Establishment of Techniques for Mass Production of Gliocladium Biofungicide and Its Application in the Disease Control

• Main Authors: Yao-Cheng Tseng, 曾耀徵
• Other Authors: Dean Der-Syn Tzeng
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/01792687016552215780

碩士 === 國立中興大學 === 植物病理學系 === 89 === By using Czapek’s broth as a basal medium, effect of various carbon and nitrogen sources on growth, sporulation and antibiotic production of Gliocladium virens SGV7 isolate were explored by a shaking flask culture system. Among 12 carbon sources tested, mannose was found the best to support the mycelial growth and conidial production. In mannose containing medium, the mycelial growth reached approximately 190 mg / 50 ml culture that was more than 4 times comparing to that in glucose containing medium. Likewise, the maximum conidia yield in mannose treatment reached approximately 5.7 X 107 /ml, whereas that of glucose treatment was only 5.1 X 106 /ml. A total of 16 nitrogen sources were screened, N-compound was among them the best for supporting the mycelial growth and as well for the chlamydospore production. In a 0.3 % N-compound amended treatment, the mycelial growth reached approximately 250 mg / 50 ml culture at 10 days after inoculation; and accompanied to that production of about 107 chlamydospore /ml was observed. The productivity of chlamydospore in this N-compound amended medium was further improved with the addition of yeast powder, oat meal and sucrose respectively. With the addition of 2 % yeast powder or 2 % oat meal, the yield of chlamydospore was increased further up to 2 X 108 / ml level. As regards to antibiotic production, addition of fructose, maltose and sucrose were of greatly stimulatory, but the increased provision of N-compound tended to reduce the activity. For the mass production of chlamydospore formulation by liquid culture system, a nature medium consisting 2-3 % (w/v) oat meal, 0.2-0.3 % (v/v) N-compound, 2% (w/v) sucrose and 2% (w/v) yeast powder is highly recommended. As an well-known antagonistic fungus existing all over the world, the potential of Gliocladium spp. in the control of soilborne diseases has been well recognized. Commercialization of this fungus as biofungicide has been successful in certain advanced countries; and the number of available commercial product is increasing. To explore the use of this fungal resource native in Taiwan, however, technique platform for the mass production is badly needed. In tradition, the fungus was produced by solid fermentation in which most of the efforts were aimed to the conidia production. The major pitfall for the conidia preparation was the limitation of poor shelf life. To cope with that, cultural system aiming for chlamydospore production has been successfully established and commercialized. The main objectives of this study were to improve the technique for the solid fermentation production of conidia and the liquid fermentation production of chlamydospores. Gliocladium virens SGV7 isolate was used as a model system for the technique platform establishment. For solid fermentation, a plastic vial system primarily used for mushroom production was adapted. Improved technology for water addition and moisture control optimization was successfully established. Direct addition of 55% (v/w) water prior to autoclaving of the oat grain medium was found appropriate to support the fungal growth and conidia production. Also found critical was the use of plastic film to provide a better moisture content during the early stage of mycelial growth. With an improved water content and moisture condition, the spore yield increased greatly. The best yield ( up to 3 X 109 conidia/g ) was observed in a system where mixture of wheat bran and fine sugarcane baggase ( 1:3, v/v ) was used to replace oat grain as a cultural substrate. The major advantage of using this substrate mixture was the loose texture and better water retaining capability. Upon harvest of the conidia preparation, formation of chlamydospores was generally observed on the residual culture substrate. As regards to the production of chlamydospores, the addition of N-compound was found greatly stimulatory, the amount of chlamydospore formation reached 107/ml in a traditional 50 L stirrer tank liquid fermentor. Moreover, the established technique platform was demonstrated working also for 2 other Gliocladium isolates and 1 Trichoderma isolate in regarding to chlamydospore production. As for the disease control efficacy, the chlamydospore preparations of G. virens SGV7 isolate and Trichoderma sp. YT-3 isolate were shown to be effective in reducing the infection of cabbage by R. solani AG4, and in improving the survival rate and growth vigor of the cabbage seedlings.