Agrobacterium Tumefaciens-mediated Transformation Of Trichoderma Viride And Correlations Between Mycoparasitism And Biological Control

Agrobacterium tumefaciens has the ability to transfer its T-DNA (transferred DNA) to the chromosomal DNA of fungi and this DNA can be integrated into recipient genome. In this study, the transformations of the biocontrol fungus Trichoderma viride LTR-2 were obtained by Agrobacterium tumefaciens-mediated transformation(ATMT)using the hygromycin B phosphotransferase (hph) gene as a selective marker. This method is characteristiced of simplicity and efficiency of transformation. The efficiency is up to 200-300 transformants per 10⁷ conidia. The transformation protocol was optimized to generate a library of insertion mutants of Trichoderma viride LTR-2. Randomly selected transformants remained mitotically stable, maintaining their hygromycin B resistance as tested by subculturing on selective and nonselective media alternately. The presence of hph in the chromosomal DNA of the transformants was confirmed by PCR. Southern blotting analysis showed that T-DNA was randomly integrated into the genome of recipient strains, and more than 90% of transformants tested contained one copy of T-DNA. About 400 mutants were obtained by this method, the mutation were mainly tested as below. (1) colony morphology: there was about 2% of colony of mutants changed greatly, including growth rate, conidia color and so on. Others change could not be seen from phenotype. (2) Inhibitory ability: mutants and wild-type strain were dually cultured with Rhizoctonia solani. Inhibitory ability of 28% of mutants declined and 56% of them increased compared with that of wild-type strain. (3) mycoparasitism ability: 36.2% of mutants declined, especially T4-31 and T4-59, and 51% became stronger in the mycoparasitism ability. This showed that ATMT method could make the wild-type strain mutate randomly and could be considered a promising tool for efficient insertional mutagenesis studies of biocontrol fungus T.viride. T₂-58, T₂-60, T₁-25, T₄-31 and T₄-59 were selected to test their biological control on Rhizoctonia cerealis of wheat. The control effects of T₄-31 and T₄-59, whose mycoparasitism ability almost lose, descended by 7-13%. On the contrary, that of T₂-58 and T₂-60 increased by 10-12%, which had a strong mycoparasitism ability. This conclusion showed that the mycoparasitism ability of T.viride had a compact correlation with their biological control.