The Biological Characteristics of Metarhizium anisopliae and Its Biological Control on Plutella xylostella

• Main Authors: Yi-Chen Chiu, 邱怡禎
• Other Authors: Chang-Hsin Kuo
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/57507948836941470391

碩士 === 國立嘉義大學 === 生物資源學系研究所 === 94 === Abstract A fungal entomopathogen was isolated from the cadaver of Allomyrina dithotomus larva found at Nantou, Taiwan. By using single spore isolation method, this fungus was identified as Metarhizium anisopliae, one of the potential entomopathogenic fungi in the world. Isolate Ma0407 was assigned for the following tests. Inoculation tests indicated three crucifer insect pests, i.e., Porthesia taiwana, Plutella xylostella, and Pieris rapae crucivora were infected by this fungus. Among them, P. xylostella was the most susceptible. However, Spodoptera litura and Crocidalomia binotalis did not show any symptom at all. The basic physiological studies of this isolate indicated that regines for the mycelial growth between 15~35oC, with optimum temperature at 30oC. But mycelial growth was inhibited below 10oC. The maximum temperature for conidial germination occurred at 30oC, and spores did not germinate below 15oC. The germination rate was decreased as the temperature decreased. The effects of culture media on the spore germination, mycelial growth and sporulation of this isolate were conducted. The data have shown the spores cultured on YPDA, 20%V8 and TSA had the highest spore germination rate than other media. TSA was the best medium for the mycelial growth, and significant higher sporulation rate was found on SMYA, YPDA, SDBY, YDA, TSA and PDA media. Eleven carbon sources and 13 nitrogen sources were chosen as nutrients for investigating their effects on the spore germination, mycelial growth and sporulation. A comparison of different carbon sources showed that spore germination rate was highest when grown on glucose, and spore germination was inhibited when grown on xylose.Better mycelial growth on glucose, mannose, xylose, maltose, saccharose, fructose and soluble starch was also found, however, growth on galactose was the worst. All kinds of nitrogen used were not significantly affect the spore germination; nevertheless, the sodium nitrite showed the most significant inhibition. In contrast, the best carbon sources for sporulation were mannose, fructose and raffinose. The mycelial growth on different nitrogen sources showed that glutamic acid, methionine, bacto peptone were better than others. The highest sporulation was found when media were added with ammonium phosphate or asparagine. The water-activity condition of medium was regulated by adding 0-1M PEG 200. The higher the concentrated PEG 200, the lower water-activity was obtained. The results indicated that spore germination could be decreased when water-activity became lower. Air-dried spores were not be able to germinate, even placed in 100% relative humility condition, and the spore needed free water to initiate the germination. P. xylostella larvae were susceptible to this isolate when the temperature ranged from 20~35oC, and the optimum temperature were 25-35oC, and no mortality was found below 15oC. Higher mortality rate of fourth-instar P. xylostella larvae was shown when inoculated with 105-107condia/ml. All the larval stages from 1st-4th instar were susceptible to this fungus but not the pupal stage. The larvae between 2nd-4th instar were more susceptible. The damage rate of potted cabbage plant infested by 3rd~4th instar of P. xylostella larvae was designed as criteria for evaluating the control efficacy. The damage rates of 3rd and 4th instar larvae were 17.3% and 17.1%, respectively. The damage rates in the group were 22.0% and 18.0%, respectively. The results indicated that control efficacy of 3rd instar larvae was significant.And the mortality of 3rd instar and 4th instar larvae was 45% and 44%, respectively. Metabolites produced by M. anisoplaie were bioassayed to estimate their virulence to P. xylostella. The items tested were the effect of incubation way, concentration and larval instar. The results showed that feeding bioassay was more sensitive than dipping; higher concentration of metabolite had led to higher mortality rate than the lower concentration; Both the 3rd and 4th instar larvae were sensitive to metabolite. The metabolite produced by M. anisoplaie was detected by shaking the YPD liquid medium for 18 days. The incubation time affected the metabolite’s production which was evaluated by high performance liquid chromatography (HPLC). The main metabolite components were eluted from HPLC at 13 min, 15 min and 18 min (named as MA, MB and MC) and variated with the incubation day. The amounts of MA and MB were increased with incubation day, and the production curves of MA and MB were similar. MC production was maximized at the 8th day, and the field of MC decreased afterward. This metabolite was defined as destruxins. However, after comparing with the standard of destruxin A, the present data have shown that this metabolite was not destruxin A. The role of the unknown metabolite in the pathogenic process needs further study.