| Sclerotinia rot of oilseed caused by Sclerotinia sclerotiorum (Lib) de Bary is a global crop distribution of intractable diseases. In recent years, biological control of S. sclerotiorum has attracted the attention of many domestic and foreign researchers. The mycoparasite Coniothyrium minitans Campbell has a promising application for biocontrol of S. sclerotiorum. Preparation of a large amount of active conidia is a prerequisite for the practical use of the mycoparasite C. minitans. With the cost-effective pulverized rape stubble as the main raw substrate, with the addition of wheat bran and cornmeal, spore production of C. minitans as biological pesticide by solid-state fermentation was studied by Aiqin Wang et al. (2007). Moreover, this procedure has simple technology and lower cost products and abroad source of raw materials, all that showed the practicality and wide value of application is confirmed.In this study, the wild type C. minitans strains Chy-1 is the researched object. And spore germination rate and conidial production by C. minitans are considered as an indicator. Based on the cost considerations, the first fermentation, the liquid and solid fermentation medium for the production of C. minitans conidia was selected in the present study. Finally the shallow dish fermentation is optimized and enlarged.Spore production of C. minitans as biological pesticide by the liquid-solid phase fermentation was studied in this research. Screening the liquid and solid medium and optimizing the conditions of fermentation were discussed. In the end, optimizing the conditions of the semi-open shallow dish fermentation, and then enlarging a shallow dish fermentation were researched. The results are as follows:The wheat grain is applied for the first fermentation medium, and C. minitans is cultured for 12～15 days. It can be used in an inoculation for the second fermented liquid, so that the concentration of spores reach to 1×106 conidia/ml. Based on conidial germination rate, under 250ml flask cultivation conditions, the carbon sources, the pH value, the charging sample volume, the culture concentration and time are optimized in the study. Then we get the results:when the conidial concentration of the liquid medium is 1×106 conidia/ml and the 250ml flask contains the liquid medium 100ml with sugar carbon source in pH 4, the conidial germination rate of C. minitans reach to 68% after culture for 24h.Under 250ml flask cultivation conditions, The optimized solid fermentation medium composition contain (w/w) wheat bran 42.31%、corn meal 36.53%、rice husk 21.16% and water content 85.12% using the whole factor experiment and central composite design experiment. The spore yield of C. minitans in the optimized solid medium reaches up to 9.5×109 spores/g initial dry matter after fermentation for 12 days.And we observed that the composition and amount of the medium had a significant influence on conidial production of C. minitans from this study.In laboratory, it is fermented in a small shallow dish (17.5 cm×14 cm×5 cm): the solid medium 120 g per shallow tray such that the thickness is 2cm, inoculated with 2×105 spores/g fermented medium and seed age for the 24h, half-opened after culture for three days, the spore yield of C. minitans reaches up to 6.8×109 spores/g initial dry matter after fermentation for 9 days.In laboratory level, it is scale-up to ferment in large shallow dish(60cm×40cm×4 7cm):the solid medium 1651g per large shallow tray such that the thickness is 2cm, inoculated with 2×105 spores/g fermented medium and seed age for the 24h, culture, half-opened after culture for three days, the spore yield of C. minitans reaches up to 5.2×109spores/g initial dry matter after fermentation for 9 days.At the same time, we analyze the factors influencing the spore production of C. minitans. The results show that the ambient temperature of the large shallow dish is maintained about 22℃. As the culture time goes by, the temperature is increased gradually in large shallow dish, and almost keeps between 22℃and 24℃. And 20℃is the optimum temperature for C. minitans. According to the repetitive measurements, the range of the above mentioned temperature coincides with the temperature range required for the spore production of C. minitans. The water content of the substrate has the significant effect on the spore production of C. minitans. Due to its respiration strengthened constantly, metabolic heat generation, and the half-open state of the shallow pan during the incubation, as well as the water evaporation continuously, and as the culture time goes by, the matrix moisture content also decreases gradually, but the changes are slow and stable relatively. In the fermentation period the consumption of the substrate by C. minitans is analyzed by weighing samples before and after drying. The results show:there are no obvious differences between the substrate reduction for 9 days to 15 days incubation by the way of ANOVA. And the change tendency is almost same between spore production of C. minitans and the substrate decrement. We can find that its growth has been slowing, metabolism weakened, the temperature and water content in the shallow dish remained unchanged basically. The usage of the matrix began to fall at the same time. With the dilution method of potato dextrose agar (PDA) plate counting in various fermentation periods (6～15days), spore germination rate of C. minitans was determined. And we find that the average germination percentage is between 85% and 90% after 24 h inoculation on PDA plate. But the final germination (spore germination rate after 36～48h) reaches up to 90-100%, there are no obvious differences each other during cultivation by the way of ANOVA. |
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