First characterization of endophytic Corynespora cassiicola isolates with variant cassiicolin genes recovered from rubber trees in Brazil Marine Deon, Ana Xavier Scomparin, Aude Tixier, Carlos R. R. Mattos, Thierry Leroy, Marc Seguin, Patricia Roeckel-Drevet, Valérie Pujade-Renaud To cite this version: Marine Deon, Ana Xavier Scomparin, Aude Tixier, Carlos R. R. Mattos, Thierry Leroy, et al.. First characterization of endophytic Corynespora cassiicola isolates with variant cassiicolin genes recovered from rubber trees in Brazil. Fungal Diversity, Springer, 2012, 54 (1), pp.87-99. 10.1007/s13225-012- 0169-6. hal-00964682 HAL Id: hal-00964682 https://hal.archives-ouvertes.fr/hal-00964682 Submitted on 29 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License Fungal Diversity (2012) 54:87–99 DOI 10.1007/s13225-012-0169-6 First characterization of endophytic Corynespora cassiicola isolates with variant cassiicolin genes recovered from rubber trees in Brazil Marine Déon & Ana Scomparin & Aude Tixier & Carlos R. R. Mattos & Thierry Leroy & Marc Seguin & Patricia Roeckel-Drevet & Valérie Pujade-Renaud Received: 28 December 2011 /Accepted: 30 March 2012 /Published online: 27 April 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Corynespora cassiicola is the causal agent of Three of four isolates were able to induce symptoms on the Corynespora Leaf Fall (CLF) disease. CLF is one of the cultivar they were isolated from in a detached leaf assay, most important fungal diseases of rubber trees in Asia and with different kinetics and intensities. One isolate had the Africa but disease outbreaks have not been reported in same pathogenicity profile as the pathogenic isolate CCP; South America. Cassiicolin, a small cysteine-rich glycopro- the other two isolates developed symptoms late during the tein secreted by the pathogenic C. cassiicola isolate CCP, course of infection, suggesting saprotrophic capabilities. was previously identified as a potential disease effector in However, no Cas3 or Cas4 transcripts could be detected rubber tree. Recently, the cassiicolin-encoding gene (Cas1) upon inoculation with the endophytic isolates, whereas the was characterized and shown to be expressed in the early reference gene Cas1 was expressed upon inoculation with phase of infection. In this study, we investigated whether the CCP isolate. This work demonstrated that C. cassiicola previously undetected strains of C. cassiicola are present in is present in South America in an endophytic form and that South American rubber plantations by examining the fungal it may evolve from an endophytic to a saprophytic or even endophyte population found in asymptomatic rubber tree potentially pathogenic life style. leaves. Four isolates were identified as C. cassiicola. Genes encoding new forms of the cassiicolin precursor Keywords Corynespora cassiicola . Rubber tree . protein (Cas3 and Cas4) were identified from these isolates. Endophyte . Brazil . Cassiicolin Electronic supplementary material The online version of this article Introduction (doi:10.1007/s13225-012-0169-6) contains supplementary material, which is available to authorized users. Corynespora cassiicola (Berk & M. A. Curtis) C.T. Wei is : M. Déon V. Pujade-Renaud (*) an anamorphic Ascomycota fungus belonging to the CIRAD, UMR AGAP, Dothideomycetes and forming a separate phylogenetic clade 63000 Clermont-Ferrand, France among the Pleosporaceae with Corynespora smithii e-mail: [email protected] (Schoch et al. 2009). It has been found on leaves, stems, : : : M. Déon A. Scomparin A. Tixier P. Roeckel-Drevet fruits and roots of more than 300 plant species primarily Clermont Université, Université Blaise Pascal, UMR 547 PIAF, in tropical and subtropical areas (http://nt.ars-grin.gov/ 63000 Clermont-Ferrand, France fungaldatabases/;FarrandRossman2011). Principally C. R. R. Mattos described as a pathogen, it causes severe damage to econom- Plantações Michelin de Bahia, ically important plants, including rubber tree, tomato, cucum- Box 02, 45435-000 Ituberá, Bahia, Brazil ber, cotton and soybean (Chee 1990;Koenningetal.2006; Oliveira et al. 2006, 2007;Schlubetal.2009). However, C. T. Leroy : M. Seguin CIRAD, UMR AGAP, cassiicola isolates were also obtained from dead organic ma- 34398 Montpellier, France terial (Kingsland 1985; Lee et al. 2004; Cai et al. 2006)and 88 Fungal Diversity (2012) 54:87–99 asymptomatic tissues (Collado et al. 1999; Suryanarayanan et containing a signal peptide at the amino terminus that is al. 2002;Gondetal.2007; Promputtha et al. 2007), and were predicted to target the protein for secretion (Déon et al. therefore designated as saprophytes and endophytes, 2012). This gene is expressed one or two days post- respectively. inoculation, suggesting a role in the early phase of infection. In the rubber tree, C. cassiicola has thus far been exclu- Only a single report mentions CLF symptoms on Hevea sively known as a necrotrophic pathogen that causes the brasiliensis growing in the American continent (Junqueira Corynespora Leaf Fall (CLF) disease, which ranks among et al. 1985). In this area, C. cassiicola remains benign on the most important fungal diseases in Asian and African rubber trees but causes significant damage to many other rubber plantations. Initially, C. cassiicola was described as a plant species. Could outbreaks of CLF disease occur in minor pathogen capable of attacking only budwood or seed- South American rubber plantations? To answer this ques- ling nursery plants (Newsam 1960; Chee 1988), but in 1975, tion, we investigated whether previously undetected strains the first epidemic outbreak on a plantation scale occurred in of the pathogen were present in rubber plantations in this Indonesia. In the 1980s, several other countries in Southeast area. The purpose of our study was to test for the presence of Asia were severely affected by disease outbreaks and C. cassiicola among fungal rubber tree endophytes from a thousandsofhectaresofrubbertreeswereuprootedin plantation in Brazil that had no history of the disease and to Malaysia, Indonesia, Thailand and Sri Lanka (Liyanage et characterize these isolates. al. 1986; Pongthep 1987; Chee 1988). By the end of the 1980s, African countries were also affected by CLF. The disease severity further increased until several important Material and methods rubber tree cultivars considered to be tolerant or resistant to CLF during the first epidemic in the mid 1980s suc- Plant material cumbed to the disease (Jayasinghe and Silva 1996; Shamsul and Shamsuri 1996; Sinulingga et al. 1996; Fungal endophytes were recovered from young Hevea bra- Wahounou et al. 1996). Currently, all Asian and African siliensis trees in nurseries consisting of 10 different cultivars rubber-producing countries, which account for 98 % of the (CDC 312, CDC 1174, FDR 5240, FDR 5665, FDR 5788, natural rubber production in the world (94 and 4 % for each GT 1, MDF 180, PB 260, PMB 1 and RRIM 600) from a continent, respectively), are affected by the disease resulting rubber tree plantation in Bahia, Brazil. The plants used for in considerable economic losses. the inoculation and gene expression experiments (cultivars CLF is characterized by necrotic lesions that develop on RRIM 600 and FDR 5788) were cultivated in a greenhouse both young and mature leaves and lead to extensive defolia- in Clermont-Ferrand (France) at 28 °C±2 °C with 80 % tion. The fungus typically causes areas of necrosis with a fish relative humidity. All of the cultivars were grafted clones. bone appearance due to the darkening of the veins adjacent to the lesions (Chee 1988; Liyanage and Liyanage 1986; Isolation of endophytic fungi from asymptomatic Brazilian Pongthep 1987). However, the symptoms vary depending on rubber tree leaves the age, type and location of the rubber tree (Jayasinghe et al. 1998). This symptom variability impedes diagnosis of the Fungal endophytes were isolated from asymptomatic mature disease in a plantation. Additionally, C. cassiicola isolates leaves that were collected in the nurseries and kept at room within the same agroclimatic zone vary widely in mor- temperature for 8 days. Leaf segments were surface- phology, colony color, growth, spore production, pathoge- sterilized through sequential immersion in 70 % ethanol nicity and genetic diversity (Darmono et al. 1996; (1 min), 2 % sodium hypochlorite solution (2 min), 70 % Jayasinghe and Silva 1996;Bretonetal.2000;Atanand (v/v) ethanol (30 s) and sterile water. Leaf pieces with Hamid 2003; Romruensukharom et al. 2005; Dixon et al. freshly cut edges were plated on Malt Extract Agar (MEA) 2009;Qietal.2009). supplemented with 0.02 % chloramphenicol and placed at Colonization of the rubber tree tissues by C. cassiicola 25 °C in the dark. The emergent fungi were isolated by involves the secretion of phytotoxic molecules (Onesirosan successive subcultures. et al. 1975; Liyanage and Liyanage 1986; Purwantara