Characterisation and pathogenicity of Neofusicoccum ribis and gene expression study of pathogenesis-related genes in rubber (Hevea brasiliensis Muell. Arg)

Rubber tree (Hevea brasiliensis) is well known as the tree of life for its by-products such as latex and wood. However, the growth and performances of H. brasiliensis are reduced substantially by Fusicoccum Leaf Blight disease, a re- emerging malady first detecte in Malaysia in 1987. This study provides a foundation for understanding the infection of healthy rubber plant tissues by Fusicoccum spp. in selected regions in Malaysia, with emphasis on pathogenicity and cross pathogenicity evaluation of associated hosts. The study provides a leaf colonisation pattern since the resistance mechanism provided by pathogenesis-related proteins (PR) and phenylalanine ammonia lyase (PAL) enzymes, both induced in infected rubber, are fairly well studied. Fungal isolates were obtained from leaf samples collected in several rubber plantations in Selangor, Perak and Johor in 2010. All isolates were identified based on cultural characteristics such as conidial production in various cultural media (PDA, MEA, CDA, CMA) and sequence data generated from the internal transcribed spacers (ITS1 and ITS2), the 5.8S rRNA gene and an unknown locus (BotF15) containing microsatellite repeats. Pathogenicity tests were conducted using representative isolates from three locations (SB30, SK10 and SJ20) for confirmation by detached leaf method and seedlings in glasshouse, and cross pathogenicity tests on detached leaves. The roles of β-1, 3-glucanase and chitinase, the two most widely studied groups of pathogenesis-related (PR) proteins (PR3 and PR2) and phenylalanine ammonia lyase (PAL) enzymes present during pathogenesis were investigated. Thirty three isolates were derived from this study. In different culture media and pH tested, significant differences were observed in spore production, dimensions of the conidia and growth rates among isolates. On PDA, pH 6 supported the best growth compared to others and optimum temperatures of growth were between 25 oC to 30 oC. The growth rate was 21.2 mm/day on PDA, 17.9 mm/day on CDA, 17.1 mm /day on MEA and 14.2 mm/day on CMA. The highest spore production of 161.33 x 10 6 spores / ml was obtained on PDA. The optimum temperature for spore germination in distilled water was 25 oC – 35 oC within a period of twenty- four hours. The fungus colony color was white to grey; dark greenish grey or greyish. Conidia were either septate or aseptate, hyaline when young and turning brown with age, oval, elliptic to ovaloid or subglobose in shape. Conidia size ranged 9.0-25.8 μm in length and 1.9-8.5 μm width with the L: B ratio of 3.4. Molecular characterisation revealed fungal sequences generated from ITS and BotF15 genes, indicating therefore that they formed a supported clade with Neofusicoccum. ribis. Tested isolates (SB30, SK10 and SJ20) were pathogenic to H.brasiliensis but varied in symptoms. Pathogenicity tests showed high susceptibility of percentage of disease severity on detached rubber leaves from clones RRIM 2024 and PB 350 (39 - 56%) and low susceptibility on clones RRIM 2002 and RRIM 2007 (6.6 - 18.8%). Some alternate hosts such as mango and guava exhibited high susceptibility (50 - 80%) while others (rambutan and papaya) were low (3 - 10%). Induction of pathogenesis–related genes in rubber leaves in the presence of fungal pathogen showed that patterns of expressions of these enzymes were up-regulated in both tolerant (RRIM 2002) and susceptible (PB 350) rubber clones over time although their expression was much higher in the latter. Generally, pathogenesisrelated proteins from H. brasiliensis could be used as molecular probes to monitor defence-response activation while the mechanism in N. ribis isolate SK10 could serve as an inducer of systemic resistance in rubber.

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