Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30986
標題: 枯草桿菌Bacillus subtilis WG6-14於檬果黑斑病防治之應用潛力與作用機制
Biological control of bacterial black spot on mango by application of Bacillus subtilis WG6-14 - potential application and mode of action
作者: 江迪蔚
Chiang, Ti-Wei
關鍵字: 檬果黑斑病
mango bacterial black spot
枯草桿菌
生物防治
Xanthomonas campestris pv. mangiferaeindicae
Bacillus subtilis
WG6-14
出版社: 植物病理學系所
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摘要: 中文摘要 檬果黑斑病 (mango bacterial black spot, MBBS ),是由Xanthomonas campestris pv. mangiferaeindicae (Manicom, 1984) 所引起,於世界主要的檬果產地普遍發生,是檬果外銷產業的主要限制因子。黑斑病在台灣最早於1969年時於台南發現,之後在台灣各主要檬果產地陸續的發生,為影響檬果栽培品質與產量最重要之細菌性病害,由於主要透過傷口及自然開口感染,在果實及葉片上形成黑色的壞疽病斑,發生時不但會造成產量的嚴重損失,並使果實品質下降,失去商品價值。檬果黑斑病對既有推廣化學藥劑抗藥性普遍,防治殊為不易,再加上農藥殘留的疑慮,亟須開發替代的防治方法。由過去的研究已證實,本實驗室所分離、保存之本土枯草桿菌菌株群 (Bacillus subtilis group, BSG) 對於由黃單胞菌屬 (Xanthomonas spp.) 細菌所引起之植物病害具有優異的防治效果,經試驗證明對於包括由X. oryzae pv. oryzae所引起的水稻白葉枯病及由X. axonopodis pv. citri所引起的柑橘潰瘍病等病害的防治應用上,均有優異的表現。本研究由包括WG6-14、TKS1-1、SP4-17、BS-1與WP8-12等5個研究室既有抗生活性與生長產孢性狀俱為優異之BSG菌株,經由對峙培養篩選,證實對檬果黑斑病菌株XN12均具有良好的拮抗性,此5個菌株繼而以SYM培養液行震盪培養,經逐日檢測其菌量及培養濾液對檬果黑斑病菌株XN12之抗生活性,進一步發現以WG6-14、TKS1-1與BS-1表現性最好。此5個BSG菌株之培養菌液經調整至菌量1×108 cfu/ml後,噴施處理於愛文檬果葉片後,按時序檢測葉表菌量殘存發現以WG6-14及BS-1存活性較佳,於處理後的第20天,菌量還能維持在7×104 cfu/cm2以上。繼而於溫室種植之愛文檬果以人工穿刺的接種方式評估在接種前一天以這5個菌株在同一噴灑濃度 (1×108 cfu/ml) 下對XN12在接種菌量為1×107 cfu/ml時之防治效果,由接種14天後調查發病情形,證實除WP8-12外其餘4個菌株對黑斑病均有防治效果,其中又以WG6-14菌株之防治效果最好,其發病率較以水處理之對照組降低達30%左右。基於上述之結果本研究遂選擇WG6-14作為檬果黑斑病防治主要應用之拮抗菌株。在後續的溫室試驗中,分別以本研究室既有先導液態發酵系統以及生產活體微生物製劑技術平台所產出之WG6-14菌株活體性內生孢子製劑 (菌量為1×1010 endospore/ml),及利用開放式系統進一步擴大量產之功能性營養發酵製劑 (FNF,菌量為1×108 endospore/ml),進行防治試驗。就上述檬果黑斑病之防治應用性而言,本研究另以人工誘變所獲得抗rifampicin而病原性與XN12無明顯差異之突變菌株XNr12-1作為挑戰接種病原,同上述溫室試驗作法於接種前24小時分別以WG6-14菌株之內生孢子製劑,經稀釋50X與100X後行噴灑處理,試驗結果顯示,接種後14天對照組發病率為83.3%,50X與100X WG6-14稀釋菌液處理組發病率則分別為12.3%、33%,防治效果與施用濃度關係甚為明顯,試驗過程中,另以rifampicin抗藥性為標誌,分析不同處理接種後葉片上XNr12-1族群之變化情形則發現,於每平方公分葉表,對照組於接種後第9天、第12天菌量分別上升達1×105 與1×106 cfu,以100X稀釋WG6-14處理組則於接種後第12天菌量仍只有5×104 cfu,第15天菌量才上升至1×105 cfu,而50X稀釋菌液處理則於接種後18天過程中菌量均維持在1×104 cfu,由此可知WG6-14能夠有效的抑制XNr12-1於葉片上之增殖作用。為進一步驗證WG6-14對檬果黑斑病防治之應用性,於民國95年2月開始,於台中縣烏日鄉之愛文檬果園,分別以100X稀釋之WG6-14發酵菌液及FNF進行噴施處理,於處理5次後調查果實之自然發病率後發現,WG6-14發酵菌液及FNF處理黑斑病之發病率分別為5.8%及8.9%,防治效果明顯優於化學藥劑及對照組之處理 (發病率為27.2%及27.4%)。利用溫室之人工接種模式系統於田間進行對黑斑病菌株XN12之防治試驗中發現,WG6-14發酵菌液及FNF俱有優異的防治效果,於接種14天後的發病率分別為21.7%及37%,優於三元硫酸銅 (發病率65.6%) 與對照組 (發病率79.4%) 之處理,可知WG6-14的發酵製劑於田間也具有等同於溫室之病害防治效果。在比較不同時間點處理對WG6-14防治效果影響之試驗當中發現,利用WG6-14 100X稀釋之發酵液於接種前24小時處理,其抑病效果最佳,與對照組相比 (發病率81.2%),抑病率達到53%,相較於接種後0.5小時處理,抑病率為26%,接種後24小時處理則無顯著之防治效果,由此可證明WG6-14應用於黑斑病的防治以接種前處理所得到的效果最佳,此一應用效果也在田間試驗中得到驗證。為強化WG6-14於檬果葉片上之固著纏據能力,本研究已證實適量之甲基纖維素與三仙膠之添加效果相當明顯,其中以1000 ppm甲基纖維素 (CMC) 對菌量的提升效果最佳,於試驗期間也發現CMC之添加處理菌量有短暫上升的趨勢,此一結果可能與WG6-14對CMC的養分利用效應有關。於處理7天後,1000 ppm之CMC添加處理菌量為對照組的5.5倍。在比較CMC之添加對於WG6-14於黑斑病防治效果影響的試驗當中,添加1000 ppm之CMC對於防治效果有提升的作用,由此可知CMC的添加對於WG6-14於檬果葉片上附著之助益性及對黑斑病防治之增益效果,值得WG6-14生物製劑配方改進上應用參考。至於就防治效果有關的抗生活性而言,本研究已証實可經由0.22μm之過濾膜過濾,Waters C18 Sep-Pak® 管柱 (1 ml) 吸附,繼而以定量60%與80% (V/V)之酒精沖提回收,獲得具抗生活性之抽出液,經減壓濃縮後以薄層色層分析顯示可於Rf值0.78至0.90處偵測到具抗生活性部分,利用呈色反應更已證實所產生之抗生物質可能為含胺基酸或脂質之衍生物。此外由此抗生物質於45℃至100℃高溫處理下其抗生活性不受影響,進而顯示有可能為較不易因高溫而被破壞之環肽類。其有關的化學屬性與生物活性仍有待進一步瞭解。綜合本研究之結果可以得知,枯草桿菌菌株WG6-14應用於檬果黑斑病之防治上具有極為優異的防治效果,由於BSG於環境中的應用其安全性廣受肯定,如能將WG6-14推廣於田間的檬果栽培管理當中,不但能降低消費者對於農藥殘留所造成之疑慮,對於維持生態的平衡也有很大的助益。
The bacterial black spot caused by Xanthomonas campestris pv. mangiferaeindicae (Manicom, 1984) is the most important bacterial disease on mango world wide. The bacteria infect mainly through natural openings and wounds which lead to the development of blackish necrotic lesion on leaves and fruits. The severe infection generally lead to great reduction of yield and quality of the fruit production. In Taiwan, the disease was first reported in 1969 and was then found widespread among the orchard allover the island. The chemical control depends mainly on the application of copper fungicides. As the efficacy of chemical control was often limited, the yield loss was enormous throughout the years. And the extensive use of chemicals have caused problems of great concern including chemical resistance, environmental deterioration, ecological imbalance and even residual toxicity threatening food safety. In Taiwan, the disease is now one of the limiting factors for mango export , and the development of biological agent for disease control is urgently in need. The main objectives of the investigation herein discussed was to explore the use of Bacillus subtilis native resources for controlling black spot disease on mango. For years, we have noticed that most of the natural strains of Bacillus subtillis group (BSG) isolated from Taiwan were strongly antagonistic against most known important phytopathogenic Xanthomonads. And the values of their uses for the control of bacterial blight of rice (caused by Xanthomonas oryzae pv. oryzae ) and bacterial canker on citrus (caused by Xanthomonas axonopodis pv. citri) have been demonstrated (Wang, 2002; Chiu, 2003). In this investigation, 5 BSG strains shown with superior antagonistic activity against X. campestris pv. mangiferaeindicae XN12 strain in a preliminary dual culture assay namely WG6-14, TKS1-1, SP4-17, BS-1 and WP8-12 were screened for effectiveness as the microbial agent for controlling black spot disease on mango. By a shaking flask system using SYM (Sucrose yeast molasses medium ) broth medium, the growth characteristics and the antibiotic activity of the test strains were compared. The cultural obtained were than evaluated by an artificial inoculation model system in greenhouse for the effectiveness in disease control. The overall performance indicated WG6-14 was among the test strains the best for the disease control application. The foliar application of WG6-14 broth culture at 100X dilution 24 hours prior multi-needle prick inoculation of X. campestris pv. mangiferaeindicae strain XN12 reduced the infection by 30% compared to that of water treated control. A serial disease control trials were later proceeded in greenhouse and in field with the use of a fermentor produced WG6-14 biofungicide preparation (contains mainly 1.0×1010 endospores/ml) and an open tank system produced functional nutritive formulation (FNF, contains mainly 1.0×108 endospores/ml) where that the fermentor produced WG6-14 bio fungicide preparation was used as starter inoculum. In greenhouse trial where that newly developed leaves of mango (Mangifera indica L. cv. Irwin) were prick inoculated with XN12 (1×107 cfu/ml), the spray treatment of a fermentor produced WG6-14 at 50X and 100X in dilution 24 hours before inoculation reduced the disease incidence (14 day after inoculation ) down to 12.3 and 33%, respectively. The efficacy of disease was rather significant since disease incidence of the compared control went up to 83.3% at the same time. In order to monitor the population dynamic of the antagonist (WG6-14) and the pathogen (XN12) on the infection court after inoculation, a refampicin resistant mutant of XN12 designated as strain XNr12-1, was developed. The mutant strain was shown with virulence unchanged comparing to the parental strain. On mango leaves, the propagule density of XNr12-1on the inoculation site increased about 100 folds (approximately 106 cfu/cm2) 12 day after inoculation. With the spray application of WG6-14 24 hours before inoculation, the population increase of XNr12-1 was greatly inhibited. On leaves with 50X diluted WG6-14 application, the propagule density of XNr12-1 was kept nearly unchanged (≒104 cfu/cm2) for 18 day. Whereas for that received 100X diluted WG6-14 treatment, the propagule density of XNr12-1 increased about 10 folds 15 days after inoculation. The efficacy of disease control was apparently the function of the WG6-14 to remain as the dominant microbial colonizer on the foliar surface. The time of application appears to be critical for the biocontrol efficacy. In a followed greenhouse trial performed, the spray application of WG6-14 (100X in dilution) reduced the disease incidence by 53% (the disease incidence of compared control was 81.2%). When the treatment time was delayed to 0.5 hrs after inoculation, the inhibition efficacy of WG6-14 was only 26%. The results obtained suggest strongly the use of WG6-14 only as preventive treatment. The effect of disease control shown in greenhouse was demonstrated to work well in the field. In the field trial proceeded in a mango (cv. Irwin) orchard at Taichung, the disease incidence of black spot on leaves of mango trees receiving weekly foliar spray of 100X diluted WG6-14 or FNF for 5 consecutive times was 5.8 and 8.9% respectively. Whereas that of the compared tribasic copper and water treated control was 27.2% and 27.4% respectively. The efficacy of disease control was again shown to be a function of WG6-14 to remain as dominant colonizer on the foliar surface, the amendment of carboxy methyl cellulose (CMC) was found of beneficial for increasing the deposition and the survival of WG6-14 on the foliar surface. With the supplementation of 1000 ppm CMC, a transient increase of WG6-14 on foliar tissue was detected and the propagule density remained at approximately 5.5X for 7 day comparing to that of non-treated control. The use of CMC as amendment for WG6-14 biofungicide formulation was thus recommended. How this may contribute to disease control effectiveness remains to be investigated. In regard to the mechanism of antagonism, extraction of antibiotic from WG6-14 culture broths were attempted by use of to Waters Sep-pek® C18 column chromatography. The antibiotics were detected mainly at the fractions eluted with 60% and 80% ethanol. When resolved by thin layer chromatography (TLC), the antibiotics were detected at Rf 0.78-0.90. And the color reaction indicated their amino nitrogen and lipid containing characteristics. The results provided in this investigation demonstrated the use of Bacillus subtilis WG6-14 strain as an effective biofungicide for controlling black spot disease on mango. As members of Bacillus subtilis group were generally considered to be safe (GRAS) for use in the environment, its commercialized application should benefit the mango production as well as the sustainability of the agricultural ecosystem.
URI: http://hdl.handle.net/11455/30986
其他識別: U0005-2808200612340300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2808200612340300
Appears in Collections:植物病理學系

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