Mutagenesis Breeding Of Chitinase-production Trichoderma Spp. Strain And Biocontrol Of Maize Sheath Blight

Maize sheath blight is a main disease of corn which is destructive soil-borne disease and can cause significant yield loss. Presently, it is difficult to select resistant plants using conventional breeding, soil-borne diseases are hard to be controlled and chemical control meets dilemma. So in this experiment, we screened and improved the Trichoderma strains in order to favor for the disease biocontrol. The results showed that:1. According to the test of anastomosis groups of Rhizoctonia solani, the pathogen of maize sheath blight, which was isolated from diseased sheath of maize in suburb of Yaan, Sichuan, was grouped into Rhizoctonia solani AG-1-IA.40 Trichoderma strains were isolated from the maize rhizosphere soils in suburb of Ya'an and identified. There were 24 isolates of T.harzianum,7 isolates of T.koningii,5 isolates of T.viride,4 isolates of T.pseudokoningii respectively. The test of hyphal growth rate showed that most of Trichoderma strains grew rapidly, and were the stronger competitor for space and nutrition resource. All the fermentation filtrates of Trichoderma strains could inhibite the growth of R.solani. T19 showed the highest inhibition rate,61.67%. Therefore, we selected it to perform the further improvement.2. Three approaches of physical mutagenesis, UV,UV+LiCl and DES, were used to obtain high yielding chitinase Rhizoctonia solani strain with stable inheritance. The result showed that the best UV mutagenic time is 120s, the LiCl strengths is 0.4% and DES mutagenic time is 40min. The chitinase activity of UV+LiCl-1, with stable heritage, increased to 1.21 U/ml which is 2.52 times compared with that the initial strain T19.3. The optimum medium and fermentation condition for UV+LiCl-1 have been established through orthogonal test design. Under the established optimal culture conditions which incoulding colloidal chitin 10.0g/L, peptone 2.0g/L, KH2PO4 2.0g/L, MgSO4.7H2O 0.6g/L and in the established optimal medium which incoulding temperature 28℃, fermentation time 5d, pH 6.0, inoculum size 7%. The chitinase activity of UV+LiCl-1 increased to 1.78U/ml which is 3.71 times compared with that the initial strain under the culture condition before optimization.4. The methord of ammonium sulfate grading precipitation was used to extract the chitinase solution. The best concentration of (NH4)2SO4, which was used to extract the Trichoderma chitinase, was 75%. Put the chitinase into the PD medium, when the concentration of the chitinase was 10%, the inhibition rate against the dry weight of Rhizoctonia mycelial was up to 68.14%, the inhibition effect is obvious.5. Trichoderma strain T19 and UV+LiCl-1 were able to colonize in maize rhizosphere soils, the surface of its roots and the leaf sheath. The colony forming units (CFU) of the T19 were 16.67×104/g,5.56×104/g, and 8.33×104/g, respectively. And the CFU of the mutant UV+LiCl-1 were 34.44×104/g,13.33×104/g, and 10.67×104/g, respectively, which was higher than the colonization ability of wild strains.6. All of the two biocontrol's Trichoderma strains have a certain effect against maize sheath blight in this experiment. The effect of mutant strain UV+LiCl-1 was 75.7%, which was significantly higher than the effect 65.06% of original strain T19. It has conrroled the incidence of maize sheath blight better.