Gene deletion of Corynespora cassiicola cassiicolin Cas1 suppresses virulence in the rubber tree.

Corynespora cassiicola is an ascomycete fungus causing important damages in a wide range of plant hosts, including rubber tree. The small secreted protein cassiicolin is suspected to play a role in the onset of the disease in rubber tree, based on toxicity and gene expression profiles. However, its exact contribution to virulence, compared to other putative effectors, remains unclear. We created a deletion mutant targeting the cassiicolin gene Cas1 from the highly aggressive isolate CCP. Wild-type CCP and mutant ccpΔcas1 did not differ in terms of mycelium growth, sporulation, and germination rate in vitro. Cas1 gene deletion induced a complete loss of virulence on the susceptible clones PB260 and IRCA631, as revealed by inoculation experiments on intact (non-detached) leaves. However, residual symptoms persisted when inoculations were conducted on detached leaves, notably with longer incubation times. Complementation with exogenous cassiicolin restored the mutant capacity to colonize the leaf tissues. We also compared the toxicity of CCP and ccpΔcas1 culture filtrates, through electrolyte leakage measurements on abraded detached leaves, over a range of clones as well as an F1 population derived from the cross between the clones PB260 (susceptible) and RRIM600 (tolerant). On average, filtrate toxicity was lower but not fully suppressed in ccpΔcas1 compared to CCP, with clone-dependent variations. The two QTL, previously found associated with sensitivity to CPP filtrate or to the purified cassiicolin, were no longer detected with the mutant filtrate, while new QTL were revealed. Our results demonstrate that: (1) cassiicolin is a necrotrophic effector conferring virulence to the CCP isolate in susceptible rubber clones and (2) other effectors produced by CCP contribute to residual filtrate toxicity and virulence in senescing/wounded tissues. These other effectors may be involved in saprotrophy rather than necrotrophy.

Endophytes from Wild Rubber Trees as Antagonists of the Pathogen Corynespora cassiicola.

The Corynespora leaf fall disease of rubber trees, caused by the necrotrophic fungus Corynespora cassiicola, is responsible for important yield losses in Asian and African plantations, whereas its impact is negligible in South America. The objective of this study was to identify potential antagonists of C. cassiicola among fungal endophytes (i.e., Pestalotiopsis, Colletotrichum, and Trichoderma spp.) isolated from wild and cultivated rubber trees distributed in the Peruvian Amazon. We first tested the endophytes in dual in vitro confrontation assays against a virulent C. cassiicola isolate (CCP) obtained from diseased rubber trees in the Philippines. All Trichoderma isolates overran the CCP colony, suggesting some antagonistic mechanism, while species from the other genera behaved as mutual antagonists. Trichoderma isolates were then tested through antibiosis assays for their capacity to produce growth-inhibiting molecules. One isolate (LA279), recovered as an endophyte from a wild Hevea guianensis specimen and identified as Trichoderma koningiopsis, showed significant antibiosis capacity. We demonstrated that LA279 was also able to endophytically colonize the cultivated rubber tree species (H. brasiliensis). Under controlled laboratory conditions, rubber plants were inoculated with three Trichoderma strains, including LA279, in combination with the pathogenic CCP. Results showed that 1 week preinoculation with the endophytes differentially reduced CCP mycelial development and symptoms. In conclusion, this study suggests that T. koningiopsis isolate LA279-and derivate compounds-could be a promising candidate for the biological control of the important rubber tree pathogen C. cassiicola.

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.

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.

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.