides complex; C. karstii and one undetermined species in the C. boninense complex; as well as one undetermined species in the C. acutatum complex. Significant differences in anthracnose severity were observed between Colletotrichum species when tested for pathogenicity on attached twigs of soursop cv. Elita. Colletotrichum theobromicola and C. tropicale were associated with high and intermediate virulence, respectively, whereas the remaining species were associated with low virulence.     

云南葡萄产区葡萄炭疽病病原鉴定及致病力分析

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