枯草桿菌 Bacillus subtilis WG6-14懸浮培養增量菌液製作及其病害管理之應用

Abstract

病蟲害綜合管理為目前各國重視且迅速發展的永續農業策略，有鑒於化學農藥的濫用已造成環境生態之浩劫，生物防治則是一種能維護農業生態系中病蟲害與益菌或天敵等族群間均衡的手段，本研究室近年來積極投入本土性微生物資源及其活體製劑量產與應用技術之開發。我們由分離自霧峰蕃石榴根圈之枯草桿菌 Bacillus subtilis WG6-14菌株，經測試證實其生長快速、產孢特性優異，對多數土壤傳播性病原真菌與黃單孢菌屬（Xanthomonas）病原細菌具抗生活性。其應用性於本研究室已成功建立內生孢子量產液體發酵培養技術，所產製之 WG6-14發酵菌液於溫室、田間試驗評估，證實對柑橘潰瘍病防治效果顯著。拮抗菌相較於病原菌族群壓倒性（inundative）優勢的維持，為影響防治效果之關鍵。為確保生物製劑在施用時菌體活性之理想狀態，並使成本可以大幅降低，本研究嘗試建立可由業者自行增量培養之方法。利用塑膠桶及定時攪拌器，配合由魚精粉、紅糖粉、乳清粉等農業常見資材，於適當配方比例下，配合培養過程相關理化條件如培養溫度、pH 値的調控，可在材料與器械不經高壓滅菌情況下，成功增量為功能性營養配方（functional nutritive formulation, FNF），WG6-14孢子活體產量達1010cfu/ml。經三個月的儲存，活菌量仍有10８cfu/ml。將此功能性營養液施用於溫室的臍橙與甘藍幼苗，觀察到明顯的促進生長效果，施用四週後可發現臍橙新梢生長勢及新葉數目的都優於對照組；施用於甘藍幼苗上也明顯的增加鮮重達51.2%。進而分析其中能促進植物生長之物質，結果發現或許與多元胺中的 putrescine 有關。而在病害防治應用上，評估功能性營養液對於溫室栽種的臍橙上柑橘潰瘍病之防治效果，結果顯示在稀釋100倍與500倍葉噴前處理下，相較於對照處理77%之發病率，處理組發病率分別只有22%與34%，防治效果顯著。進而施用於大林田間葡萄柚及柳丁上評估對於潰瘍病的防治作用，於每週定期噴灑施施用下，能將發病度壓制在43.8%，相較於對照組58.9%的發病度有明顯的防治效果。本研究所建立之功能性營養配方於作物栽培上提供病害防治兼有促進生長之效果，此於病蟲害綜合管理上之應用性值得推薦。

The major objective of the investigation was to establish a non-sterile propagation system for the production of a functional nutritive formulation (FNF) which consists mainly viable endospores of Bacillus subtilis WG6-14 and the microbial metabolites which appeared to be nutritive for plant growth. The tested biological control agent WG6-14, originally isolated by Lee (2002) from guava rhizosphere, has been shown with great potential for biofungicide application and superior antagonistic effectiveness against most soil-borne fungal pathogens and phytopathogenic Xanthomonads. As the maintenance of an inundative advantageous status of antagonistic microorganisms is known to be critical for the key point of effectiveness on disease control, a pilot scale liquid fermentation technique platform for the production of WG6-14 endospore formulation was established. Using the endospore formulation as a seed inoculum, a broth culture amplification system was attempted with the use of common agricultural waste as major constituent of growth medium and the cultures were performed with a self-constructed open tank system where in a timer controlled stirring device was equipped. The culture medium was freshly prepared and used without autoclave sterilization. The establishment of a FNF in this investigation was aimed to provide a grower do-it-yourself system for the mass propagation and reactivation of the fermentor produced endospore biomass right before application and thus to reduce the cost of the biofungicide application. With the optimization of the cultural substrate formulation, the yield of endospore of WG6-14 in the FNF prepared with 200L and 500L scale plastic tanks reached 101０cfu/ml 10-12 days. The viable biomass of FNF dropped to 108 cfu/ml level gradually after 3 months. With the application by soil drenching and/or foliar spray of FNF each at appropriate concentration, substantial growth promotion on the cabbage seedling and Navel orange were demonstrated. In the case of Navel orange, the foliar spray with FNF at 100X dilution led to 25.8% increase in numbers of new leaves. Whereas for cabbage seedling, the same treatment led to 51.2% increase in fresh weight 4 weeks after treatment. The analysis by high performance liquid chromatography (HPLC) revealed the presence of polyamines-mainly putrescine and spermidine in the FNF prepared by an optimized protocol. The possible connection of the polyamine production to the observed growth promotion and antimicrobial activity deserves future attention. The application of the prepared FNF in disease control was tested on the green house grown Navel orange by an artificial inoculation model system previously established for citrus canker disease control evaluation. The pretreatment by foliar spray with 100X and 500X diluted FNF 24 hours before artificial inoculation resulted in the reduction of disease severity down to 22% and 34% respectively, the significance of disease control was clearly indicated by that the disease severity of the compared control was 77%. In field trial recently conducted, the foliar spray of 100X diluted FNF was shown to reduce disease severity on grapefruit by 40% comparing to that of non-treated control. The success of FNF development warrants the application of the inundative strategy where in the application of viable, active WG6-14 propagable for biocontrol is attempted.