The physiological response of the white-rot fungus, Schizophyllum commune to Trichoderma viride, during interspecific mycelial combat

摘要

Fungal species compete for space and nutrients in organic matter, resulting in strong morphological and biochemical reactions in the interacting mycelia. Interspecific mycelial interactions have attracted extensive studies because of their potential applications in biological control, bio-pulping, screening for novel bioactive metabolites and enhancement of extracellular enzyme production. Studies of interspecific mycelial combat have also contributed to the understanding of the structure and development of fungal communities. Although the behaviour of interacting mycelia has been understood, mainly at the morphological level, the biochemical aspects have yet to be fully elucidated. The main aim of this study was to endeavour to understand the underlying cellular and molecular response patterns and adaptations of the white-rot fungus, Schizophyllum commune to a highly antagonistic strain of Trichoderma viride, by correlating the expression patterns of metabolites, proteins and selected genes of Schizophyllum commune in response to the antagonist. The study also investigated the implication of oxidative damage in these response patterns. Microscopic examination of stained and unstained mycelia of S. commune confronted by the mycelia of T. viride, revealed cell wall lysis, protoplasmic degeneration, hyphal expansion and subsequent hyphal disintegration, hence, cell death in the mycelia of S. commune, after 7 days of mycelial contact. Metabolite patterns of both species near the interaction zone were profiled by HPLC and GC/MS, in comparison to their self-paired mycelia. Sugar alcohols, phenolic compounds and organic acids were up-regulated in the interacting mycelia of both species, while �-aminobutyric acid, myo-inositol phosphate, pyridoxine and N-acetylglucosamine, were up-regulated in S. commune mycelia with a concurrent decrease in the levels of fatty acids detected in the latter. Expression patterns of selected genes of S. commune confronted by T. viride were investigated by RT-PCR, relative to patterns in its self-paired cultures. Genes encoding proteins involved in the synthesis of cell wall polymers, protein synthesis and protein quality control, signalling, and stress response were up-regulated. On the other hand, genes that code for proteins associated with glycolysis, nitrogen assimilation, membrane transport, mitochondrial ATP-synthetic machinery, and cellular multiplication/growth were down-regulated. Changes in protein expression were profiled in the mycelia of both species paired against each other using 2- Dimensional gel electrophoresis, and differentially expressed proteins were identified by MALDI-TOF-MS/MS, following peptide fragmentation. Proteins involved in protein synthesis and assembly, unfolded protein response, response to cellular injury, synthesis of phenolic compounds, recycling of carbon and nitrogen were up-regulated in the confronted mycelial domain of S. commune. Proteins involved in glycolysis and heat shock response were predominantly down-regulated in the mycelia of S. commune paired against T. viride. Proteins associated with antagonism, cellular metabolism, glycolysis, and ATP generation and protein synthesis were up-regulated in the mycelia of T. viride interacting with S. commune with a decline in the detected levels of proteins involved in cytoskeleton organisation. Biochemical assays revealed increases in the activity levels of antioxidant enzymes, superoxide dismutase, catalase, succinic semialdehyde dehydrogenase, glucose-6-phosphate dehydrogenase and in the levels of indicators of oxidative stress and secondary metabolism, such as lipid peroxidation, protein carbonylation, superoxide anion and phenolic levels in the mycelia of S. commune paired against T. viride. Similarly, the activities and protein levels of phenol-oxidising enzymes, namely laccase and manganese peroxidase increased in the confronted mycelial domain of S. commune. Chitinase activity increased in mixed liquid cultures of both fungi. Protein, and gene expression patterns, in the confronted mycelia of S. commune suggest an increase in the flux through the protein synthetic machinery, possibly resulting in endoplasmic reticulum stress, which may have activated the unfolded protein response. These are strong indications of oxidative stress induction and switch of mycelial growth to secondary metabolism. There was little evidence of antagonism by S. commune towards T. viride, suggesting that the patterns reported herein, may be a response rather than an attack mechanism towards the latter.