| In order to improve the control efficiency of biocontrol strain MT-06and reduce the usage dose of pesticide and keep control effect or even be strengthened, we mixed the biocontrol strain with several fungicides.Firstly, the selective toxicity to MT-06of six chemical fungicides was evaluated by testing their growth inhibition rate to MT-06on Petri dish. Results showed that thiophanate methyl, iprodione and carbendazim had strong inhibitory effect on MT-06, so these three kinds of fungicides were abandoned. Toxicity of chlorothalonil, metalaxyl mancozeb and mancozeb to the indicating strains were determinated. The linear regression equations and EC50were obtained, the toxicity selective ratios (TSR) of various fungicides to these pathogens were calculated. The results showed that the EC50values of mancozeb against biocontrol agent MT-06is0.0595mg/ml, and Alternaria solani, Rhizoctonia solani and Didymella biyoniae were0.01295mg/ml,0.01336mg/ml and0.026mg/ml. Those TSR values were4.5946,4.4536and2.2885, respectively, ranged from1to10which meant positive regulation. The TSR values of chlorothalonil against R. solani and D. bryoniae were12.9743and18.2633, with a moderate positive regulation. The TSR values of metalaxyl mancozeb agaist A. solani, Helminthosporium zizaniae, R. solani and D. bryoniae were13.9277,13.9277,2.89and2.4596, respectively. These R values of three kinds of fungicides were0.91,0.95and0.85, respectively, which meant that above data with high reliability.For the detection of toxicity determination, The pot experiment and field tset were carried out by using1×10conidia/ml MT-06mixed with chemical fungicide half usual dose or less than the positive control to verify the toxicity determination. Firstly, the protective control efficiency of MT-06mixed with chemical fungicide on A. solani in pot experiment was better than their curative control efficiency. The treatment of0.25mg/ml metalaxyl mancozeb mixed with1×106conidia/ml MT-06got the best protective control efficiency, reached95.45%, higher than that of positive control of2mg/ml metalaxyl mancozeb, but was not significant difference. The concentration0.5mg/ml of mancozeb mixed with1×106conidia/ml MT-06achieved highest protective control efficiency of mancozeb treatments, was90.83%. In addition, the protective control efficiency of1mg/ml mancozeb mixed with1×106conidia/ml MT-06is86.54%, higher than the positive control. All of the mixture treatments have significantly higher control efficiency than the treatment of only1×106conidia/ml MT-06. The evaluation of the curative control efficiency of MT-06mixed with fungicides on A. solani showed the treatment of2mg/ml mancozeb had the highest curative control efficiency, any formulations of MT-06mixed with mancozeb have failed to achieve better control effect than only fungicide. However, the curative control efficiency of the mixture of0.5mg/ml metalaxyl mancozeb and1×106conidia/ml MT-06, reached73.31%, higher than the positive control. The curative control efficiency of mixture of mancozeb and MT-06spores was significantly lower than that of mancozeb fungicide positive control.(2) The protective control efficiency of MT-06mixed with fungicides on D. bryoniae showed the treatments1×106conidia/ml MT-06mixed with0.25mg/ml or0.5mg/ml mancozeb got higher control efficiency than the positive control, were75.40%and71.91%. As the same of protective experiment, curative control efficiency of the treatments of0.25mg/ml or0.5mg/ml mancozeb mixed with1×106conidia/ml MT-06were higher than the positive control. The results of the mixture of MT-06and metalaxyl mancozeb to control wheat head blight in the field test showed the control efficiency of MT-06mixed with metalaxyl mancozeb was higher compared with the use of metalaxyl mancozeb alone, the best is the treatment10times diluted liquid including MT-06hyphal with conidia, reached74.70%, better than the positive control of4mg/ml metalaxyl mancozeb,71.79%. All of the mixture treatments have significantly higher efficiency than the1×106conidia/ml MT-06alone, which demonstrated the promising future of the strain MT-06as a biocontrol agent.To investigate the biocontrol mechanism of MT-06, degenerate PCR primers were designed according to the conserved amino acid sequence of several filamentous fungus PMK1class homologueto amplify the MAPK internal DNA fragment from MT-06. Using Thermal Asymmetric Interlaced-PCR (TAIL-PCR) and RT-PCR the complete TMK1DNA and cDNA sequences were obtained. The total length of TMK1was2145bp, contained3introns and encoded a355amino acid protein. The deduced amino acid sequence showed highly homology to M.grisea PMK1, Colletotrichum lagenarium CMK1, Botryotinia fuckeliana BMP1, Gaeumannomyces graminis GMK1and Pyrenophora teres PTK1. Then a complement vector containing the complete cDNA of TMK1was constructed and introduced into△PMK1mutant of M. oryzae. Six randomized transformants were selected and analyzed.The result showed that TMK1partially recovered the ability of appressorium formation and infection of△PMK1on barley leaves. These data suggested that TMK1is a functional homologue PMK1gene. |
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