Corynespora Leaf Fall Disease Research Articles

CCK1, a PMK1-type MAP kinase is required for hyphal growth, pigmentation, conidiation, enzyme activity, osmotic stress response, and pathogenicity in Corynespora cassiicola

Corynespora cassiicola is a fungal pathogen causing Corynespora Leaf Fall disease (CLF) in rubber trees (Hevea brasiliensis). CLF has great economic impact in rubber-producing countries. However, the molecular mechanisms involved in the pathogenicity of C. cassiicola remain poorly understood. Mitogen-activated protein kinase (MAPK) is one of the major signaling pathways responsible for regulation of fungal development and virulence in many plant fungal pathogens. The C. cassiicola cck1 gene encodes a MAPK that is homologous to the rice pathogen Magnaporthe grisea MAPK Pmk1 protein. In this study, targeted cck1 gene disruption and complementation experiments were carried out in C. cassiicola. Compared to the wild-type and CΔcck1 complement, the Δcck1 mutant had more pigmentation and exhibited dramatically reduced aerial mycelial mass and mycelial growth rates, along with the inability to produce conidia or conidiophores. In addition, the Δcck1 hyphae were darker in color and had less branches and septae with visibly condensed protoplasm. The Δcck1 mutant was more resistant to hyperosmotic stress, and was able to produce greater amount of laccase and lesser cellulase in vitro; however, the production of laccase was delayed. Importantly, Δcck1 mycelia and crude toxin were less virulent upon inoculation of rubber tree leaves. These findings indicate that the C. cassiicola CCK1 MAP kinase is a versatile regulator involved in nearly every aspect of plant fungal pathogenicity including mycelial growth, development, differentiation, pigmentation, conidiation, production of extracellular degradation enzymes, osmoregulation, and virulence.

Genetic Determinism of Sensitivity to Corynespora cassiicola Exudates in Rubber Tree (Hevea brasiliensis).

An indirect phenotyping method was developed in order to estimate the susceptibility of rubber tree clonal varieties to Corynespora Leaf Fall (CLF) disease caused by the ascomycete Corynespora cassiicola. This method consists in quantifying the impact of fungal exudates on detached leaves by measuring the induced electrolyte leakage (EL%). The tested exudates were either crude culture filtrates from diverse C. cassiicola isolates or the purified cassiicolin (Cas1), a small secreted effector protein produced by the aggressive isolate CCP. The test was found to be quantitative, with the EL% response proportional to toxin concentration. For eight clones tested with two aggressive isolates, the EL% response to the filtrates positively correlated to the response induced by conidial inoculation. The toxicity test applied to 18 clones using 13 toxinic treatments evidenced an important variability among clones and treatments, with a significant additional clone x treatment interaction effect. A genetic linkage map was built using 306 microsatellite markers, from the F1 population of the PB260 x RRIM600 family. Phenotyping of the population for sensitivity to the purified Cas1 effector and to culture filtrates from seven C. cassiicola isolates revealed a polygenic determinism, with six QTL detected on five chromosomes and percentages of explained phenotypic variance varying from 11 to 17%. Two common QTL were identified for the CCP filtrate and the purified cassiicolin, suggesting that Cas1 may be the main effector of CCP filtrate toxicity. The CCP filtrate clearly contrasted with all other filtrates. The toxicity test based on Electrolyte Leakage Measurement offers the opportunity to assess the sensitivity of rubber genotypes to C. cassiicola exudates or purified effectors for genetic investigations and early selection, without risk of spreading the fungus in plantations. However, the power of this test for predicting field susceptibility of rubber clones to CLF will have to be further investigated.

Cultural and Reproductive Variability of Corynespora cassiicola from Different Host Plants

Natural rubber industry plays an important role in the economy of Sri Lanka. Therefore, improvement of productivity levels has become important. Corynespora leaf fall disease (CLFD) is the most destructive disease of rubber caused by Corynespora cassiicola. In Sri Lanka two epidemics occurred in 1986 and 1995. C. cassiicola has a wide host range including vegetables, ornamentals, weeds, yams, medicinal and other cash crops. Hence, there is a high risk of cross infection possibilities. The objective of this study was to examine the diversity of the pathogen isolates from several host plants; rubber-RRISL202 (A), RRISL 110 (B), RRISL201 (C), Eggplant (D), Papaya (E), Tomato (F). Diseased leaf samples were collected and isolated on potato dextrose agar (PDA). Single spore isolates were obtained and the cultural variations were studied. Except flat textured D and E, all the other isolates showed puffy growth. Colony appearance varied from olivaceous green colour to different shades of grey. Except A, B and C all the other isolates showed concentric rings on lower surface. Except A and B, the other isolates showed uneven margins. In microscopic view, all rubber isolates showed thread like hyphae with a width of (3.20-3.58) μm. However, in the isolates of alternative hosts, hyphae were wider (6.00-7.80 μm). Generally mycelia were branched and septate with conidiophores at the tips of hyphae. Isolates A, B and C were fast growers, D and E were moderate while F was slow. In reproductive studies, generally conidia of C. cassiicola were segmented and showed characteristic hilum at the pointed tips. There was no significant variation in the dimensions of spores among the isolates. The conidial length varied from (15-275 μm) and width varied from (3.75-12.50 μm). Straight, curved, cylindrical and obclavate shapes were observed. Mostly slender, straight conidia were observed in rubber isolates. Rubber isolates were poor sporulators compared to the alternative host isolates. Present study revealed that there is a significant variation in the morphology and reproductive characteristics among the selected C. cassiicola isolates. Further investigations will be carried out to examine the genetic and pathogenicity variations among the isolates. Keywords: Corynespora cassiicola, Alternative hosts, Morphology and reproductive Characteristics

Infection process of Corynespora cassiicola tagged with GFP on Hevea brasiliensis

Corynespora leaf fall (CLF) disease, caused by the fungal pathogen Corynespora cassiicola, is a major disease of rubber tree (Hevea brasiliensis) that affects the production of natural rubber. To study its pathogenesis, we developed a C. cassiicola transformant tagged with green fluorescent protein and studied it using fluorescence and confocal laser scanning microscopy. The transformed isolate retained its pathogenicity and grew similarly to the wild-type. Conidia germinated and produced germ tubes 4 h after inoculation, after which the fungus infected leaves through wounds; extended into veins, cells, and intercellular spaces within 24 h; produced abundant mycelia on the surfaces of diseased leaves within 48 h; and began to form conidiophores and conidia within 72 h. The infection process of C. cassiicola on H. brasiliensis was complete within 72 h. Our findings may be useful for predicting the path of CLF disease and evaluating H. brasiliensis resistance to C. cassiicola via leaf colonization studies.

Diversity of the cassiicolin gene in Corynespora cassiicola and relation with the pathogenicity in Hevea brasiliensis

Corynespora cassiicola is an important plant pathogenic Ascomycete causing the damaging Corynespora Leaf Fall (CLF) disease in rubber tree (Hevea brasiliensis). A small secreted glycoprotein named cassiicolin was previously described as an important effector of C. cassiicola. In this study, the diversity of the cassiicolin-encoding gene was analysed in C. cassiicola isolates sampled from various hosts and geographical origins. A cassiicolin gene was detected in 47 % of the isolates, encoding up to six distinct protein isoforms. In three isolates, two gene variants encoding cassiicolin isoforms Cas2 and Cas6 were found in the same isolate. A phylogenetic tree based on four combined loci and elucidating the diversity of the whole collection was strongly structured by the toxin class, as defined by the cassiicolin isoform. The isolates carrying the Cas1 gene (toxin class Cas1), all grouped in the same highly supported clade, were found the most aggressive on two rubber tree cultivars. Some isolates in which no Cas gene was detected could nevertheless generate moderate symptoms, suggesting the existence of other yet uncharacterized effectors. This study provides a useful base for future studies of C. cassiicola population biology and epidemiological surveys in various host plants.

First characterization of endophytic Corynespora cassiicola isolates with variant cassiicolin genes recovered from rubber trees in Brazil

Corynespora cassiicola is the causal agent of Corynespora Leaf Fall (CLF) disease. CLF is one of the most important fungal diseases of rubber trees in Asia and Africa but disease outbreaks have not been reported in South America. Cassiicolin, a small cysteine-rich glycoprotein secreted by the pathogenic C. cassiicola isolate CCP, was previously identified as a potential disease effector in rubber tree. Recently, the cassiicolin-encoding gene (Cas1) was characterized and shown to be expressed in the early phase of infection. In this study, we investigated whether previously undetected strains of C. cassiicola are present in South American rubber plantations by examining the fungal endophyte population found in asymptomatic rubber tree leaves. Four isolates were identified as C. cassiicola. Genes encoding new forms of the cassiicolin precursor protein (Cas3 and Cas4) were identified from these isolates. Three of four isolates were able to induce symptoms on the cultivar they were isolated from in a detached leaf assay, with different kinetics and intensities. One isolate had the same pathogenicity profile as the pathogenic isolate CCP; the other two isolates developed symptoms late during the course of infection, suggesting saprotrophic capabilities. However, no Cas3 or Cas4 transcripts could be detected upon inoculation with the endophytic isolates, whereas the reference gene Cas1 was expressed upon inoculation with the CCP isolate. This work demonstrated that C. cassiicola is present in South America in an endophytic form and that it may evolve from an endophytic to a saprophytic or even potentially pathogenic life style.

The Current and Future Potential Geographic Range of Corynespora Leaf Fall Disease in China

The Current and Future Potential Geographic Range of <i>Corynespora</i> Leaf Fall Disease in China

Characterization of a cassiicolin-encoding gene from Corynespora cassiicola, pathogen of rubber tree (Hevea brasiliensis)

Corynespora Leaf Fall (CLF) is a major disease of rubber tree (Hevea brasiliensis) caused by the Ascomycota Corynespora cassiicola. Here we describe the cloning and characterization of a gene encoding cassiicolin (Cas), a glycosylated cystein-rich small secreted protein (SSP) identified as a potential CLF disease effector in rubber tree. Three isolates with contrasted levels of aggressiveness were analyzed comparatively. The cassiicolin gene was detected – and the toxin successfully purified – from the isolates with high and medium aggressiveness (CCP and CCAM3 respectively) but not from the isolate with the lowest aggressiveness (CCAM1), suggesting the existence of a different disease effector in the later. CCP and CCAM3 carried strictly identical cassiicolin genes and produced toxins of identical mass, as evidence by mass spectrometry analysis, thus suggesting conserved post-translational modifications in addition to sequence identity. The differences in aggressiveness between CCP and CCAM3 may be attributed to differences in cassiicolin transcript levels rather than qualitative variations in cassiicolin structure. Cassiicolin may play an important role in the early phase of infection since a peak of cassiicolin transcripts occurred in 1 or 2 days after inoculation (before the occurrence of the first symptoms), in both the tolerant and the susceptible cultivars.

Susceptibility of different leaf stages of Hevea to Corynespora cassiicola

Corynespora leaf fall disease caused by Corynespora cassiicola is considered one of the most destructive foliar diseases of rubber. The pathogen affects the leaves causing leaf fall throughout the year. According to the results of this study the young leaf stages, copper brown and late copper brown, were the most susceptible to CLFD. With maturity, the lesion sizes reduced and it was not possible to artificially inoculate mature leaves from any clone. The observations were confirmed by results from the field experiments. The results of this investigation suggests that the fungicide spray rounds could be minimized if spraying programme is scheduled targeting the refoliation period.

Screening of fungicides against Corynespora leaf fall disease of rubber under nursery conditions

Quality planting material is a basic requirement for the increase in productivity levels and one of the key indicators of quality is the health of nursery plants. Corynespora leaf fall disease (CLFD) affects all clones of rubber under nursery condition. In this investigation the most effective treatment to manage CLFD in nurseries was the introduction of overhead shading together with application of the fungicide mancozeb. The fungicide mixture (carbendazim + mancozeb) applied at 7-d intervals, and carbendazim sprayed at 2-week intervals together with shade showed equally good results. Carbendazim (0.5 g l–1) sprayed at 7- or 10-d intervals, the application of other fungicides, mancozeb, metalaxyl 8% + mancozeb 64%, captan and propineb, at 3 g l–1 at 7-d intervals was also effective. Though the systemic fungicide, carbendazim efficiently controlled the disease, tebuconazole and hexaconazole were found to be much less effective.

Evaluation of screening methods against Corynespora leaf fall disease of rubber (Hevea brasiliensis)

Identification of resistant clones is a major strategy in the management of Corynespora leaf fall disease (CLFD) of rubber. Several methods to identify resistance/susceptibility were examined in this study. There were significant variations in the mean lesion scores of different clones when evaluated by laboratory based in vitro assays. When screened in a bud wood nursery, the lesion types agreed with the symptoms observed in field plants. Analysis of results of clones screened under natural conditions showed that 85% of the clones in the Rubber Research Institute of Sri Lanka (RRISL) recommendation list can be ranked as resistant. The results of this investigation suggest that laboratory based in vitro screening methods should be used only for preliminary data. Field experiments, though dependable, take a considerably longer period. As such the bud wood nursery experiments which takes a much shorter time can be recommended to screen planting material for resistance or susceptibility to CLFD.

Nested PCR assay for detection of Corynespora leaf fall disease caused byCorynespora cassiicola

Corynespora leaf fall disease of Hevea brasiliensis caused by Corynespora cassiicola has been a major cause of severely reduced rubber latex production in Asia and Africa. The aim of this study was to determine whether physiological races of C. cassiicola exist in the Hainan and Yunnan provinces, China, and to accelerate and simplify the process of diagnosis. We conducted pathogenicity tests with 17 C. cassiicola isolates on detached leaves of four different Hevea rubber clones (genotypesPR107, Dafeng 95,RRIM600 and Reyan 7-33-97). Pathogenicity tests showed that 16 of the isolates were pathogenic to RRIM 600, which confirms these isolates as C. cassiicola race 1. An initial PCR was performed to amplify a portion of the ITS1-5.8S-ITS2 region using a C. cassiicola species-specific primer set (CCF and CCR-2).DNA fragments of 272 bp in length indicated the presence of C. cassiicola. To improve the sensitivity, a nested PCR assay was developed to obtain DNA fragments of the expected length (152 bp) using diluted (1 : 100) amplified product from the initial PCR as the template and species-specific oligonucleotides CCF and CCR-1 as primers. The lowest concentration of fungal DNA that could be detected by nested PCR was 168 fg, while its concentration in the initial PCR was 16.8 pg. Therefore, the sensitivity of detection was enhanced 100-fold using nested PCR. In addition, the pathogen could be detected from artificially infected Hevea rubber trees 3 days after inoculation. Due to its high sensitivity, specificity, and reliability, nested PCR is useful for the screening and certification of young C. cassiicola-free Hevea rubber plants for distribution to commercial growers.

Detection of Corynespora cassiicola in Hevea rubber tree from China

Corynespora cassiicola, which causes Corynespora leaf fall disease, is one of the most destructive and economically important fungal pathogens of Hevea rubber trees in Asian and African countries. This fungus is present in some Hevea rubber plantations and nurseries in Hainan and Yunnan provinces of South China. We describe the development of a PCR assay for C. cassiicola. With this test, of the 32 leaf samples obtained from 28 Hevea rubber trees in 15 rubber plantations and nurseries of Hainan and Yunnan provinces, 21 tested positive for the fungus. This assay is useful for the screening and certification of young C. cassiicola-free Hevea rubber plants (for distribution to commercial growers) due to its high sensitivity, reliability and efficacy.

First record of Corynespora leaf fall disease of Hevea rubber tree in China

Corynespora cassiicola has been found for the first time in China on Hevea rubber tree (Hevea brasiliensis in south China during a survey in 2006. Symptoms on leaves included fishbone necrosis and spots. Sequence data from the ribosomal ITS operon placed the isolates within the Corynespora cassiicola complex.

Structural Analysis of Cassiicolin, a Host-selective Protein Toxin from Corynespora cassiicola

Cassiicolin is a host-selective toxin (HST) produced by the fungus Corynespora cassiicola (strain CCP). It is responsible for the Corynespora leaf fall (CLF) disease, which is among the main pathologies affecting rubber tree ( Hevea brasiliensis). Working on purified cassiicolin and using electron microscopy, we have demonstrated that this 27-residue O-glycosylated protein is able to induce cellular damages identical to those induced by the fungus on rubber tree leaves and displays the same host selectivity. The solution structure and disulfide pairing of cassiicolin have been determined using NMR spectroscopy and simulated annealing calculations. Cassiicolin appears to have an original structure with a prolate ellipsoid shape. It adopts an over-all fold consisting of three strands arranged in a right-handed twisted, antiparallel β-sheet knitted by three disulfide bonds. Its conformation resembles that found in small trypsine-like inhibitors isolated from the brain, the fat body and the hemolymph of locust grasshoppers. But cassiicolin has no sequence homology with these protease inhibitors, and lacks their characteristic substrate-binding loop. Probably, this motif represents one of the few highly stabilized “minimal” scaffolds, with a high sequence permissiveness, that nature has selected to evolve over different phyla and to support different functions. The knowledge of the 3D structure opens the way to the delineation of the mechanism of action of the toxin using site-directed mutagenesis.