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Mycoparasitic role of extracellular chitinase in Trichoderma virens
Quantitative competitive polymerase chain reaction (QC-PCR) was developed for specific detection and enumeration of genetically modified strains of Trichoderma virens in the soil environment. The PCR technique was much more sensitive than the traditional dilution plating method especially for the detection of fungal spores. An efficient procedure for extraction of DNA from nonsterile soil was determined. The procedure, a combination of SDS-heating and bead-beating, resulted in a 85-95% relative efficiency of recovery of T.virens DNA from soil samples. This QCPCR technique was also used to study the interaction between two strains of T. virens in which a chitinase gene was either disrupted or overexpressed. A homologous transformation system was developed for T. virens based on complementation of an arginine auxotrophic strain. The mutation was found in the arg2 gene encoding the small subunit of carbamoyl phosphate synthetase. The wild-type arg2 gene was cloned and used as a selectable marker in a transformation system that yielded as many as 370 transformants per mg of DNA. The predicted amino acid sequence of the arg2 gene showed 56 - 82% identity with homologous polypeptides of Neurospora crassa, Magnaporthe grisea and yeast. A 42-kDa extracellular chitinase was found to be the major source of endochitinase activity when T. virens was cultured in a fungal cell wall medium. The role of this extracellular chitinase in the biocontrol activity and rhizosphere competence of T. virens was determined. The gene encoding the 42-kDa extracellular chitinase was cloned from T. virens and disrupted (KO) or constitutively overexpressed (COE) in the fungus through genetic transformation. KO and COE transformants were selected for similarity to the wildtype strain in growth rate, sporulation and antibiotic production. These strains demonstrated similar colonization efficiency on cotton roots and growth/survival in nonsterile soil amended with different carbon sources. However, KO and COE strains showed decreased and enhanced biocontrol activity, respectively against result suggests that the 42-kDa extracellular chitinase is an important component of the mycoparasitic ability of T. virens.
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Includes bibliographical references: pages 105-119.