Study On Solid Cultivation Of Coniothyrium Minitans

Sclerotinia sclerotiorum (Lib.) de Bary, a pathogen of Sclerotinia stem rot, can infect 408 plant species, and also results in great oilseed rape production loss in China. A widely used agent nowadays to control Sclerotinia rot, Carbendazim belonging to benzimidazole, is facing the restricted use for its chemical residue and environment pollution. Thus, new biological agent with same control efficacy but without pollution and other bad effects is urgently needed in the crop production. Many efforts have been made on biological control of Sclerotinia stem rot. Since the discovery of Coniothyrium minitans showing the control efficacy on Sclerotinia rot, many proceedings have been achieved in the fields of physiological characteristics, bio-control mechanism, mass cultivation and spores production of C. minitans, etc.The conidium is main type of C. minitans for propagation and long-term conservation. The conidia of C. minitans can invade and germinate on S. sclerotiorum and, by which Sclerotinia stem rot can be bio-controlled. Availability of adequate conidia is an essential prerequisite of mass production and utilization of C. minitans. Thus, efficient and mass culture of C. minitans conidia becomes an important research direction, and need to be resolved. Based on above, this dissertation was to study the technologies related to solid culture of C. minitans, including culture conditions, culture patterns, nutrient addition, and cheap medium selection for mass conidia production. The expected results will benefit to the practical utilization of C. minitans as a biological control agent.A series of systemic study was conducted with a solid film culture experiment combined inert supporting medium and film culture, and C. minitans was served as the model strain of filamentous bacteria. The results showed that, in solid film culture system, mycelia and medium could be easily separated, which had no bad effects on mycelia growth and sporulation, moreover, which could yield more spores. The usage of a new kind of petri dish was very convenient to measure quantitatively the related indicators during the culture process, such as mycelia growth, spores yield, substrate consumption, and pH value. The semi-continuous culture method could meet the demand to explore efficacy of recycle fermentation under the conditions of solid-like culture. The film culture showed an enormous potential in the research field of mycelia growth characterization and continuous /semi-continuous fermentation. Diverse concentrations of tryptophon with were added into the solid film culture medium by different amounts at various time to evaluate the effect on spores yield, to determine the tryptophan addition strategy for yield gain of C. minitans spores. The results indicated that, at the range of concentration, adding tryptophan benefited to spores yield of C. minitans. When the initial concentration of tryptophan in medium was above 0.50g/L, inhibition effect of tryptophan on sporulation of C. minitans was detected, and 0.10g/L was the optimal concentration for tryptophan addition. The sporulation and maturation stage (3.5-4 days after conidia inoculation) was the recommended time for tryptophan addition which could strengthen sporulation of C. minitans.The glucose was mid-term supplemented into the bran solid medium to explore its effect on C. minitans culture, especially, on spores yield. The results showed that different initial concentration of glucose in medium exhibited detectable effect on the conidia growth at the first culture stage, and the suitable initial concentration of sugar should be 4% - 8%. Mid-term supplement of sugar powder could extend the stable growth stage and increase spores yield. The dynamic curve of reducing sugar in culture medium and growth curve of C. minitans spores indicated that the most significant effect on spores yield was detected in the treatment of adding 1.5% glucose powder/dry weight of medium on third-day.The technology pattern of solid culture for C. minitans was investigated in the medium of apple pomace, and the tested factors were including washing times of apple pomace, initial pH value, added urea, calcium carbonate, inoculum age of C. minitans. The results showed the optimal technology pattern for C. minitans was to wash apple pomace once with 4-multiple waterâ†'to adjust the initial pH 6.0â†'to add 15g/kg calcium carbonate or 25g/kg ureaâ†'to sterilizeâ†'to inoculate C. minitans spores pre-cultured in PDB medium for three daysâ†'to culture at 20℃for 10 daysâ†'to obtain spore culture of C. minitans which contained about 1×109 spores /g culture.