Improvement Of Spore Production By Coniothyrium Minitans On Solid Substrates Of Stubble Of Oilseed Rape

Sclerotinia stem rot caused by Sclerotinia sclerotiorum(Lib) de Bary was stubborn disease of several important crops with a worldwide distribution. The sclerotial mycoparasite Coniothyrium minitans Campbell had already shown promising potenti-al for biocontrol of S. sclerotiorum. To develop a method to produce conidia in masses for future practical use as a biocontrol agent, these conidia should be efficient and consistent for biological control of S. sclerotiorum. Yingchao Wang et al.(2005) reported that the oilseed rape stalks were desirable substrate for conidial production because of their abundant nutrition. Furthermore, that research had simple technology and abroad source of raw materials and lower cost products, all showed the practicality and wide value of application were confirmed.With the cost-effective pulverized rapeseed stubble as main raw substrates, with the addition of cornmeal and wheat bran, spore production of C. minitans as biological pesticide by solid-state fermentation were studied. Screening of The medium composition and optimization of the fermentation condition, as well as conidial quality assessment of C. minitans produced by solid-state fermentation during storage were discussed. Finally its effect to control S. sclerotiorum was also observed. The results were as follows:Based on the yield of conidia and cost considerations, solid-state fermentation medium for the production of C. minitans conidia was selected in the present study, which the wild type C. minitans strains Chy-1 was research object. Under 250ml flask cultivation conditions, The optimized medium composition contained(w/w) pulverized rapeseed stubble 71%、corn meal 23%、wheat bran 6% using one-factor experiment and orthogonal designs. From this study, we observed that the medium component and amount or quantity had a significant influence on conidial production of C. minitans. In addition, pulverized rapeseed stubbles only took up less space than before.In laboratory studies, 250ml flask cultivation(containing 15g optimized fermented media per flask): When the ration(g:ml) of dry material and water content was 1:3～1:5, inoculated with 10~6～10~7 spores/g initial dry matter, the spore yield of C. minitans obtained reaches up to 3.3×10~9 spores/g initial dry matter after fermentation for 15 days; The sterilize-able polypropylene plastic bags(17cm diameter×33cm length)(ID17cm×L33cm) is used to enlarged culture: 50g optimized fermented media with the initial water content 1:3 are placed in the bags especially designed for scale-up fermentation, conidial production reaches up to 2.5×10~9 spores/g initial dry matter after fermentation for 20 days. Conidial production under these conditions(with substrates based on rapeseed stubble/cornmeal/wheat bran) is 5-fold higher than with rapeseed stubble alone(0.88×10~9spores/g initial dry matter). So this is a considerable improvement as compared to the previous study.At the same time, several affecting factors during sporulation of C. minitans are analyzed. The result shows: when the surrounding environmental temperature is maintained about 22.5°C after the fermented bags is piled up, the temperature increases gradually in the fermented bags along with prolongation of the incubation time and almost keeps between 22℃and 24℃. And that the optimum temperature for C. minitans is 20℃, the range of temperature is similar to that fitted to growth and spore production of C. minitans according to repetitive measurements. The water content in the fermented medium shows the strong effect on sporulation of C. minita-ns. Due to its respiration constantly strengthen and metabolic heat generation, as well as the water evaporation continuously in the fermented bags during the incubation, the matrix moisture content also decreases gradually. But changes were relatively slow and stable. The substrate consumption by C. minitans is analyzed by weighing samples before and after drying in the fermentation period. Results showed that there are no obvious differences between the Substrate reduction for 20 days incubation and 25 days or 30 days by the way of ANOVA. And that the change tendency is almost same between the substrate decrement and spore production of C. minitans. This explained that its growth had been slowing, metabolism weakened, the temperature and water content in the fermented bags remained basically unchanged. Simultaneously, the usage of substrate nutrients also began to decline in later period(30 days). Spore germination of C. minitans was determined by dilution method of potato dextrose agar(PDA) plate counting in various fermentation periods(10～30 days). The average germination percentage was around 80% after 24 h inoculation on PDA plate. But yet the final germination(spore germination rate after 36～48h) reached up to 90～100%, there were no evident discrepancy each other during cultivation by the way of ANOVA.The purpose of this thesis was aimed at large numbers of infective spores of C. minitans required to be able to fully control sclerotinia disease. Inocula quality was assessed using both an existing conidial germination test and novel laboratory assays which determine the ability of the conidial inocula to infect and reduce sclerotial viability under different storage conditions(4℃、20℃、28℃). Firstly, the conidial germination of two different treatments at varied temperatures after storage were compared(24h and 36h tested on PDA plate). It’s obvious from the curve of spore germination that percentage germination of non-dried conidia(fresh conidial suspensions) harvested from the fermentation bags and tested immediately on PDA plates(36h) was higher than of dried conidia collected from the fermentation bags before storage(0d), and no significant difference was found between them. They maintained higher germination level after longer time(5～6 months) at 4℃: percentage germination of dried conidia was 43.4%, But there was no significant difference compared with the non-dried conidia(57.5%); as storage time increased the percentage germination was noticeably low storaged at 20℃and 28℃. Moreover, the percentage of dried conidia was higher than of non-dried conidia(24h and 36h), after 4 months, germination ability of non-dried conidia was almost lost. At the same time, rot index of the sclerotia of S. sclerotiorum parasitized by the two different C. minitans conidia produced in solid substrates were determined during storage. The results showed that: the sclerotia in moist sand treated with C. minitans conidia had been rotten at 20℃for 2 months. During the storage the rot index was significantly higher at 4℃than at 20℃and 28℃, after 6 months the dried conidia had lower germination and parasitic ability. Therefore, the dry spores survived than non-dried.C. minitans spore germination rate and the parasitic ability had been gradually declining, the rot index of sclerotia was higher after treated with 10~7conidia/ml and 10~5 conidia/ml than 10~3conidia/ml, this showed 10~5～10~7conidia/ml was the optimal.The relative efficacy of C. minitans inocula were tested in pot bioassays to evaluate their effect on carpogenic germination of buried sclerotia and their survival and ability to infect and rot sclerotia of S. sclerotiorum. C. minitans inocula applied to soil as conidial suspensions or as solid-form inocula could infect sclerotia and make them rot when the same dose(10~5 conidia/g soil), and the rapeseed production harvest-ed from the soil had improved.In this study, C. minitans spore was directly applied into the soil after solid-state fermentation, which had not to be purified or prepared into agent, so it not only reduces costs and makes straw, but also causes no environmental pollution, also importantly it reaches a persistent disease control purposes. In general, production of C. minitans conidia by solid state fermentation based oilseed stubble as main material had broad prospects for development.