Solid Fermentation And Formulation Of Conidia For The Mycoparasite Coniothrium Minitans

Sclerotinia sclerotiorum (Lib) de Bary is an important panthogen, responsible for leaf blight, stem rot and pod rot of oilseed rape (Brassica napus L.), resulting in the great losses to this crop, especially in the canola-growing areas along the Yangtae River. In recent years, biocontrol of sclerotinia disease using the mycoparasite Coniothyrium minitans has attracted attention of many researchers. Spore production of C.minitans as a bio-pesticide was optimized using oil seed rape stalks as solid-state fermentation substare. Wettable powder formulation of C. minitans was studied in this thesis.Stalks of oil seed rape were collected from filed and prepared as the basic substrate (water-saturated stalk pieces) for fermentation of C. minitans. Baed on the yield of conidial production, tolerance of conidia to high temperature (40*C) and ultraviolet, conidial germination, Chy-1 was selected as the best among 5 different C. minitans strains suitable for solid fermentation. Water content has a significant effect on conidial production. The optimum water content ranged from 1 : 3 to 1 : 5 for the dry weight of rapeseed stalks : water. The highest yield of conidia (2×10⁹ spores/g) produced by C. minitans on the basic substrate occurred at the 12th day after inoculation. Inoculant quantity ranged from 5×10⁵ to 2.6×10⁷ conidia/g substrate showed no significant effect on conidial production. Addition of carbon sources or nitrogen sources to the substrate did not result in any significant effect on conidial production as expected. However, addition of soluble starch and yeast extract can enhance the quality of conidia.Wettable powder formuation of conidia of C. minitans was established. The ingredients included: Conidia of C. minitans (>10⁸/g), sodium dodecyl benzenesulfonate (1-5%), calcium or sodium lignosulfonate (5-10%), xanthan (1%), ZnSO₄(0.1%), vitamin C (10%), and alginate (74.9-83.9%). The quality of the formulation included water content, moisturizing time, pH can meet national standards.Temperature was found to be the key factor affecting conidial survival in the formulation of C. minitans. After storage for 210 d, viable conidia were detected at 4"C, but not at 20℃ and 30℃.Potting experiments for testing efficacy of C. minitans preparation in suppression of carpogenic germination of S sclerotiorum were conducted under natural conditions. The treatments included directly inoculation (DI) of C. minitans onto sclerotia of S. sclerotiorum, which were then buried in soil, or spraying sclerotia-infested soil (SS) with suspensions of C. minitans preparation. The inoculations were arranged at the beginning of seeding of oilseed rape (October of 2003 and 2004). In the 2003 experiment, the totalnumber of apothecia produced in each pot was 37 > 30 and 10 for the DI treatment with 4 x 104connidia/mL -. 4x105connidia/mL and 4x106connidia/mL, respectively. The number of healthy sclerotia was 18> 11 and 8, respectively, for these treatments. A significant difference between these treatments and the control (62 apothecia/pot? 27 healthy sclerotia/pot). In the 2004 experiment, the total number of apothecia produced in each pot was 0> 0*. 0 for the DI treatment with 4xl04connidia/mL> 4xl05connidia/mL and 4><106connidia/mL,respectively. The number of healthy sclerotia was 2K 24 ? 18, respectively.for these treatments A significant difference between these treatments and the control (9 apothecia/pot, 31 healthy sclerotia/pot).For the SS treatment, in the 2003 experiment, the total number of apothecia produced in each pot was 8, 6> 1 for the SS treatment with 4xl06connidia/mL%*7 ft8x10 connidia/mL and 2x10 connidia/mL,respectively. The number of healthy sclerotia was 18> 16-. 5, respectively. A significant difference between these treatments and the control (67 apothecia/pot, 35 healthy sclerotia/pot). In the 2004 experiment,the total number of apothecia produced in each pot was 94 > 70, 37 for the SS treatment with 4xl06connidia/mLN 8xl07connidia/mL and 2xl08connidia/mL,respectively. The number of healthy sclerotia was 3K 34> 28, respectively. A significant difference between these treatments and the control (219 apothecia/pot. 50 healthy sclerotia/pot).Field experiments in 2004 and 2005 showed that application of the wettable powder of C. minitans (108conidia/g) on oilseed rape at the flowering time could significantly suppress sclerotinia stem rot. In the 2004 trial, the incidence for the treatments of spraying at 3.0xl07, 8.0xl08, and 2.4x109conidia/m2 was 14.6, 10.8, 8.5%, repectively, and the disease index for these treatments was 5.9,3.7, and 4.3, respectively. These values were significantly lower than those for control (incidence was 39.5%, disease index was 12.9). In the 2005 trial, the incidence for the same treatments of C. minitans was 27.9, 23.9 and 21.2%, respectively, and the disease index was 8.3, 5.6 and 4.7, respectively. These values were significantly lower than those for control (incidence was 51.2%, disease index was 19.3).These results suggest that there was feasibility for production of C. minitans conidia using oilseed stalks as substrate, and it is possible to use the wettable powder formulation of C. minitans for control of S. sclerotiorum and diseases caused by this pathogen.