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International audience; 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.

Fusarium root rot is a major cryptogamic disease in olive trees caused by the soil-borne fungus Fusarium solani. Controlling this disease requires the extensive use of chemicals. However, using BCAs such as some Trichoderma strains may be an opportune alternative to fungicides in protecting olive plantations. A new isolate (Fso14) was isolated from young olive trees showing severe dieback symptoms. The objective of this work was to analyze the biocontrol behavior of a Tunisian strain of T. harzianum (Ths97) on olive trees against Fso14 by assessing both mycoparasitic activity (in planta and in vitro) and ability to locally modulate different gene-related defenses of the plant. Ths97 was found to inhibit Fso14 growth in vitro. Optical microscopic analysis at the confrontation zone between hyphae showed that Ths97 grew alongside Fso14 with numerous contact points suggesting parasitic activity. On olive trees, Ths97 developed a strong protective role against root infestation by Fso14, whether inoculated before or after the pathogenic agent. When inoculated alone, Fso14 and Ths97 did not modulate (or only slightly with inhibitions or inductions, respectively) the expression of genes involved in plant immunity (oxidative stress, phenylpropanoid pathway, PR-proteins and JA/Et-SA hormonal status). However, when Ths97 was inoculated in combination with Fso14, several defense-related genes were highly up-regulated, indicating probable primed-plant events. These promising results provided valuable information on using Ths97 as a beneficial agent to control fusarium root rot disease caused by F. solani in olive trees.

The Corynespora leaf fall disease of rubber trees, caused by the necrotrophic fungus

International audience; 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.

Major intrinsic proteins (MIP) are characterized by a transmembrane pore-type architecture that facilitates transport across biomembranes of water and a variety of low molecular weight solutes. They are found in all parts of life, with remarkable protein diversity. Very little is known about MIP from fungi. And yet, it can legitimately be stated that MIP are pivotal molecular components in the privileged relationships fungi enjoy with plants or soil fauna in various environments. To date, MIP have never been studied in a mycoparasitism situation. In this study, the diversity, expression and functional prediction of MIP from the genus Trichoderma were investigated. Trichoderma spp. genomes have at least seven aquaporin genes. Based on a phylogenetic analysis of the translated sequences, members were assigned to the AQP, AQGP and XIP subfamilies. In in vitro and in planta assays with T. harzianum strain Ths97, expression analyses showed that four genes were constitutively expressed. In a mycoparasitic context with Fusarium solani, the causative agent of fusarium dieback on olive tree roots, these genes were up-regulated. This response is of particular interest in analyzing the MIP promoter cis-regulatory motifs, most of which are involved in various carbon and nitrogen metabolisms. Structural analyses provide new insights into the possible role of structural checkpoints by which these members transport water, H2O2, glycerol and, more generally, linear polyols across the membranes. Taken together, these results provide the first evidence that MIP may play a key role in Trichoderma mycoparasitism lifestyle.

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca, and Botrosphaeria dothidea) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector-based classification was found to be highly consistent with the phylogenomic trees. Identification of lineage-specific effectors is a key step toward understanding C. cassiicola virulence and host specialization mechanisms.

Genome-wide analysis of Corynespora cassiicola putative effectors involved in the CLF disease of rubber tree.. 12th EFPP-10th SFP conference: Deepen Knowledge in Plant Pathology for Innovative Agro-Ecology

Plant Breed.ISI Document Delivery No.: AJ4WPTimes Cited: 0Cited Reference Count: 31Cited References: Argout X, 2008, BMC GENOMICS, V9, DOI 10.1186/1471-2164-9-512 BESSE P, 1994, THEOR APPL GENET, V88, P199, DOI 10.1007/BF00225898 Conesa A, 2005, BIOINFORMATICS, V21, P3674, DOI 10.1093/bioinformatics/bti610 Cubry P, 2013, BMC GENOMICS, V14, DOI 10.1186/1471-2164-14-10 Ellis JR, 2007, HEREDITY, V99, P125, DOI 10.1038/sj.hdy.6801001 Feng SP, 2009, MOL BREEDING, V23, P85, DOI 10.1007/s11032-008-9216-0 Garcia D, 2011, PHYSIOL MOL PLANT P, V76, P126, DOI 10.1016/j.pmpp.2011.07.006 Gupta PK, 2005, PLANT MOL BIOL, V57, P461, DOI 10.1007/s11103-005-0257-z Gupta S, 2013, PLANT BREEDING, V132, P367, DOI 10.1111/pbr.12070 Kumar Yadav H., 2010, TREE GENET GENOMES, V7, P207 Le Guen V., 2008, EXPLORATION DIVERSIT Le Guen V, 2011, PLANT BREEDING, V130, P294, DOI 10.1111/j.1439-0523.2010.01774.x Le Guen V, 2009, TREE GENET GENOMES, V5, P673, DOI 10.1007/s11295-009-0218-9 Lespinasse D, 2000, THEOR APPL GENET, V100, P127, DOI 10.1007/s001220050018 Li DJ, 2012, BMC GENOMICS, V13, DOI 10.1186/1471-2164-13-192 Liu KJ, 2005, BIOINFORMATICS, V21, P2128, DOI 10.1093/bioinformatics/bti282 Ohyama A, 2009, MOL BREEDING, V23, P685, DOI 10.1007/s11032-009-9265-z Perovic D, 2009, MOL BREEDING, V23, P641, DOI 10.1007/s11032-009-9262-2 Perrier Xavier, 2003, P31 Perrier X., 2006, DARWIN SOFTWARE Pootakham W, 2012, PLANT BREEDING, V131, P555, DOI 10.1111/j.1439-0523.2012.01982.x Rafalski JA, 2010, CURR OPIN PLANT BIOL, V13, P174, DOI 10.1016/j.pbi.2009.12.004 Rozen S, 2000, Methods Mol Biol, V132, P365 SAITOU N, 1987, MOL BIOL EVOL, V4, P406 Seguin Marc, 2003, P277 Souza LM, 2009, CONSERV GENET RESOUR, V1, P75, DOI 10.1007/s12686-009-9018-7 Triwitayakorn K, 2011, DNA RES, V18, P471, DOI 10.1093/dnares/dsr034 Tyrka M, 2008, J APPL GENET, V49, P127, DOI 10.1007/BF03195605 Varshney RK, 2005, TRENDS BIOTECHNOL, V23, P48, DOI 10.1016/j.tibtech.2004.11.005 Vigouroux Y, 2005, GENETICS, V169, P1617, DOI 10.1534/genetics.104.032086 Wen Mingfu, 2010, BMC Res Notes, V3, P42, DOI 10.1186/1756-0500-3-42Cubry, Philippe Pujade-Renaud, Valerie Garcia, Dominique Espeout, Sandra Le Guen, Vincent Granet, Francoise Seguin, MarcFrench National Research Agency (Agence Nationale pour la Recherche; contrat ANR/Genoplante) [GPLA07017C]This work was performed as part of the CIRAD - Michelin collaborative project 'Genesalb' supported by a grant from the French National Research Agency (Agence Nationale pour la Recherche; contrat ANR/Genoplante no GPLA07017C). We thank the Montpellier Languedoc-Roussillon Grand plateau technique regional for hosting the SSR genotyping activities. ESTtik database was made available by the SouthGreen bioinformatics platform at http://southgreen.cirad.fr/. We thank Ronan Rivallan for technical help, Xavier Argout for computational procedures and data retrieving, Gerald Oliver for keeping some vegetal material in a greenhouse in Montpellier, Jean-Marc Thevenin and Fabien Doare for providing us with some fresh material from CIRAD's Pointe Combi Centre (French Guyana) collection.Wiley-blackwellHoboken; Despite its economic importance and recent genome release, the need for molecular tools for Hevea brasiliensis is high. In the frame of a disease resistance study, EST sequences were retrieved from public database or generated by sequencing SSH libraries. Sequences were trimmed and microsatellite motifs searched using an ad hoc bioinformatic pipeline, and pairs of primers for the amplification of candidate markers were generated. We found a total of 10499 unigenes from both sources of sequences, and 673 microsatellites motifs were detected using the default parameters of the pipeline. Two hundred sixty-four primer pairs were tested and 226 (85.6%) successfully amplified. Out of the amplified candidate markers, 164 exhibited polymorphism. Relationships based on dendrograms using simple matching index and diversity statistics based on EST-SSRs were compared with Genomic SSRs, showing the potentialities of EST-derived microsatellites for resistance studies but also for population genetics approaches.

X-Intrinsic Proteins (XIP) were recently identified in a narrow range of plants as a full clade within the aquaporins. These channels reportedly facilitate the transport of a wide range of hydrophobic solutes. The functional roles of XIP in planta remain poorly identified. In this study, we found three XIP genes (HbXIP1;1, HbXIP2;1 and HbXIP3;1) in the Hevea brasiliensis genome. Comprehensive bioinformatics, biochemical and structural analyses were used to acquire a better understanding of this AQP subfamily. Phylogenetic analysis revealed that HbXIPs clustered into two major groups, each distributed in a specific lineage of the order Malpighiales. Tissue-specific expression profiles showed that only HbXIP2;1 was expressed in all the vegetative tissues tested (leaves, stem, bark, xylem and latex), suggesting that HbXIP2;1 could take part in a wide range of cellular processes. This is particularly relevant to the rubber-producing laticiferous system, where this isoform was found to be up-regulated during tapping and ethylene treatments. Furthermore, the XIP transcriptional pattern is significantly correlated to latex production level. Structural comparison with SoPIP2;1 from Spinacia oleracea species provides new insights into the possible role of structural checkpoints by which HbXIP2;1 ensures glycerol transfer across the membrane. From these results, we discuss the physiological involvement of glycerol and HbXIP2;1 in water homeostasis and carbon stream of challenged laticifers. The characterization of HbXIP2;1 during rubber tree tapping lends new insights into molecular and physiological response processes of laticifer metabolism in the context of latex exploitation.

Mechanical wounding and jasmonic acid (JA) treatment have been shown to be important factors in controlling laticifer differentiation in Hevea brasiliensis (rubber tree). With the long-term aim of potentially modifying the endogenous levels of JA in H. brasiliensis by gene transfer, we describe in this paper the molecular cloning of a H. brasiliensis aliene oxide synthase (AOS) cDNA and biochemical characterisation of the recombinant AOS (His6-HbAOS) enzyme. The AOS cDNA encodes a protein with the expected motifs present in CYP74A sub-group of the cytochrome P450 super-family of enzymes that metabolise 13-hydroperoxylinolenic acid (13-HPOT), the intermediate involved in JA synthesis. The recombinant H. brasiliensis AOS enzyme was estimated to have a high binding affinity for 13-HPOT with a K. value of 4.02 ± 0.64 ttM. Consistent with previous studies, mammalian cycloxygenase (COX) and lipoxygenase (LOX) inhibitors were shown to significantly reduce His6-HbAOS enzyme activity. Although JA had no effect on His6-HbAOS, salicylic acid (SA) was shown to significantly inhibit the recombinant AOS enzyme activity in a dose dependent manner. Moreover, it was demonstrated that SA, and various analogues of SA, acted as competitive inhibitors of His6-HbAOS when 13-HPOT was used as substrate. We speculate that this effect of salicylates on AOS activity may be important in cross-talking between the SA and JA signalling pathways in plants during biotic/abiotic stress. © 2007 Elsevier Masson SAS. All rights reserved.

The effectors of Corynespora cassiicola virulence in rubber tree. In IRRDB International Rubber . IRRDB International Rubber Conference

A procedure has been established for Agrobacterium tumefaciens-mediated genetic transformation of Hevea brasiliensis embryogenic friable calli. Precultivation of tissues on a CaCl(2)-free maintenance medium dramatically enhanced the transient activity of the reporter gene, gusA encoding beta-glucuronidase (GUS). The increase was first noticed in highly active cells (undifferentiated or/and embryogenic), in tissues precultured for 2-8 weeks. Beyond 8 weeks of preculture, GUS activity increased again, but this time in tissues consisting of differentiated cells accumulating polyphenols. Out of five Agrobacterium strains cocultivated with CaCl(2)-free precultured tissues, only inoculation with EHA105pC2301 led to high transient GUS activity. Paromomycin proved more effective than kanamycin for the selection of transformed cells, as it inhibits the growth of non-transformed cells more radically. Five paromomycin-resistant callus lines were established. The presence of gusA and neomycin phosphotransferase ( nptII) genes in the plant genome was confirmed by DNA amplification, and by Southern hybridization. These results confirmed that A. tumefaciens is an effective system for mediating stable transformation of rubber tree calli with a low copy number of transgenes. Transgenic callus lines constitute a useful tool for studying genes of interest on a cellular level and for regenerating transgenic rubber trees.

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.

The fungus Corynespora cassiicola is an important pathogen that causes necrotic lesions in several plant species. Saprophytism and parasitism habits of plants are common survival strategies for this pathogen. Few studies referred to the formation of chlamydospores by C. cassiicola. The objective of this study was to test the formation of chlamydospores by several C. cassiicola isolates from different agronomic crops and weeds. Fifteen isolates were analysed by in vivo and in vitro tests. Six isolates from four host plants (Cucumis sativus, Lantana camara, Malpighia glabra and Vernonia cinerea) were able to produce chlamydospores, both in vitro and in vivo. In vitro, chlamydospore production was highest for the Malpighia glabra isolate and one Cucumis sativus isolate, intermediate for two other C. sativus isolates, and lowest for the Vernonia cinerea and Lantana camara isolates. However, no difference in the relative number of chlamydospores produced among isolates was observed in vivo.

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R. Leroy, Thierry Seguin, Marc Roeckel-Drevet, Patricia Pujade-Renaud, Valerie IFC (Institut Francais du Caoutchouc, Paris, France); Michelin (Clermont-Ferrand, France); SIPH ("Societe Internationale de Plantations d'Hevas", Courbevoie, France); SOCFIN ("Societe Financiere des Caoutchoucs", Bruxelles, Belgium) This work was supported in part by a grant from the IFC (Institut Francais du Caoutchouc, Paris, France) and the companies Michelin (Clermont-Ferrand, France), SIPH ("Societe Internationale de Plantations d'Hevas", Courbevoie, France) and SOCFIN ("Societe Financiere des Caoutchoucs", Bruxelles, Belgium). We thank Boris Fumanal and Jean-Stephane Venisse for their valuable comments. Springer New york Si; 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 isolate CCP, was previously identified as a potential disease effector in rubber tree. Recently, the cassiicolin-encoding gene () was characterized and shown to be expressed in the early phase of infection. In this study, we investigated whether previously undetected strains of are present in South American rubber plantations by examining the fungal endophyte population found in asymptomatic rubber tree leaves. Four isolates were identified as . 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 or transcripts could be detected upon inoculation with the endophytic isolates, whereas the reference gene was expressed upon inoculation with the CCP isolate. This work demonstrated that 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.

UMR 1334 AGAP : Equipe AFEF 'Architecture et Fonctionnement des Espèces fruitières' ; Team AFFS 'Architecture and Functioning of Fruit Species' Contact: valerie.pujade-renaud@cirad.fr; 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.

In this report, we assess by suppression subtractive hybridization (SSH) the biological process of the South American leaf blight (SALB) that is an important disease of rubber tree caused by Microcyclus ulei (P. Hem.) v. Arx. Leaves from MDF180 (a partially resistant cultivar from Peruvian origin) and PB314 (a susceptible cultivar) were inoculated with M. ulei . Leaf fragments were collected from 6 h post-infection (hpi) to 58 days post-infection (dpi) and five cDNA libraries enriched for genes induced by M. ulei in order to identify up-regulated genes at the different stages of the host-parasite interaction. We sequenced 8027 expressed sequence tags (EST). After control of their quality, trimming and assembling, we obtained 1165 singlets and 458 contigs. The EST redundancy within each SSH library varied from 23% to 87%. Based on sequence similarity 49% of the sequences could be assigned to functional categories, whereas 21% corresponded to unknown function and 30% did not show any significant similarity with other sequences. The profile of gene expression was different for each library and gave a comprehensive picture of the transcriptome dynamics of rubber tree in response to M. ulei throughout the infection process. Most of the ESTs described in this study (80%) are specific to leaf tissues since they did not show any homology with ESTs previously identified in latex transcriptome. The occurrence of ESTs whose functional annotation was related to “stress” or “defence” was compared between libraries and their specific function is discussed in relation to the corresponding stage of leaf lesions and to histological observations. As far as we known, this is the first report on ESTs from Hevea brasiliensis collected during a biotic stress. It provides a significant contribution to the understanding of the molecular mechanism of the resistance and the susceptibility to South American leaf blight and it represents a new molecular resource for the development of polymorphic markers and marker-assisted selection.

International audience; Hevea brasiliensis is an important industrial crop for natural rubber production. Latex biosynthesis occurs in the cytoplasm of highly specialized latex cells and requires sucrose as the unique precursor. Ethylene stimulation of latex production results in high sugar flow from the surrounding cells of inner bark towards the latex cells. The aim of this work was to understand the role of seven sucrose transporters (HbSUTs) and one hexose transporter (HbHXT1) in this process. Two Hevea clones were used: PB217 and PB260, respectively described as high and low yielding clones. The expression pattern of these sugar transporters (HbSUTs and HbHXT1) was monitored under different physiological conditions and found to be maximal in latex cells. HbSUT1, one of the most abundant isoforms, displayed the greatest response to ethylene treatment. In clone PB217, ethylene treatment led to a higher accumulation of HbSUT1B in latex cells than in the inner bark tissues. Conversely, stronger expression of HbSUT1B was observed in inner bark tissues than in latex cells of PB260. A positive correlation with HbSUT1B transcript accumulation and increased latex production was further supported by its lower expression in latex cells of the virgin clone PB217.

Correspondance: sunderasan@lgm.gov.my. UMR DAP équipe DGB; International audience; Corynespora leaf disease poses a serious threat to rubber cultivation because infected leaves develop necrotic lesions and abscise, leaving the tree unproductive. The destructiveness of Corynespora cassiicola has been largely attributed to cassiicolin, a protein toxin secreted by the fungus. Recombinant antibody technology offers hope to curtail the disease whereby single-chain variable fragments (scFv) specific to cassiicolin could bind and deactivate the toxin in genetically modified rubber trees that harbour the antibody gene. A scFv phage library was constructed from heavy and light variable chains of IgG from cassiicolin immunized Balb/C mice spleen. Biopanning of the phage library yielded a scFv clone with high specificity to cassiicolin. The nucleotide sequence and deduced amino acid sequence information of the scFv were obtained. Hemagglutinin (HA)-tagged scFv expressed in Escherichia coli is discerned as a band at ca. 30 kDa on SDS-PAGE, and the corresponding band was detected by anti-HA IgG on a Western immunoblot. Deactivation of cassiicolin by the affinity-purified scFv was demonstrated in a detached-leaf bio-assay on selected susceptible Hevea clones (PB 260, RRIM 2020, RRIM 901 and RRIM 929). The assay was also performed on clones that are relatively more resistant to the fungus (RRIM 600 and GT-1), and the results were as expected. Thus, we have successfully demonstrated that the cassicolin-specific scFv can effectively reduce cassicolin toxicity

Hevein is involved in the coagulation of rubber particles and is known to be highly expressed in latex cells from Hevea brasiliensis. As all HEVEIN genes are highly conserved in their transcribed region, a promoter functional analysis was carried out in transgenic rubber tree in order to evaluate the expression of HEV2.1, one member of the HEVEIN multigenic family. Three out of the 14 established independent transgenic lines carrying the HEV2.1::GUS construct were selected and set to regenerate plantlets. In situ hybridization of transgenic and wild-type plant tissues was respectively carried out with gusA and HEVEIN antisense mRNA probes. This showed co-localized-expression of the HEV2.1 gene with the overall HEVEIN gene expression. Laticifer-specific expression was observed in roots and stems, but not in leaves where HEVEIN genes were expressed in all cell types. Since HEV2.1::GUS callus lines responded significantly to light stimulation, up-regulation of the expression of HEV2.1 genes by light was suggested in leaves.

Correspondance: valerie.pujade-renaud@cirad.fr; International audience; 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 beta-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

UMR DAP - Equipe DGB; International audience; Cassiicolin, a phytotoxin produced by the necrotrophic fungus Corynespora cassiicola, was purified to homogeneity from a rubber tree isolate. The optimized protocol involves reverse phase chromatography followed by size exclusion chromatography, with monitoring of the toxicity on detached rubber tree leaves. Cassiicolin appeared to be a peptide composed of 27 amino acids, glycosylated on the second residue, with a N-terminal pyroglutamic acid and 6 cysteines involved in disulfide bonds. Its molecular mass was estimated to be 2885 Da. No significant sequence homology with other proteins could be found. The availability of pure toxin in sufficient amount is a prerequisite for its structure determination, which is a key step in the understanding of the aggression mechanism

The cloning of hevein genes from Hevea brasiliensis was undertaken with the objective to isolate useful promoters to drive transgene expression in genetically engineered rubber tree. Four different full length genes were cloned by library screening and a fifth, a partial gene, by adaptor-anchored PCR. Sequence alignment revealed that hevein genes, although highly conserved in their transcribed region, diverged in two groups, with major differences in their promoter region, suggesting a more rapid evolution of the upstream regulatory functions of the genes than the downstream functions of their protein products. The promoter regions from two hevein genes representative of each group were isolated and analyzed in rice. Although both were functional, only the longest promoter sequence (PHev2.1) conferred a high level of expression to the transgene in various tissues of this heterologous host. It was in addition up-regulated by mechanical wounding and fungal infection in leaves. A number of potential cis-regulatory elements were identified in silico and are discussed in view of the expression profiles observed in rice.

The tapping panel dryness (TPD) syndrome of rubber is characterized by the reduction or ultimately total cessation of latex flow upon tapping, due to physiological disorders in the bark tissue. The protein pattern in the cytoplasm from healthy and TPD tree latex cells was compared by electrophoresis. Two polypeptides (P15 and P22) of 15 and 22 kDa, respectively, were found to accumulate in the cytosol of the TPD-affected trees, whereas a 29 kDa polypeptide (P29) appeared de novo. P15 and P22 were identified as REF (Hev b1) and SRPP (Hev b3), respectively, two proteins proposed to be involved in rubber biosynthesis. P29 appeared to be a new member of the patatinlike protein family. Specific molecular probes were designed for a detailed characterization of REF and SRPP gene expression and RFLP mapping. This allowed the demonstration that REF and SRPP display very similar expression profiles. They are highly over-expressed by the tapping-induced metabolic activation, although not by wounding per se, or ethylene or ABA. In addition to this similarity in gene expression, they were found to share one common locus in the genome. No significant difference in REF and SRPP gene expression was observed between healthy and TPD trees, indicating that their TPD-related accumulation in the cytosol was not transcriptionally regulated. Western blot analysis demonstrated that osmotic lysis of the sedimentable organelles (lutoids) in vitro caused the release of REF and SRPP from the rubber particle membrane into the cytosol. A mechanism of cellular delocalization as a consequence of the lutoids instability is proposed to explain REF and SRPP accumulation in the cytosol of TPD trees. (Resume d'auteur)

Under the effect of ethylene, there is a close parallel between increased latex production and the alkalinisation of laticiferous cytosol, which is parallel with activation of tonoplast ATPase and vacuole (lutoid) acidification. The alkalinisation of the cytosol by several tenths of a pH unit considerably enhances the activity of certain enzymes, including invertase, the key enzyme in glucidic catabolism. Ethylene increases the ATP content, the pool of adenine nucleotides and the myokinase balance shifts towards ATP synthesis. Increase in the proton motive force, related to that of the cytosol/vacuole pH gradient, enhances membrane transport including an H+/sucrose symport on laticiferous cell plasmalemma. The sink effect of the latificers for sucrose increases in proportion to over-production of latex. The polyribosome level in latex is increased and several enzymes are activated (post-translation or de novo synthesis?) Hevein transcripts analogous with certain agglutinins and heveamines with chitinase/lysozyme activity are more abundant after mechanical stress, addition of ABA or ethylene. Ethylene causes changes in calmodulin distribution between cytosol and organelles and an increase in protein phosphorylation. Repeated treatment with large amounts of Ethrel results in imbalance in latex between toxic oxygen production (O2·-, H2O2) and protective systems (superoxide dismutase, catalase, thiols, etc.); this is followed by in situ coagulation of latex and degeneration of the laticiferous system. The laticiferous system in Hevea is totally dependent on ethylene stress and forms an ad hoc model for understanding the mechanisms of the action of ethylene.

Tropical tree plantations are rapidly expanding to meet the growing demand for wood and nonwood forest products. Current research-combining ecophysiology, genetics and functional genomics-aims to enhance the climate change mitigation role of these plantations and their adaptation. The positive effect of the increase in atmospheric carbon dioxide concentration on photosynthesis could boost biomass production, but the higher water and nutrient needs will have to be fulfilled to ensure the sustainability of this positive effect. Multispecies plantations benefitting from positive interactions between species could help maintain production in tropical tree plantations against a background of increasing biotic and abiotic stress.

The rubber tree (Hevea brasiliensis) is the primary commercial source of natural rubber worldwide. In Asia and Africa, H. brasiliensis is affected by the Corynespora leaf fall (CLF) disease, caused by the broad-spectrum necrotrophic fungus Corynespora cassiicola. During severe attacks, massive fall of young leaves can occur in susceptible cultivars. Early evaluation of the susceptibility of rubber clones in breeding programs is required to avoid developing highly susceptible clones that would amplify the disease. An indirect phenotyping procedure envisaged consists in testing the sensitivity to the fungal toxins (or effectors) rather than the susceptibility to the fungus itself (toxin test). Among all putative effectors identified in silico, only cassiicolin Cas1 has been purified and characterized to date. This small secreted glycoprotein was for long suspected to play a role in the early phase of infection by inducing tissue necrosis. Strains carrying the Cas1 gene are the most aggressive on tested rubber clones. However, strains without cassiicolin gene (called Cas0) still show moderate aggressiveness, suggesting the existence of effectors other than cassiicolin. The objectives of this study are (i) to determine if susceptibility to cassiicolin Cas1 is a relevant selection criterion to eliminate the rubber clones most susceptible to CLF disease, and (ii) identify molecular factors involved in the sensitivity to Cas1, in rubber tree. We have thus analyzed the typology of a large set of C. cassiicola isolates collected from various rubber plantations in West Africa. Our results show that isolates carrying the cassiicolin isoform Cas1 are widely represented, but that the most represented type (A/Cas0) are isolates without cassiicolin gene. Here we show that deletion of the cassiicolin gene in the isolate CCP resulted in a total loss of virulence. This clearly demonstrated that cassiicolin is indeed a necrotrophic effector required for the virulence of isolate CCP in rubber tree. Finally, we have investigated susceptibility factors to cassiicolin Cas1 on rubber tree with two different approaches. We identified about thirty candidate proteins that could physically interact with the toxin, through the two-hybrid assay. A transcriptomic approach allowed us to identify the rubber genes differentially expressed in response to the purified cassiicolin, comparing a susceptible clone (PB260) and a tolerant clone (RRIM600). In conclusion, we think that the necrotrophic effector Cas1 can be an interesting tool for effector-based selection of tolerant clones for African plantations; however, efforts should also be placed on A/Cas0 isolates, in order to identify potential necrotrophic effector(s) responsible for their virulence. This would enlarge the potential of effector-based selection.; L'hévéa (Hevea brasiliensis) est la seule source de caoutchouc naturel commercialisé à travers le monde. En Afrique et en Asie, la maladie 'Corynespora Leaf Fall' (CLF), causée par le champignon nécrotrophe Corynespora cassiicola, affecte les plantations hévéicoles en provoquant des défoliations massives sur les clones les plus sensibles. L’évaluation précoce de la sensibilité des clones dans les programmes de sélection est un enjeu majeur pour écarter les individus les plus sensibles des programmes de développement et ainsi réduire la pression de la maladie. Une des méthodes d’évaluation envisagée consiste à tester indirectement la sensibilité des clones aux effecteurs fongiques responsables de la virulence (test toxinique). Parmi tous les effecteurs potentiels du champignon identifiés in silico, seule la cassiicoline Cas1 a été purifiée et caractérisée à ce jour. Il s’agit d’une petite glycoprotéine sécrétée qui jouerait un rôle dans les phases précoces de l’infection en induisant la nécrose des tissus. Les souches porteuses du gène Cas1 sont parmi les plus agressives sur les clones d'hévéa testés. Néanmoins, certaines souches de C. cassiicola ne produisant pas de cassiicoline présentent tout de même une agressivité modérée, suggérant l'implication d'autres effecteurs dans l'établissement de la maladie CLF. Les objectifs de cette thèse sont (i) de déterminer si la sensibilité à la cassiicoline Cas1 est un critère de sélection pertinent pour identifier les clones d’hévéa les plus sensibles à la maladie CLF, et (ii) d’identifier chez l’hévéa des facteurs de sensibilité à la cassiicoline Cas1. Nous avons d'abord analysé l’inoculum naturel et montré que les souches porteuses du gène Cas1 représentent un quart de la population de C. cassiicola dans les plantations d’hévéa d’Afrique de l’Ouest, le reste étant majoritairement constitué de souches dépourvues du gène codant la cassiicoline (Type A/Cas0). Nous avons ensuite créé un mutant de délétion du gène Cas1 pour la souche de référence CCP et comparé sa virulence à celle de la souche sauvage. Nous avons ainsi pu montrer que la cassiicoline Cas1 est bien un effecteur de nécrotrophie déterminant pour la virulence de C. cassiicola chez l’hévéa puisque la souche délétée perd toute virulence sur les clones testés. Enfin, nous avons recherché chez l'hévéa des facteurs de sensibilité à la cassiicoline Cas1 à travers deux approches. La technique de "double hybride en levures" nous a permis d'identifier une trentaine de protéines candidates qui pourraient interagir physiquement avec la toxine. Une approche transcriptomique, nous a permis d’identifier les gènes d’hévéa dont l’expression est modifiée suite à l’application de la cassiicoline purifiée, en comparant un clone sensible (PB260) et un clone tolérant (RRIM600). En conclusion, ces travaux ont permis de mieux comprendre les mécanismes impliqués dans l'interaction compatible entre C. cassiicola et l'hévéa et ouvrent la voie de la sélection assistée par effecteur.

Résumé : L'hévéa (Hevea brasiliensis) est la seule source de caoutchouc naturel commercialisé à travers le monde. En Afrique et en Asie, la maladie 'Corynespora Leaf Fall' (CLF), causée par le champignon nécrotrophe Corynespora cassiicola, affecte les plantations hévéicoles en provoquant des défoliations massives sur les clones les plus sensibles. L’évaluation précoce de la sensibilité des clones dans les programmes de sélection est un enjeu majeur pour écarter les individus les plus sensibles des programmes de développement et ainsi réduire la pression de la maladie. Une des méthodes d’évaluation envisagée consiste à tester indirectement la sensibilité des clones aux effecteurs fongiques responsables de la virulence (test toxinique). Parmi tous les effecteurs potentiels du champignon identifiés in silico, seule la cassiicoline Cas1 a été purifiée et caractérisée à ce jour. Il s’agit d’une petite glycoprotéine sécrétée qui jouerait un rôle dans les phases précoces de l’infection en induisant la nécrose des tissus. Les souches porteuses du gène Cas1 sont parmi les plus agressives sur les clones d'hévéa testés. Néanmoins, certaines souches de C. cassiicola ne produisant pas de cassiicoline présentent tout de même une agressivité modérée, suggérant l'implication d'autres effecteurs dans l'établissement de la maladie CLF. Les objectifs de cette thèse sont (i) de déterminer si la sensibilité à la cassiicoline Cas1 est un critère de sélection pertinent pour identifier les clones d’hévéa les plus sensibles à la maladie CLF, et (ii) d’identifier chez l’hévéa des facteurs de sensibilité à la cassiicoline Cas1. Nous avons d'abord analysé l’inoculum naturel et montré que les souches porteuses du gène Cas1 représentent un quart de la population de C. cassiicola dans les plantations d’hévéa d’Afrique de l’Ouest, le reste étant majoritairement constitué de souches dépourvues du gène codant la cassiicoline (Type A/Cas0). Nous avons ensuite créé un mutant de délétion du gène Cas1 pour la souche de référence CCP et comparé sa virulence à celle de la souche sauvage. Nous avons ainsi pu montrer que la cassiicoline Cas1 est bien un effecteur de nécrotrophie déterminant pour la virulence de C. cassiicola chez l’hévéa puisque la souche délétée perd toute virulence sur les clones testés. Enfin, nous avons recherché chez l'hévéa des facteurs de sensibilité à la cassiicoline Cas1 à travers deux approches. La technique de "double hybride en levures" nous a permis d'identifier une trentaine de protéines candidates qui pourraient interagir physiquement avec la toxine. Une approche transcriptomique, nous a permis d’identifier les gènes d’hévéa dont l’expression est modifiée suite à l’application de la cassiicoline purifiée, en comparant un clone sensible (PB260) et un clone tolérant (RRIM600). En conclusion, ces travaux ont permis de mieux comprendre les mécanismes impliqués dans l'interaction compatible entre C. cassiicola et l'hévéa et ouvrent la voie de la sélection assistée par effecteur

Hevea brasiliensis is the only commercial source of natural rubber. The CLF (Corynespora Leaf Fall) disease caused by the fungus Corynespora cassiicola is an important foliar pathology of the rubber tree in Asia and Africa. The disease progression is related with the development of new highly sensitive clonal varieties. A phenotyping method based on the measurement of electrolyte leakage in response to C. cassiicola exudates (culture filtrates or purified toxin) was developed and characterized. This test, without direct contact with the fungus itself, can be implemented without risk for plantations. After application of exudate samples on detached leaflets, conductivity is measured before (C1) and after (C2) autoclaving, and the percentage of electrolyte leakage %FE=100*C1/C2 is calculated. The influence of different factors such as leaf stage or the duration of incubation has been clarified. A positive correlation was found between the responses to the toxin test and to conidial inoculation. The pertinence of C1 correction by C2 was analyzed.Two toxin test studies have been carried out for a set of rubber clones, in France, with potted plants in greenhouse, and also in Ivory Coast, under the conditions normally considered for this test, with leaflets from budwood gardens. Repeatability between these two experiments was not perfect but nevertheless satisfying. The %FE character was found more sensitive and accurate than surface necrosis (SN) measurement. A significant variability in clone susceptibility and treatment virulence was observed, with clone x treatment interactions. The genetic determinism of sensitivity to C. cassiicola exudates was analyzed using the toxin test, by QTL detection on two populations of the biparental rubber family PB260 x RRIM600, in two field trials in Ivory Coast. A genetic map was previously built, using 308 SSR markers. Phenotyping of the two progeny populations revealed a polygenic determinism, without major monolocus effect. Over all the fungal exudates, 13 QTL distributed over 10 different linkage groups were identified, with percentages of explanation R2 varying from 10 to 20 %. On both sites, two QTL have been identified jointly for both the culture filtrate CCP and the toxin Cas1 purified from CCP. Positive correlations have been observed between some of the filtrates, with similar response profiles and common QTL.The toxin test could usefully replace the conidial inoculation method. However, the value of the test for predicting rubber clones susceptibility to the CLF disease in field conditions remains to be better characterized. Better understanding of the epidemiology of the H. brasiliensis x C. cassiicola pathosystem would contribute to the breeding objective, and more broadly to the design of control or avoidance methods.; Hevea brasiliensis est la seule source commerciale du caoutchouc naturel. La maladie CLF (Corynespora Leaf Fall) provoquée par le champignon Corynespora cassiicola est une pathologie foliaire de l’hévéa importante en Asie et en Afrique. Les épidémies sont liées au développement de nouvelles variétés clonales très sensibles. Une méthode conductimétrique de phénotypage de la réponse de l’hévéa aux exsudats de C. cassiicola (filtrats de culture ou toxine purifiée) a été développée et caractérisée. Ce test toxinique, sans contact direct avec le champignon lui-même, peut être mis en œuvre sans risque sur les plantations. Après application de gouttes d’exsudats sur des folioles détachées d’un génotype d’hévéa, deux mesures de conductivité C1 et C2 sont réalisées avant et après autoclavage, et le pourcentage de fuite d’électrolyte dû au traitement, %FE = 100*C1/C2, est calculé. L’influence de différents facteurs tels que le stade foliaire et la durée d’incubation, a été précisée. Une corrélation positive a été trouvée entre les réponses au test toxinique et à l’inoculation par des conidies du champignon. La pertinence de la correction de C1 par C2 a été analysée.Deux études du test toxinique ont été réalisées pour une série de clones d’hévéa, d’une part en France avec des plantes en pots cultivées en serre, et d’autre part en Côte d’Ivoire, dans les conditions normalement envisagées pour ce test, avec des folioles provenant de jardins à bois de greffe. La répétabilité entre ces deux études est imparfaite mais néanmoins satisfaisante. Le critère %FE est apparu plus sensible et plus précis que la mesure de la surface de nécrose induite (SN). Une variabilité importante de la sensibilité des clones et de l’agressivité des exsudats a été observée, avec l’existence d’interactions. L’analyse du déterminisme génétique de la réponse au test toxinique a été menée par détection de QTL sur deux populations de la famille biparentale d’hévéa PB260 x RRIM600, analysées sur deux sites en Côte d’Ivoire. Une carte génétique a été construite au préalable avec 308 marqueurs SSR. Le phénotypage des deux populations a révélé un déterminisme polygénique, sans effet monolocus majeur, avec, pour l’ensemble des exsudats fongiques étudiés, 13 QTL répartis sur 10 groupes de liaison et présentant des pourcentages d’explication R2 variant de 10 à 20 %. Sur les deux sites, deux QTL ont été identifiés conjointement pour le filtrat CCP et pour la toxine Cas1 purifiée à partir du filtrat CCP, démontrant l’importance de Cas1 pour la toxicité de ce filtrat. Des corrélations positives entre certains filtrats ont été observées, avec des profils de réponse similaires et des QTL communsLe test toxinique pourrait remplacer avantageusement la méthode d’inoculation conidienne. Cependant, la valeur prédictive du test pour la sensibilité des clones d’hévéa à la maladie CLF en plantation reste à caractériser. Une meilleure connaissance de l’épidémiologie du pathosystème H. brasiliensis x C. cassiicola contribuerait à l’objectif de sélection, et plus largement à la conception de méthodes de lutte ou d’évitement.

Rubber tree (Hevea brasiliensis) is the only source of commercial natural rubber. The “Corynespora Leaf Fall” (CLF) disease, caused by the fungus Corynespora cassiicola, has become over the last 50 years a very serious problem in the Asian and African rubber producing countries. Currently, the main methods to face the problem are the uprooting of the most susceptible cultivars and chemical treatments in case of severe outbreaks. However, the number of cultivars affected by the disease keeps increasing, and the selection of new cultivars, both tolerant and high yielding, is urgent. In this study, the gene encoding cassiicolin, a toxic glycosylated protein secreted by C. cassiicola, was characterized and its diversity analyzed. The analysis of three C. cassiicola isolates with contrasted levels of aggressiveness revealed the presence of identical cassiicolin genes in the highly and moderately aggressive isolates but none in the isolate of mild aggressiveness. The levels of aggressiveness were correlated to the cassiicolin gene transcript levels. The cassiicolin gene was preferentially expressed in the early phase of the infection. Analysis of the cassiicolin gene diversity among isolates from various host and various geographical origins revealed the existence of at least six protein isoforms (Cas1 to Cas6). The genetic structure of the isolates based on neutral markers was closely related to the genetic structure based on the cassiicolin gene. The isolates sampled on rubber tree were grouped in several specialized clades, including one clade regrouping all the Cas1 isolates, which were the most aggressive on rubber tree. However, 58 % of the isolates seemed to be deprived of cassiicolin gene, although some of them generate moderate symptoms on rubber tree, suggesting the existence of other effectors. Endophytic C. cassiicola isolates were found in asymptomatic rubber tree leaves from Brazil, a region were CLF outbreak was never reported. No expression of the cassiicolin genes carried by these isolates could be detected in interaction with rubber tree. In addition, we found that the Trichoderma and Xylaria species, all fungal endophytes of rubber tree, were antagonists of C. cassiicola, in vitro. This work opens new perspectives for the control of CLF (through early diagnosis, selection of tolerant clones, or biocontrol)., L’hévéa (Hevea brasiliensis) est actuellement la seule source commerciale de caoutchouc naturel. Parmi les maladies affectant l’hévéa, la CLF (« Corynespora Leaf Fall ») causée par le champignon Corynespora cassiicola, est devenue en une cinquantaine d’années un fléau pour l’ensemble des pays hévéicoles d’Asie et d’Afrique. Actuellement, la gestion du problème consiste à arracher les clones les plus sensibles et à traiter les arbres avec des fongicides en cas d’épidémie. Cependant, le nombre de clones touchés par la maladie ne cessant d’augmenter, il devient urgent de sélectionner de nouveaux clones à la fois tolérants et aptes à la production. Nos travaux ont permis de caractériser le gène codant la cassiicoline, toxine protéique glycosylée secrétée par C. cassiicola, et d’analyser sa diversité. Une étude comparative portant sur trois isolats de C. cassiicola d’agressivité contrastée a montré la présence du gène Cas1 chez les isolats de forte et moyenne agressivité, alors qu’il n’est pas détecté chez l’isolat de faible agressivité. Les niveaux d’agressivité des isolats sont corrélés aux niveaux de transcrits du gène de cassiicoline. Le rôle de la cassiicoline serait prépondérant dans les phases précoces de l’infection. L’analyse de diversité du gène de cassiicoline à partir d’une collection d’isolats provenant de différents hôtes et d’origines géographiques variées, a révélé l’existence d’au moins six isoformes protéiques (Cas1 à Cas6). La structuration génétique globale des isolats basée sur des marqueurs neutres est similaire à la structuration basée sur le gène de cassiicoline. Les isolats prélevés sur hévéa se regroupent en clades spécialisés, dont un correspondant aux isolats porteurs du gène Cas1, identifiés comme étant les plus agressifs sur hévéa. Cependant, 58 % des isolats testés semblent dépourvus de gène de cassiicoline, bien que certains génèrent des symptômes modérés sur hévéa, ce qui suggère l’existence d’autres effecteurs. Des formes endophytiques de C. cassiicola ont été isolées à partir de feuilles asymptomatiques provenant du Brésil, zone encore indemne de CLF. Les gènes de cassiicoline portés par ces souches (isoformes Cas3 et 4) ne semblent pas exprimés lors de l’interaction avec l’hévéa. Nous avons montré par ailleurs que les champignons endophytes de l’hévéa appartenant aux genres Trichoderma et Xylaria présentent une forte activité mycoparasitaire sur C. cassiicola, in vitro. Ces travaux ouvrent de nouvelles perspectives pour le contrôle de la maladie (diagnostic précoce, sélection de clones tolérants, lutte biologique).