- 學位論文
拮抗微生物防治Rhizoctonia solani引起甘藍幼苗立枯病之探討
Study on the control effects of antagonistic microrgamisms against cabbage seedling damping-off caused by Rhizoctonia solani
摘要
當前農業栽培管理系統主要仍依賴化學合成農藥來防治植物病蟲害, 造成病原產生抗藥性、生態環境汙染及農產品食物安全等問題，為減少化學農藥的使用，利用微生物的生物防治法是一種可能策略之一。Rhizoctonia solani kuhn是一重要之土壤傳播性植物病原真菌，具有生長迅速、寄主範圍廣泛，以及腐生能力強等特性，可危害台灣地區栽培之大部分農作物，為引起甘藍幼苗立枯病(damping - off)的主要病原之一。本研究探討以拮抗微生物防治甘藍幼苗立枯病之效果，以供試細菌Bacillus subtilis LB5、Bacillus amyloliquefaciens PMB01及Pseudomonas putida FPP5與真菌Trichoderma koningii 進行評估其防治甘藍幼苗立枯病之可行性。由立枯病病原菌培養資材之篩選及不同接種量於塑膠袋生長試驗，獲得以紅高梁資材最適病原菌R. solani菌絲生長，可做為病原菌紅高梁菌種。以玻璃紙抗生法測試T. koningii 對R. solani菌絲生長之拮抗作用，得知所有供試木黴菌菌株皆對R. solani菌絲生長有抑制作用，其中以TDS菌株對病原菌菌絲生長抑制效果最為佳。利用玻片觀察T. koningii對R. solani菌絲之拮抗機制，以顯微鏡(200X)觀察發現，T. koningii TFS、TDS及TAS.2等3菌株，具有導致R. solani菌絲發生斷裂之能力，此外，TFS另具有穿生於病原菌菌絲中之能力。以拮抗作用測試用玻璃環平板培養基測試各種供試細菌菌液對R. solani菌絲生長之拮抗作用，發現B. amyloliquefaciens PMB01及B. subtilis LB5，具有抑制病原菌菌絲生長能力，P. putida FPP5則無。測試以peptone PDB或PSB之B. subtilis LB5培養菌液及其過濾液對R. solani菌絲生長之拮抗效果，獲得LB5培養菌液對病原菌之菌絲生長有抑制效果，但過濾液則無。以B. amyloliquefaciens PMB01菌液於試管內對R. solani引起甘藍幼苗立枯病之初步防治測試，發現PMB01可降低甘藍幼苗罹病株率達70%，具有防治立枯病潛力。測試B. subtilis LB5、B. amyloliquefaciens PMB01及P. putida FPP5對T. koningii TDS之拮抗作用，獲得 LB5及PMB01皆對T. koningii TDS之菌絲生長具有拮抗作用，但FPP5則無。由溫室試驗R. solani接種量對甘藍幼苗發病之影響結果得到，R. solani紅高梁菌種與滿地王泥炭介質以1：2.5 (W：W)混合之病土，引起甘藍幼苗植株死亡率及罹病度較高，分別為45%及62.5%。於溫室進行各供試細菌對甘藍幼苗生長之影響測試，發現各種細菌對滿地王泥炭介質中之甘藍幼苗生長無明顯負面影響。而於溫室進行，各種供試微生物對R. solani引起甘藍病害之生物防治效果試驗，結果顯示單獨處理B. subtilis LB5及P. putida FPP5於病土中對甘藍幼苗立枯病病害不具防治效果，但B. amyloliquefaciens PMB01具防治效果。應用複合供試微生物B.amyloliquefaciens PMB01、P. putida FPP5 及B. subtilis LB5及T. koningii TDS對R. solani引起甘藍病害之防治效果評估，發現於移植同時再接種各供試細菌之處理，無論是單獨接種T. koningii TDS或T. koningii TDS混合各供試細菌培養菌液之接種處理組，甘藍幼苗罹病度皆相較其他處理組低，顯示木黴菌T. koningii TDS施用於土壤中防治R. solani引起甘藍幼苗立枯病，比施用拮抗細菌於土壤之防治效果好。而比較單獨T. koningii TDS或T. koningii TDS混合各供試細菌培養菌液之處理，與單獨各供試細菌培養菌液之處理，發現各供試細菌甘藍幼苗立枯病防治效果可因混合T. koningii TDS而提升。綜合本研究所有試驗，發現所有供試拮抗菌B. subtilis LB5、B. amyloliquefaciens PMB01及T. koningii 都具有進行拮抗R. solani之能力，亦可抑制其生長；但施用於病土中防治R. solani引起之甘藍立枯病效果，木黴菌T. koningii較明顯，拮抗細菌B. subtilis LB5、B. amyloliquefaciens PMB01較不明顯，亦說明木黴菌培養於木屑介質施用於土壤中優於拮抗細菌在土壤中對R. solani所引起立枯病之防治效果。
並列摘要
Bacillus subtilis LB5, Bacillus amyloliquefaciens PMB01, Pseudomonas putida FPP5 and Trichoderma koningii TFS, TDS, TAS.2 isolates were utilized as bio-control agents to evaluate the biological control efficiency against damping-off of cabbage seedling caused by Rhizoctonia solani. Red sorghum was selected as R. solani spawn material for inoculum mass production based on screening resnets from various grain materials. The experimental disease soil was mixed with R. solani pathogen spawn and peat moss material in the ratio of 1 : 2.5 (W/W). All isolates of T. koningii were found that they were all antagonists containing inhibition ability to mycelia growth of R. solani on cellophane membrane method. In microscopy observation, T. koningii TFS, TDS, TAS.2 isolates were found that they had hyphal breaking ability to R. solani. In addition, TFS isolate could invade and grow its hyphae within the hyphae of R. solani. In grass-ring agar plate test, B. subtilis LB5 and B. amyloliquefaciens PMB01 from cultured liquid of PSB or peptone PDB, could inhibit mycelial growth of R. solani with antagonistic substances based on the inhibition zone shown between Bacillus bacteria and Rhizoctonia solani, but the filtrates of cultured liquid of two Bacillus species had no inhibition effect. The infected plant rate of damping-off could be reduced to 70 % by treating with B. amyloliquefaciens PMB01 in preliminary tube test. Dual culture on agar plate between B. subtilis LB5, B. amyloliquefaciens PMB01, P. putida FPP5 and T. koningii TDS showed that B. subtilis LB5 and B. amyloliquefaciens PMB01 could inhibit mycelial growth of T. koningii, whereas P. putida FPP5 had no inhibition effect. B. subtilis LB5, B. amyloliquefaciens PMB01 and P. putida FPP5 could not influence the growth of cabbage seedlings grown in peat moss in greenhouse preliminary test. In diseased soil, B. subtilis LB5 and P. putida FPP5 could not express the control efficiency against cabbage seedling damping-off. But only B. amyloliquefaciens PMB01 could reduce disease severity to 28.13 % comparing 89% of check treatment if the tested seedlings were nursed from soil poured with PMB01 bacterial suspension. However, all seedlings in healthy soil could grow better in fresh weight and plant height than seedlings grown in diseased soil. On the greenhouse experiments of control efficiency evaluation used combined experimented microorganisms against damping-off of cabbage seedling, the T. koningii TDS treatment in post-transplantation reduced the disease severity to 20.31 % was only better than that of FPP5 and PMB01 treatments on the seedlings were nursed from soil poured with P. putida FPP5 suspension. T. koningii TDS could increase the control efficiency of LB5 or PMB01 bacterial isolate when LB5 or PMB01 bacterial isolate was used with T. koningii TDS, comparing the treatments of LB5 or PMB01 individually.
參考文獻
延伸閱讀
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