拮抗性桿菌屬(Bacillus spp.)於水稻白葉枯病防治之應用及其作用機制

Abstract

近來水稻白葉枯病已成為台灣及亞洲主要稻米栽培國家之重要病害之一，其對水稻之生長與產量造成嚴重的影響。由於化學防治殊為不易，本病害之防治管理仍有待抗病性水稻品種之發展。不幸地，由於自然界病原族群新生理小種的演化快速，加上栽植環境的不適宜，使得既有抗病品種之抗病性表現常未盡理想，因此迫切需要有效的病害管理模式來防治此病害，而生物防治顯然是一不錯的防治策略。本研究旨在開發本土分離之拮抗性桿菌菌株於水稻白葉枯病防治之應用並檢測其可能之防治作用機制。由本研究室微生物種原庫所收集共計326個桿菌屬菌株，經測試篩共選出8個對真菌與細菌具有廣泛拮抗族譜 (spectrum)且具有優異之生長與產孢能力之菌株，用來測試其對防治水稻白葉枯病之效果。此8個供試菌株經對峙培養測試，證實其均對白葉枯病菌XF13菌株均具有良好的拮抗能力，在溫室中將培養菌液噴佈施用於水稻葉片亦已證實菌量均可維持104 cfu/cm2至少20天。為瞭解這些菌株防治白葉枯病之功效本研究利用剪葉接種方式將拮抗菌培養液與白葉枯病原菌株XF13菌液同時處理於台梗8號水稻植株以評估其應用潛力。測試結果證實此8個菌株中有五個對白葉枯病的感染與病斑的擴展具有良好的抑制效果，其中又以TKS1-1與WG6-14菌株防治效果最為顯著。此兩菌株經利用本研究室既有的搖瓶培養系統，證實其在SYM培養液中培養五天其內生孢子產量均可達2.5×109 cfu/ml以上，且伴隨其內生孢子之形成並有抗生活性明顯提高之現象。此外培養基中 citrulline、 ornithine 與 histidine 等胺基酸氮素源添加已證實均可顯著提昇其抗生活性。此兩拮抗菌株對於白葉枯防治之應用性，經利用溫室水稻剪葉接種方式進行測試，結果發現兩拮抗菌於包括TK8、TN67、TCS10與TN1等不同品種水稻之白葉枯病皆有顯著之防治效果，此一防治應用性繼而於苗栗苑裡田間同法進行檢測也證實效果同樣顯著。而經過重複之測試顯示此防治效果具有濃度效應，以10~100倍稀釋之WG6-14菌液施用其防治效果比1000倍稀釋效果佳，其防治效果可明顯由剪葉切口處拮抗菌抑制壞疽病斑的情形清楚觀察到。另於 WG6-14 1000倍稀釋處理組雖白葉枯病病原菌於水稻上之菌量於初期有明顯減少現象，但到第五天後病原菌族群有回升之趨勢。利用離心過濾方式分離菌體和培養液分別測試，發現施用 WG6-14對水稻白葉枯病之防病成效乃為菌體 (主要為內生孢子) 本身及培養液內菌體所產生之代謝產物共同加成之效果；試驗中發現以不含培養液之菌體懸浮液或以濾液進行防治，效果皆明顯劣於包含菌體之培養液直接施用。另於接種前三天或接種後三天以10倍稀釋WG6-14菌液用以浸漬方式處理剪葉切口亦證實可有效防治葉片切口所產生之病徵。除剪葉傷口上的施用成效外，試驗中也發現WG6-14以澆灌方式也可有效防治此病害，以100及500倍稀釋之WG6-14菌液處理溫室種植之TK8水稻發現與處理水之對照組比較可減輕罹病度達37%左右。雖田間防治測試效果評估仍須未來延續田間試驗方可證實，但目前所獲得的數據已證實WG16-14與TKS1-1為相當具有潛力防治水稻白葉枯病之生物防治菌株。另為了瞭解本試驗中病害之防治是否和抗病性之誘導有關，本研究中亦選殖水稻 PAL (phenylalanine ammonia lyase) 及PR-1 (pathogenesis related protein) 基因片段用以偵測水稻抗病性表現之情形，試驗中利用北方轉漬雜合反應，已證實供試水稻在施用100倍稀釋之WG6-14後，於3-4天後PAL基因有誘導大量表現之現象，而施用100倍稀釋之WG6-14菌液澆灌水稻植株於接種後三天也具有明顯PAL與PR-1表現的情形。此一結果是否即顯示試驗中所見施用拮抗菌之病害防治成效為phenylpropanoid代謝路徑相關反應被活化所致，仍有待更進一步之研究證實。

Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) has recently become one of the most devastating diseases of rice cultivated in Taiwan and in most of Asian rice growing countries. Because the lack of effective chemicals, the disease management depend greatly upon the use of resistance cultivars. Unfortunately, the performance of disease resistance is quite often unsatisfactory due to the fast evolution of pathogen traits and/or the unsuitable environmental conditions. Measurement for effective disease control is urgently in need, and biological control appears to be the best among the known alternative strategies. The main objectives of this investigation were to explore the potential of antagonistic bacilli native in Taiwan for the control of leaf blight on rice, and to learn the possibly involved mechanism. A total of 8 Bacillus spp. isolates, putatively chosen from 375 antagonistic bacilli isolate collection based on the understanding of their superior performance in antagonisity against wide spectrum of fungal and bacterial pathogens and good growth and sporulation characteristic, were screened for their competence in leaf blight control. By dual culture assay, all these isolates showed strong antagonisity against the targeted Xoo isolate XF13. And on greenhouse grown paddy rice, all of them survived well on the foliar tissue up to 20 days after spray application. The disease control efficacy was evaluated by greenhouse test in which culture broths of each test bacterium were applied to leaves of TK8 cultivar rice simultaneously with Xoo isolate XF13 by clip inoculation. Among the 8 isolates tested, 5 appeared to be effective in counteracting the infection by XF13. And among the 5, isolates TKS1-1 and WG6-14 consistently performed the best and were thus used for continued disease control screening trials. By shaking culture, the two isolates both appeared to grow well in SYM broth medium; the yield of endospore achieved 2.1 X 109 cfu/ml 5 days after inoculation and in parallel to that was the increase of the antagonisity. The antagonistic activity was significantly enhanced by supplementation of citrulline, ornithine, and histidine. The efficacy of leaf blight control of both isolates was demonstrated by a greenhouse test by the clip-inoculation described. The application of both isolates successfully controlled the leaf blight infection on TK8, TN 67, TCS 10, and TN 1 rice; the efficacy was manifestated by the significantly reduced disease severity and percent infection. Similar results were later obtained in a field trial conducted in Yun-Li, Miao-Li, indicating both isolates are of great value as regard to biological control of the bacterial leaf blight. The efficacy appeared be concentration dependent; satisfactory control efficacy was consistently observed in 10 to 100X diluted WG6-14 treated plants comparing to that treated by 1000X diluted sample. The disease control efficacy appeared to due mainly to the complete inhibition by the applied antagonist on the propagation of Xoo on the clipping wounds. In 1000X diluted WG6-14 treated plants, although the propagation of Xoo was significantly reduced at the early phase of infection, its propagule number was later increased at 5th day after inoculation to the level comparable to that of water treated control plants. The observed inhibitory effect on Xoo was apparently a combined function of applied WG6-14 propagules (endospores primarily) and the accumulated metabolites in the broth culture. The application of WG6-14 bacteria per se and the culture filtrate-each respectively obtained by centrifugation separation of the culture broth, were both less effective in inhibiting Xoo infection as comparing to that by the whole broth culture. By submerging treatment of the cutting wounds with a 10 X diluted WG6-14 culture broth, effective control of Xoo infection was succeeded even with the submerging treatment performed 3 days before or after the Xoo inoculation. It was also noted that the effectiveness of disease control by WG6-14 was prominent even by drenching application. On TK8 rice grown in greenhouse, drenching application of WG6-14 culture broth at 500X and 1000X in dilution reduced the disease severity approximately 37% as comparing to that of water treated control. Although more extensive field trial are needed in order to conclude the disease control efficacy observed, the data presented clearly demonstrated the great potential of WG6-14 and TKS1-1 as biofungicide for the management of bacterial leaf blight of rice. In order to learn whether or not the observed disease control efficacy may be connected to induction of disease resistance, partial sequence of PAL (phenylalanine ammonia lyase) and PR-1 (pathogenesis related protein) genes were cloned by PCR amplification from TK8 rice and used as the molecular tool for monitoring the expression of these resistance pertained genes. Analysis by northern hybridization has detected a transient increase of PAL gene in foliar tissue 3-4 days after spray application with WG6-14 at 100X in dilution. Whether or not this suggests the enhancement of phenylpropanoid pathway in observed disease control efficacy remains to be elucidated.