生物製劑多元防治能力快速測試系統之研究

Abstract

由於糧食不足問題日漸嚴重，進行適當的作物病害防治以減少產量損失也成為重要議題，而生物防治則是最佳解決方法之一。數十年來，投入生物防治的相關單位漸多，在台灣登記上市的產品也逐漸增加，顯示其具有極大發展潛力。另一方面，許多宣稱具有防治能力的生物製劑也不斷推陳出新，而這些產品是否確實具有所標榜之效力則還需要再確認。常見對於生物製劑採取的檢測方法，如對峙培養、玻璃環法、田間試驗等等，存在一些諸如無法確切表示生物製劑之拮抗能力、測試時間較長等缺點。因此本研究擬針對生物製劑之多元防治能力測試尋求更加妥善快速之方法。本研究選用台灣重要的十二種植物病原菌：鏈格孢菌Alternaria brassicicola、灰黴病菌Botrytis cinerea、炭疽病菌Colletotrichum gloeosporioides、鐮孢菌Fusarium oxysporum、疫病菌Phytophthora nicotianae、腐霉菌Pythium spinosum.、立枯絲核菌Rhizoctonia solani、菌核病菌Sclerotinia sclerotiorum、白絹病菌Sclerotium rolfsii、軟腐病菌Erwinia carotovora subsp. carotovora、青枯病菌Ralstonia solanacearum、黃單胞菌Xanthomonas campestris pv. campestris等，進行各種測試方法之實驗，目標是設計出多元快速之測試系統。實驗首先採用三分格毒食法，再改良成三分格抑制環法。另外亦一併測試瓊脂膜法、玻璃紙法、雙層培養基法等三種方法。其中三分格抑制環法係結合系統將不同生長速度之病原真菌分成三組之三分格，加上細菌一組，即可一次使用四皿測試十二種病原，是一創新技術，其在八天內即可獲得結果，未來應可將此法應用於市面上生物製劑之測試，並快速驗證其所宣稱之效力。另針對本研究室自行篩選之拮抗性鏈黴菌Streptomyces sp. YU-01進行培養，篩選出紅豆及米糠煎汁為較有利其抗生活性表現之培養資材，將此發酵產物以三分格抑制環法進行測試，確實發現其對於多數病原均具有良好抑制效果。利用上述多元快速測試系統配合三分格抑制環法，已對市售四種生物製劑B01~B04進行拮抗成效之驗證，發現其效果仍多紛歧，但也確認本研究多元快速測試系統之可行性。

Since food shortage has become an increasingly serious problem, the prevention and control of crop diseases in order to reduce the yield loss is also now an important issue. The biological control is at present one of the best solution for the diseases or pests control. For decades, there has been more and more scientists devoted themselves in biological control research. The biopestcide products registered in Taiwan is also increasing, showing its great potential for future application. On the other hand, many biocontrol agents that claim to have the ability to prevent plant disease continued to go to market, and their effectiveness need to be checked or confirmed. Therefore, we intiate this study on rapid biocontrol testing system. There are many methods for testing the efficiency of the biocontrol agents, such as the dual culture, glass ring method, and field trials. However, they have some disadvantages such as not so acurate, need longer test times and the other shortcomings. Therefore, this study intends to seek proper and rapid methods to screen the anti-phytopathogen ability of biocontrol agents. In this study, 12 important phytopathogens has been gathered into the testing system: Alternaria brassicicola, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum, Phytophthora nicotianae, Pythium spinosum, Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii, Erwinia carotovora subsp. carotovora, Ralstonia solanacearum, Xanthomonas campestris pv. campestris. A variety of testing methods has tried. We first tried the Y-plate poison food method and then improve it to develop the 3-way ring-plate method. We’ve also tried the agar film method, cellophane method, and two-layer medium method. Among these methods, the 3-way ring-plate method in combination with the system divides the pathogenic fungi into three groups and the pathogenic bacteria for another. Therefore, we can screen all the 12 pathogens in 4 plates at a time. This system is a brand new one, and we can get the whole results within 8 days. The 3-way ring-plate method in combination with the system may provide a good way to screen the commercial biocontrol agents in the future. In addition, we also stuty on the culture of Streptomyces sp. YU-01, find out that red beans and rice bran are better materials for the antibiotic performance of YU-01. The fermentation products have showed good antibiotic activity to the majority of pathogens in our study, using 3-way ring-plate method in combination with the system. Finally, we have actually applied the 3-way ring-plate method in combination with the system to test 4 commercial biocontrol agents, finding that these products have various antibiotic abilities. Our conclusion is that 3-way ring-plate method in combination with the system is a reliable method for the rapid biocontrol testing.