Breeding Of Trichoderma Harzianum Carbendazim Resistance And The Study Of β-tubulin Mutant Site

In the current agricultue production, chemical fungicide still plays an important role incontrolling of plant fungal diseases. Plant biocontrol microorganisms have various advantagesthat chemical fungicides do not have, but they are also sensitive to chemical fungicide. Thesetwo methods have certain complementary function in plant disease control. So it is important tobreed biological microorganism that have the tolerance ability to chemical fungicide in theagriculture.Trichoderma harzianum starting strain hc was mutated by UV and LiCl complex mutationmethod in the resistance ability on Carbendazim，315strains of resistant mutants was obtained.Using PCR method, the gene sequence of its β-tubulin was analysised. Three mutation strainswith the replacement of amino acid in β-1tubulin gene coding region were screened out. Thefurther research on hc-9strain with the strongest fungicide resistance was preceded.By fungicide plates testing, the change of effective concentration (EC50) of carbendazim onmutation strain hc-9was tested, result showed: EC50on strain hc-9increased285%, comparedwith that on the starting strain hc, the resistance of the mutation strain to carbendazim wasrelatively stable after transfer12generations; By confrontation cultivation and slide culturemethod, the restrain effect of mutation strain to different pathogenic fungi was studied, there wasno significant difference between starting strain hc and the mutation strain hc-9, and thehyperparasitic ability of mutation strain hc-9was still existence. Czapek–Dox Medium was usedas the basic medium, changing the carbon source, nitrogen source, temperature and pH, andusing different concentration of NaCl and PEG to simulate salt stress and drought stress，thechange of physiological characteristics of mutation strain hc-9were studied. Result showed thatunder those conditions described as before, the mycelium growth rates of mutation strain hc-9were all slower than that of the starting strain hc, but the trend was consistent under differentconditions.Taking soybean as material, using pot experiment，several physiological indexes of differentsoil treatment samples in leaves and roots at the stage of soybean seedling were determined(threeantioxidant enzymes activity and the content of malondialdehyde (MDA), soluble protein) toresearch the application prospect of T. harzianum starting strain hc and the mutant strain hc-9. Result shows that compared with the treatment of pathogen，the anti-enzyme activity in theroots and leaves treated by T. harzianum starting strain hc and the mutation strain hc-9were allincreased, but the content of MDA was reduced. Among them, SOD enzyme, CAT enzymeactivitiy in leaves and roots treated by pathogenic fungi and T. harzianum mutation strain wasimproved in a higher degree, the content of MDA treated by pathogenic fungi and carbendazimwas decreased the most among all the treatments, for example, the content of MDA in leaves was0.022μmol/g，decreased by43.59%; But the result treated by both carbendazim and T. harzianummutation strain hc-9was different to that treated only by T. harzianum or carbendazim, forexample, SOD enzyme activity in leaves or roots was lower than that of other treatmens. Afterinoculation with Sclerotinia sclerotiorum on leaves, the changes of antioxidant enzymes indifferent treatments were detected, the degree of SOD and POD enzyme activities were increasedhigher than that of CK, the content of MDA was lower than that of CK.The results of our study will provid a scientific basis for the research and comprehensiveapplication of T. harzianum in the future.