Isolates Of Colletotrichum Acutatum Research Articles

Detection of Colletotrichum spp. Resistant to Benomyl by Using Molecular Techniques.

Colletotrichum species is known as the major causal pathogen of red pepper anthracnose in Korea and various groups of fungicides are registered for the management of the disease. However, the consistent use of fungicides has resulted in the development of resistance in many red pepper-growing areas of Korea. Effective management of the occurrence of fungicide resistance depends on constant monitoring and early detection. Thus, in this study, various methods such as agar dilution method (ADM), gene sequencing, allele-specific polymerase chain reaction (PCR), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were applied for the detection of benzimidazole resistance among 24 isolates of Colletotrichum acutatum s. lat. and Colletotrichum gloeosporioides s. lat. The result of the ADM showed that C. gloeosporioides s. lat. was classified into sensitive and resistant isolates to benomyl while C. acutatum s. lat. was insensitive at ≥1 µg/ml of benomyl. The sequence analysis of the β-tubulin gene showed the presence of a single nucleotide mutation at the 198th amino acid position of five isolates (16CACY14, 16CAYY19, 15HN5, 15KJ1, and 16CAYY7) of C. gloeosporioides s. lat. Allele-specific PCR and PCR-RFLP were used to detect point mutation at 198th amino acid position and this was done within a day unlike ADM which usually takes more than one week and thus saving time and resources that are essential in the fungicide resistance management in the field. Therefore, the molecular techniques established in this study can warrant early detection of benzimidazole fungicide resistance for the adoption of management strategies that can prevent yield losses among farmers.

In vitro sensitivity of Colletotrichum acutatum isolates from strawberry to tebuconazole, prochloraz, fludioxonil and thiophanate-methyl

The objective of this study was to determine the Colletotrichum acutatum isolates sensitivity to several chemical compounds in vitro and to find their possibilities as a potential control agents for anthracnose disease on strawberry. C. acutatum J.H. Simmonds, a causing agent of anthracnose, is a very important pathogen of strawberry, which leads to devastating losses in its production. The pathogen is effectively controlled by the fungicides. Thus their application is necessary for achieving high yield and fruit quality. The sensitivity of 14 isolates of C. acutatum, collected from commercial strawberry plantations in Serbia, to four fungicides, was examined by an in vitro sensitivity assay. Based on the results of morphological, pathogenic and molecular characterization (up to the complex level), all 14 isolates were determined as C. acutatum. The commercial formulation of tebuconazole, fludioxonil, prochloraz and thiophanate-methyl were used for the sensitivity test. The mycelial growth assay method was used to investigate isolates sensitivity to fungicides. The tested isolates were very sensitive to prochloraz and fludioxonil, with mean EC50 values of 0.067?0.062 mg L-1 and 0.093?0.043 mg L-1, respectively. Significantly higher mean EC50 values were observed for tebuconazole (1.473?0.878 mg L-1) and thiophanate-methyl (1.718?1.592 mg L-1). The toxicity of tested fungicides in the mycelial growth assay of C. acutatum isolates indicates the potential implementation of these fungicides in the protection programs against strawberry anthracnose disease.

Short Communication: Histopathology of red chilli fruit (Capsicum annuum) infected with Colletotrichum acutatum of Java, Indonesia isolates

Abstract. Muljowati JS, Soesanto L, Nugroho LH. 2021. Short Communication: Histopathology of red chilli fruit (Capsicum annuum) infected with Colletotrichum acutatum of Java, Indonesia isolates. Biodiversitas 22: 874-880. Colletotrichum acutatum isolates from Malang, Temanggung, Kulonprogo, Brebes, Garut, and Pandeglang in Indonesia varied in their ability to produce pigments. In the present study, the histopathological status of red chilli was investigated during the early phase of infection by C. acutatum Java isolate. The results included a description of the histopathological features of red chillies (Capsicum annuum) in the early phase of infection by C. acutatum isolates and the relationship between the origin of the isolates and the time of onset of infection. The red chilli fruits were inoculated with fungal conidia suspension and then the histopathology of chilli fruits was observed at 0 hours (uninfected control), 8 hours, 16 hours, 24 hours, and 32 hours after inoculation. The results showed that C. acutatum isolates from Kulonprogo caused host damage within 8 hours, while the other isolates (from Malang, Brebes, Garut and Pandeglang) began to produce similar symptoms at 16 hours or 24 hours. This study revealed for the first time that pathogenic activity begins at the onset of infection, and the resulting anatomical damage to red chillies begins at different times.

Baseline sensitivity of Colletotrichum acutatum isolates from Brazilian strawberry fields to azoxystrobin, difenoconazole, and thiophanate-methyl

Anthracnose fruit rot, caused by Colletotrichum acutatum, is an important disease affecting strawberry fields in Brazil where fungicide applications are frequently needed for disease control. Isolates of C. acutatum were collected in conventional and organic strawberry fields in the states of Sao Paulo and Espirito Santo, Brazil, from 2013 to 2015. Sensitivity to azoxystrobin, difenoconazole and thiophanate-methyl was evaluated based on mycelial growth, spore germination, detached fruit assays and molecular characterization of genes targeted by these fungicides. The effective concentration needed to reduce mycelial growth by 50% (EC50) was determined for 78 isolates. Mean EC50 values for isolates collected in organic fields were 0.44 and 0.10 μg/ml, and in conventional areas were 0.62 and 0.09 μg/ml for azoxystrobin and difenoconazole, respectively. Mean EC50 values, determined using spore germination test, for 43 isolates were 0.04 and 0.13 μg/ml for isolates from organic and conventional fields, respectively, for azoxystrobin. Azoxystrobin- and difenoconazole-resistant isolates were not observed. Populations of C. acutatum showed insensitivity rather than resistance to thiophanate-methyl and EC50 values could not be determined. Molecular analyses of the cytb, cyp51 and β-tub genes did not reveal any of the most common point mutations associated with fungicide resistance.

In Vitro Evaluation of the Resistance of Seven Isolates of Colletotrichum acutatum to Thiram and Copper

Seven Isolates of Colletotrichum acutatum have been collected from strawberries attacked with anthracnose in the Loukous territory, Morocco. The study of the effect in vitro of 3 fungicides with Thiram as active substance and another one with copper on seven isolates of C. acutatum was studied. Thiram has a potent effectiveness on germination and is moderately effective on mycelial growth and sporulation. Copper has shown effectiveness on germination as variable activity in other strain’s life stage.

Effects of Phenolic Compounds on Growth of Colletotrichum spp. In Vitro.

Colletotrichum acutatum is responsible for anthracnose fruit rot, one of the most devastating diseases in strawberry. Phenolic compounds have been described as contributors to anthracnose resistance in strawberry (Fragaria x ananassa, Duch.). Six isolates of Colletotrichum acutatum and four isolates of three other Colletotrichum species, C. gloeosporioides, C. fragariae, and C. graminicola, associated with disease symptoms were investigated in this study. The potential inhibitory effect of phenolic acids (gallic acid, caffeic acid, chlorogenic acid, ferulic acid, trans-cinnamic acid, p-coumaric acid, salicylic acid), flavonoids (catechin, quercetin, naringenin), and ellagic acid, which are naturally found in strawberry, were screened against two different spore suspension concentrations of the Colletotrichum isolates at 5, 10, 50mM in vitro. Among the phenolic acids and flavonoids tested in this study, only trans-cinnamic acid, ferulic acid, and p-coumaric acid inhibited fungal growth. The inhibitory effects were concentration-dependent but also varied with the spore suspension concentration of the isolates. The results demonstrated that trans-cinnamic acid had the greatest inhibitory effect on all Colletotrichum spp. isolates tested.

Phenotypic and genotypic responses of chili (Capsicum annuum L.) progressive lines with different resistant genes against anthracnose pathogen (Colletotrichum spp.)

Anthracnose disease caused by Colletotrichum spp. is a serious disease of chili (Capsicum annuum), particularly in tropical countries. Host plant resistance breeding is one of the most effective disease management strategies. It requires identification of parents with genes resistant to Colletotrichum species predominant in a given region. For phenotypic evaluation, 35 chili lines consisting of progressive lines of C. annuum derived from C. baccatum PBC80 (34 lines) and from C. chinense PBC932 (one line) were inoculated with two aggressive isolates of Colletotrichum acutatum (Ca) and C. capsici (Cc) by injection into the pericarp of green and red-ripe fruit. Lesion diameters were scored at seven days after inoculation. Two simple sequence repeat (SSR) and one sequence characterized amplified region (SCAR) markers were used for validation in four progressive lines and three each of susceptible and resistant checks for genotypic response. The progressive lines were classified into 10 groups based on their responses to two pathogens and at the two fruit stages. Four progressive lines (101, 205, 210 and 215) were selected and used for developing crosses to combine resistance genes from two sources resistant to Ca and Cc at both fruit stages. Progressive lines derived from PBC80 showed DNA fragment 231 bp amplified by primer HpmsE032 associated with Cc at green fruit stages. Hence the HpmsE032 marker could be considered useful in the selection of resistant genotypes derived from PBC80.

Virulence of Colletotrichum acutatum isolates to several host plants

Colletotrichum acutatum belongs to polyphagous fungal pathogens and is widespread in many countries on all continents. C. acutatum causes the most serious economic damage in strawberry (Fragaria x ananassa Duch.). Considering the wide variability of the pathogen may be assumed spread to other areas which constitutes danger not only for strawberry, but also other economically important fruit crops, vegetables and fruits.The main objective of our study was to verify the cross infection of eleven C. acutatum isolates from different host plants (strawberry, safflower, lupine, pepper and Hypericum perforatum) to selected host plants (strawberry, pepper and safflower). Two varieties from each of the experimental plant species were selected and virulence of isolates C. acutatum was evaluated.Based on results of statistical evaluation, virulence of C. acutatum isolates was different on strawberry, pepper and safflower. The strawberry variety Pegasus was more susceptible to C. acutatum than the variety Elkas. Isolate 710 from H. perforatum showed the highest virulence for both varieties in terms of index of infection intensity. The pepper variety Pirouet was more susceptible than the variety Cynthia. The highest degree of virulence was found for isolate 29267 from pepper in the variety Cynthia, the highest virulence was proved for isolate 231 from strawberry in the variety Pirouet. No statistical difference was confirmed between susceptibility of the safflower varieties. Isolate 1209 from safflower showed the most important effect on tested plants of safflower. Isolates 710 from H. perforatum, isolate 1209 from safflower, isolate 29267 from pepper and isolate 231 from strawberry showed different virulence for tested host plants.

Characterization of three Colletotrichum acutatum isolates from Capsicum spp.

Colletotrichum acutatum causes anthracnose on peppers (Capsicum spp.), resulting in severe yield losses in Taiwan. Fungal isolates Coll-153, Coll-365 and Coll-524 collected from diseased peppers were found to differ in pathogenicity. Pathogenicity assays on various index plants revealed that Coll-524 was highly virulent and Coll-153 was moderately virulent to three commercially available pepper cultivars. Both isolates induced anthracnose lesions and produced abundant conidia. Coll-365 was only weakly virulent on pepper fruit, where it caused small lesions and hardly produced conidia on pepper fruit. However, Coll-365 was highly pathogenic to tomato fruit and mango leaves, where it caused anthracnose lesions and formed acervuli and conidia. All three isolates showed similar abilities in the attachment and germination of conidia, formation of highly branched hyphae and appressoria, penetration of cuticles, and infection of epidermal cells on chili peppers. Coll-365 accumulated less turgor pressure in appressoria but produced higher levels of cutinase and protease activity than Coll-153 and Coll-524 did. All three isolates invaded the neighbouring cells through plasmodesmata in chili peppers and showed similar pectinase or cellulase activities in culture. However, the most virulent strain Coll-524 expressed stronger laccase activity and was more resistant to capsaicin compared to Coll-153 and Coll-365. The three isolates are different in numbers and sizes of double-stranded RNAs. Depending on the cultivar genotypes, cellular resistance of chili pepper to C. acutatum might rely on the ability to restrict penetration, colonization, or conidiation of the pathogen. We conclude that the differences in pathogenicity among the three C. acutatum isolates of pepper are attributed to their ability to colonize the host plant.

Vegetative Compatibility Groups and Parasexual Segregation in Colletotrichum acutatum Isolates Infecting Different Hosts

Heterokaryosis is an important mechanism which provides genetic variability increase in filamentous fungi. In order to assess the diversity of vegetative compatibility reactions existing among Colletotrichum acutatum isolates derived from different hosts, complementary nit mutants of each isolate were obtained and paired in all possible combinations. Vegetative compatibility groups (VCG) were identified among the isolates according to their ability to form viable heterokaryons. Seven VCGs were identified among the isolates, one of which contained isolates from different hosts. VCGs 2 and 6 contained two and three members, respectively; VCG-3 contained four members, and four VCGs (1, 4, 5, and 7) contained a single one. This study shows, for the first time, the isolation and the parasexual segregation of a heterozygous diploid sector derived from the heterokaryon formed with nit mutants from VCG-6. Diploid, named DE-3, showed nit+ phenotype and growth rate similar to the parental wild isolate. When inoculated in the presence of the haploidizing agent benomyl, the diploid strain produced parasexual haploid segregants exhibiting the nit phenotypes of the crossed mutants. Since viable heterokaryons and diploid may be formed among vegetative compatible isolates of C. acutatum, this study suggests that the parasexual cycle may be an alternative source of genetic variability in C. acutatum isolates.

First Report of Anthracnose Caused by Colletotrichum acutatum on Persimmon Fruit in the United States.

Persimmon trees are important for their fruit as well as their colorful fruit and foliage in the fall. Persimmon fruit (Japanese persimmon, Diospyros kaki cv. Fuyu) were collected in November 2008 from a tree in Windsor, NC, located in the Coastal Plain. Fruit were not symptomatic on the tree but developed dark lesions after harvest. Isolations from six fruit yielded seven isolates of Colletotrichum acutatum J. H. Simmonds. After incubation at 25°C under continuous light for 15 days on potato dextrose agar (PDA), all isolates had gray aerial mycelium, but the inverse sides of the plates of six isolates were maroon and one was beige. Masses of salmon-colored conidia were formed first in the center of the colonies, then were observed scattered across the colonies in older cultures. Conidia were hyaline, one-celled, elliptic with one or both ends pointed, and measured 8.1 to 16.3 × 3.1 to 5 μm. Setae and sclerotia were not observed. There were also dark structures measuring 1 to 10 mm that were partially embedded in the agar that contained conidia. Cultural and conidial characteristics of the isolates were similar to those of C. acutatum (3). PCR amplification was performed with the species-specific primer pair CaInt2/ITS4 (2) and genomic DNA from the original isolates and isolates obtained from inoculated fruit. An amplification product of approximately 490 bp, which is specific for C. acutatum, was observed. To fulfill Koch's postulates, persimmon fruit obtained from the grocery store were surface disinfested with 0.5% sodium hypochlorite and sterile filter paper disks dipped in conidial suspensions (1 × 105 conidia/ml) of two C. acutatum isolates (maroon and beige reverse) or sterile, deionized water were placed on the fruit. Three fruit were inoculated per treatment and the disks were placed on four locations on each fruit. Parafilm was wrapped around the diameter of the fruit to keep the filter paper disks moist and in place. Fruit were placed in moist chambers and incubated at 25°C. After 3 days, the Parafilm was removed and the fruit returned to the moist chambers. Small, dark lesions were observed on fruit inoculated with each isolate of C. acutatum when the filter paper disks were removed. Ten days after inoculation, dark lesions and acervuli with salmon-colored masses of conidia were observed on fruit inoculated with both isolates of C. acutatum and the fruit were soft. After 12 days, there were abundant masses of conidia and the inoculated areas were decayed. Control fruit remained firm and did not develop symptoms. Cultures obtained from the fruit and the conidia produced were typical of the isolates used to inoculate the fruit. C. acutatum has been reported to cause fruit rot on persimmon fruit in New Zealand (1). To our knowledge, this is the first report of C. acutatum on persimmon fruit in the United States.

Influence of Fruit Maturity, Cultivar Susceptibility, and Inoculation Method on Infection of Olive Fruit by Colletotrichum acutatum.

Detached olive (Olea europaea) fruit inoculated with isolates of Colletotrichum acutatum, causal agent of olive anthracnose, were used to study host-pathogen interactions. Fruit susceptibility increased with increasing fruit maturity. Wounded fruit were more severely affected than nonwounded fruit; however, the wound effect depended on cultivar and inoculation method. Severity of fruit infection increased with inoculum density, although this effect also depended on fruit maturity and cultivar susceptibility. The susceptibility of selected olive cultivars to anthracnose under field conditions correlated well with the response of immature fruit under controlled conditions. As fruit matured, there were fewer differences among cultivars. Based on these results, an inoculation method using immature green fruit and high inoculum densities (105 to 106 conidia/ml) sprayed on the fruit has been proposed to evaluate olive cultivars for anthracnose resistance under controlled conditions.

Lifestyles of Colletotrichum acutatum.

Lifestyles of Colletotrichum acutatum.

고추 탄저병균의 배양형 변이 그리고 병원성 차이

The dominant isolates of Colletotrichum gloeosporioides from the red pepper anthracnose(both of the diseased plants and debris) was more pathogenic than the isolates of Colletotrichum acutatum of minorly isolated from Gyeong-buk, Korea. There were both of the G and R strains of cultural variants of Colletotrichum gloeosporioides, the G strain was more pathogenic than R strain. The cultivars of red-pepper, cv. `Kumsegi` was the most susceptible and cv. `papet` was the least susceptible in the pathogenicity test. The isolates of Colletotrichum gloeosporioides from other host plant such on sesame, safflower, yam, strawberry could infect to the red pepper plant also.

Modified AFLP analysis method for species with small genomes

A modified method for detecting amplified fragment length polymorphism (AFLP) has been developed for species with small genomes. We successfully used a combination of primers with a reduced number of selective bases to differentiate and fingerprint isolates ofColletotrichum acutatum, the causative agent of strawberry black spot, an important strawberry disease.

Almond improvement in Australia

Introduction. The Australian almond [Prunus dulcis (Miller) D.A. Webb] improvement program commenced five years ago with the main aims of developing scion and rootstock cultivars that are better adapted to local conditions, and that provide a superior product for Australian and overseas markets. Approaches used. The program has a number of approaches including breeding, virus detection and elimination, and biotechnology. The classical hybridisation approach aimed at generation of diversity is combined with research into the more targeted techniques of plant tissue culture, genetic fingerprinting, genome mapping and transformation. Cryopreservation research is important for genebank storage, and tissue culture for micropropagation of new rootstocks and for transformation. Material is screened for Prunus Necrotic Ringspot (PNRV) and Prune Dwarf (PDV) Viruses. In addition, work has commenced into identifying Australian isolates of Colletotrichum acutatum , the pathogen causing anthracnose disease of almonds. The work is conducted in collaboration with overseas research groups, to take advantage of the long experience of these programs, and to contribute to the international effort in Prunus improvement. Outcomes from the almond project. The important outcomes achieved after the first five years of the project by the research team are listed.

Restriction landmark genomic scanning (RLGS) in fungi

RLGS is a technique to detect DNA polymorphism using restriction sites as landmarks. It identifies the landmarks through direct end-labeling, two-dimensional electrophoresis and autoradiography, giving a profile with many spots to allow the scanning of numerous DNA loci. We successfully performed the technique on fungi using isolates ofColletotrichum acutatum andC. gloeosporioides in anamorphic Ascomycotina,Rhizopus oryzae in Zygomycotina,Phytophthora nicotianae in Mastigomycotina (or Oomycota) andRhizoctonia solani in anamorphic basidiomycotina. RLGS of total genomic DNA digested with three restriction enzymes,Not, I,EcoR V andMbo I, reproducibly gave specific profiles of ca. 400 to 1.600 spots for each isolate. A polymorphic spot appearing to reflect a genetic difference between the twoColletotrichum species was found in the profiles of the isolates. No other common spots were found in any combination of isolates of the twoColletotrichum species, and thus the other spots on the profiles were regarded as unique to each isolate. These results indicated that RLGS could be applied, as a powerful fingerprinting technique based on genetic information from the whole genomic DNA, to search for useful DNA markers for taxonomic and genomic studies on many fungal species.

Factors affecting spore production, germination and viability of Colletotrichum acutatum isolates from Hevea brasiliensis

Colletotrichum acutatum is cosmopolitan and causes diseases in many crops including Hevea brasiliensis. It sporulated freely on PDA at 10–40 °C with peaks at around 15° and 25°. Self-inhibition of spore germination occurred at concentrations above 8 x 106 spores ml−1. Ultraviolet radiation (especially 254 nm) inactivated the spores. Spore germination was around 90% between 15–35°. Free water promoted spore germination but was not essential; high relative humidity (95%) was sufficient. Spores could also withstand temperatures up to 35°. It is concluded that the most favourable conditions for the spread of C. acutatum are prevalent in the major rubber growing areas in Sri Lanka throughout the monsoons.

Growth at different temperatures and on fungicide amended media: two characteristics to distinguish Colletotrichum species pathogenic to rubber.

Slower growth at temperatures ranging from 15 to 32.5 degrees C and high tolerance of three fungicides, benomyl, carbendazim and thio-phanate methyl, were shown to be reliable characteristics in distinguishing rubber isolates of Colletotrichum acutatum from Colletotrichum gloeosporioides in addition to the characteristics reported previously.

Differentiation of Colletotrichum species responsible for anthracnose of strawberry by arbitrarily primed PCR

A collection of 39 isolates of Colletotrichum acutatum, C. fragariae and C. gloeosporioides , which cause anthracnose on strawberry, was grouped into species based on the arbitrarily primed polymerase chain reaction (ap-PCR). All isolates used had previously been identified according to classical taxonomic morphology. Ap-PCR amplification of genomic DNA using four different primers allowed for reliable differentiation between isolates of C. acutatum, C. fragariae and two genotypes of C. gloeosporioides . Fifteen of the 18 C. acutatum isolates were very similar, although three isolates which produced a red pigment had distinctly different banding patterns. Nearly identical banding patterns were observed for all nine isolates of C. fragariae . The 12 C. gloeosporioides isolates were more diverse and two separate genotypes, Cgl-1 (six isolates) and Cgl-2 (five isolates) were distinguished by ap-PCR. An additional isolate did not conform to either the Cgl-1 or Cgl-2 genotypes. The utility of ap-PCR compared with other molecular techniques for reliable identification of Colletotrichum isolates pathogenic on strawberry is discussed.