GFP-labeling And Biocontrol Analysis Of Chaetomium Globosum And Trichoderma Viride

Both Chaetomium globosum and Trichoderma viride are important biocontrol fungi. Despite of increasing application, the biocontrol mechanism still leaves a lot to be studied. Investigation of their biocontrol characteristics can not only be beneficial to deeper research into the biocontrol mechanism, but also to the control of relative fungal diseases and further study of other biocontrol agents. Construction of reporter gene-labeled transformants is an effective pathway to accomplish this objective.This paper optimized preparation and regeneration of C. globosum. Types of lysing enzymes, enzymatic digesting temperature and duration, kinds and concentrations of osmotic stabilizers, age of mycelia and lysing buffer were examined for protoplast liberation, while types of regeneration media, kinds and concentrations of osmotic stabilizers both in lysing solution and in regeneration media, enzymatic digesting duration were tested for protoplast regeneration. Through enumeration of protoplasts released, examination of viability and measurement of regeneration frequency, the optimal protocol was determined as follows: 10~8 spores of C. globosum were shaken in 250ml CM broth at 25 ℃, 150rpm for 2-2.5d, then the mycelia collected were digested in 0.6M KCl, phosphate buffer (pH=6.0), with 1% lywallzyme and 1.5% snailase as lysing enzyme, at 30℃, 100rpm for 2h.The resulted protoplasts were washed in 0.6M KCl for three times and then regenerated in OCM with 0.76M sucrose as osmotic stabilizer. The quantity and quality of protoplasts produced satisfied the demand of protoplast-mediated transformation, with a yield of 10~8 /g mycelia (wet weight), a viability of 98.15% and a regeneration frequency of 62.93%.Several fluorescence-expression vectors were constructed. Recombinant vector pBES and promoter-trapping vector pES were transformed into the protoplasts of C. globosum, while recombinant vector pCSN43-hph-EYFP was transformed into those of T. viride, both resulted in fluorescence-labeling transformants. After several rounds of screening and verification, including screening with selective plates, PCRamplification of targeted fragment, Southern blotting and examination with fluoroscope, it was definite that EGFPIEYFP had been inserted and expressed in the genome of C. globosum and T. viride in several transformants. One fluorescing transformant of each fungus was randomly chosen to do biocontrol analysis. The mechanism of antagonism between C. globosum or T. viride and pathogenic fungi was preliminarily studied in the way of co-culture on plate and confrontation on slide. It seems the main suppression of pathogens by T. viride is resulted from the competition for nutrition and growth space and mycoparasitism. When it comes to C. globosum, the suppression is previously reported to be result of antibiotic substances and competition. However, in this study, it is found that due to the moderate rate of growth, C. globosum has no distinct advantage over pathogens through competition, and it has apparent process of mycoparasitism during the confrontation with R. solani. A binary vector with SUR gene as selecting label was constructed and the conditions suitable for Agrobacterium tumefaciens-mediated transformation (ATMT) of C. globosum were optimized, resulting in a transformation frequency as high as 72 transformants /105 ascospores. Moreover, the protocol for extraction fungal genomic DNA from soil samples and its subsequent PCR amplification was also investigated, which might lay a basis for further molecular ecological study on the effect of biocontrol agent on pathogenic fungi.