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|標題:||拮抗性桿菌屬 (Bacillus spp.) 之分離、培養與抗生活性之改進以及病害防治之應用
Antagonistic Bacillus spp.- isolation, culture and antagonisity improvement, and the application in disease control
為探討本土桿菌屬（Bacillus spp.）細菌於農業上之實際應用，由收集之田間土壤、栽培介質與有機堆肥中，利用加熱前處理，大量篩選菌落生長形態類似桿菌屬之細菌菌落，共分離出326株革蘭氏陽性桿狀菌，藉由對峙培養檢測其抗生活性，其中123株對Pythium aphanidermatum具有抗生活性，125株對Rhizoctonia solani AG4具有抗生活性，58株對Xanthomonas axonopodis pv. citri（Xac）具有抗生活性，以及58株對Xanthomonas axonopodis pv. vesicatoria (XVT12) 具有抗生活性。上述菌株經綜合其生長強勢與抗生活性之結果，篩選出HSP1、TKS1-1、OF3-17、SP4-17、WG6-14、TLB7-7與WP8-12等七株具實際應用潛力之菌株。菌株BS001為本研究室應用已久之桿菌屬細菌，並以此菌株為模式菌株，建立起液態發酵產孢技術，於本試驗中作為菌株改進之對照菌株。將上述八株菌株進行種之鑑定工作，根據Bergey’s Manual所記載之生理生化特性，除TLB7-7歸屬為B. pumilus外，其餘菌株皆歸屬於B. lichniformis ，並以Biolog 細菌鑑定系統作輔助確認，其中TLB7-7歸為B. pumilus，TKS1-1與WP8-12 歸為B. subtilis，BS001與SP4-17歸為B. megaterium，WG6-14歸為B. amyloliquefaciens，而OF3-17則無法確認其種之歸屬，雖然兩者鑑定結果有所差異，根據Priest（1993）所採行之分類方法皆歸屬為B. subtilis group。以本研究室已建立之液體發酵培養模式，將上述八株菌株培養於SYM培養液中並於溫室進行初步篩選工作，培養液澆灌於甘藍種子，發現依序以WG6-14、TLB7-7、SP4-17、WP8-12與BS001等菌株對甘藍幼苗具有較佳之生長促進效果，值得注意的是此八株測試菌株中並無抑制甘藍植株生長之現象。上述八株測試菌株於SYM培養液搖瓶培養下，菌株生長與內生孢子形成均良好，而隨著內生孢子的形成，抗生物質亦伴隨著產生，當內生孢子形成量逐漸增加時，培養過濾液之抗生物質逐漸減少，主要為內生孢子之菌體所產生之抗生物質逐漸增加，顯見存在著由不同代謝路徑所產生之抗生物質。就BS001及WG6-14兩測試菌株所產生之抗生物質而言，以XVT12及Xac作為抗生活性標的菌株，兩者培養液經調整酸鹼值後，酸鹼值對於不同標的菌株其抗生活性強度具有不同之影響，對XVT12於pH7-9之間表現出最高之抗生活性，而對Xac最高之抗生活性範圍則於pH3-6之間，而丙酮具抑制對Xac抗生物質之結果，而對於XVT12則無明顯之差異，另藉由C18逆向層吸管柱以不同濃度甲醇提洗，對XVT12之抗生物質可由70%甲醇提洗出，而對Xac之抗生物質則可由10-100%甲醇提洗出。去除菌體之BS001發酵濾液，對XVT12具有致死作用，XVT12菌液(1×109 cfu/ml)經發酵過濾液處理24小時後菌量降至100倍以下。由試驗結果顯示，對Xanthomonads產生作用之抗生物質屬為胜肰類抗生物質，且至少包含兩種抗生物質以上。為促進抗生物質之產生，於Modified Czapek’s Hoagland基礎培養基中分別添加19種胺基酸與8種無機氮，其中除胱胺酸外，胺基酸的添加相較於無機氮添加，對生長與內生孢子形成具有較佳之效果，而其中以丙胺酸、精胺酸、瓜胺酸、谷胺酸與甘胺酸的添加對菌量生長最好，可達1×108 cfu/ml以上，而於此培養基中對內生孢子的形成普遍不佳，孢子量只達1×107 cfu/ml左右，無機氮的提供無法有效促進菌量之生長，除磷酸銨及硝酸銨外，上述之無機態氮並不利於生長，然而硝酸鈣對內生孢子形成具有促進作用，為所有氮素源中促進內生孢子形成最佳者。氮素源的添加對XVT12與Xac之抗生活性促進方面，其中以組胺酸、鳥胺酸與瓜胺酸的添加處理促進效果最佳，而硝酸鉀與硝酸鈉亦有利於產生對Xac之抗生物質，然對XVT12之抗生物質則無促進之作用。而於所試驗之12種糖類中，所有糖類對BS001與WG6-14等供試菌株皆生長良好，而其中以葡萄糖、蔗糖、果糖、棉子糖、麥芽糖、甘露糖與甘露糖醇等添加處理對抗生活性具有助益之效果，除此外甘露糖醇亦具有促進內生孢子形成之作用。外加胺基酸於SYM培養液並非必須的，然而在BS001菌株接種後6或14小時添加組胺酸對產孢是有所助益的。為實際應用桿菌屬細菌於田間以防治植物病害，將篩選出具實際應用潛力之菌株先行於溫室與田間進行病害防治試驗，供試菌株BS001發酵液經200或500倍稀釋，每月於田間澆灌柑橘植株一次，有效抑制田間柑橘植株Xac之感染。而由下述兩者試驗觀察結果，桿菌屬細菌除可有效防治病害外，並可促進植物之生長與分櫱，菌株培養液經稀釋於田間澆灌使用時，對植株之生長促進效果非常顯著。茭白莖基腐病乃由腐生性細菌所感染，於埔里地區進行茭白莖基腐病害防治試驗，WG6-14發酵液經500倍稀釋，每分地施用250公升，觀察結果顯示施用WG6-14發酵液除可有效抑制茭白莖基腐病外（數據未顯示），亦可增加茭白分櫱數與茭白筍之全年產量達56%。另外於埔里地區進行蟲草白絹病害防治試驗時，BS001發酵液經500倍稀釋澆灌施用，試驗結果顯示除可增加植株存活外，並可增加塊莖之產量。雖然BS001與WG6-14兩供試菌株於病害防治方面結果仍須以試驗更進一步加以證實，但由上述試驗結果明白顯示，兩者具有實際應用田間之潛力與價值。
In order to explore the use of native bacilli for agricultural application, isolation and mass screening of antagonistic bacilli from natural resources including field soil, culture substrates, and natural composts was conducted. A total of 326 isolates of bacteria with bacilli colony characteristics was obtained by a thermal sensitized screening method previously established. The bacteria were all shown to be Gram positive rods; and among them 123 showed antagonisity against Pythium aphanidermatum (Pa), 125 against Rhizoctonia solani (AG4), 58 against Xanthomonas axonopodis pv. citri (Xac), and 58 against X. axonopodis pv. vesicatoria (XVT12). For exploring the potential of practical application, 7 isolates namely HSP1, TKS1-1, OF3-16, SP4-17, WG6-14, TLB7-7, and WP8-12, were chosen as targets for further evaluation among these isolate collection based on the overall performance of growth vigor and antagonisity. BS001, a Bacillus sp. Isolate long being used in the laboratory as a model isolate for establishing endospore production liquid fermentation technique, was also included in the test as a comparison for strain improvement. Attempted identification of these chosen isolates according to morphological and physiological characteristics shown in Bergey's Manual indicated that all of them (including BS001) can be identified as B. lichniformis except that TLB7-7 was identified as B. pumilus. However, a double check of the identification by Biolog○R system indicated that TKS1-1 and WP8-12 should be B. subtilis, BS001 and SP4-17 should be B. megaterium, and WG6-14 should be B. amyloliquefaciens. The system also confirmed the species identity of TLB7-7 as B. pumilus, but it failed to identify isolate OF3-16. The species identified, although different, all belong to B. subtilis group of Priest (1993). A preliminary greenhouse screening test using culture broth of all 8 isolates grown in SYM medium demonstrated that by drenching treatment, WG6-14, TLB7-7, SP4-17 WP8-12, and BS001 were in the order among these tested isolates the best in stimulating growth of cabbage seedlings. Also worth to mention is that none of the tested isolates showed deleterious effect on the plant growth and development.
The tested isolates all grow well and transform well into endospores in SYM broth medium under continuous shaking. In accompany to the endospore formation, a paralleled increase of antibiotics was detected which generally appeared about one day before the detection of endospore formation. It was also noted that during the period of endospore formation, there was a decrease of antibiotic in culture filtrate and in accompany to that was the increment of spore extractable antibiotics indicating the existence of different types of antibiotics and the involvement of different pathways for the production. For antagonisity characterization, effect of pH on that of BS001 and WG6-14 was tested with Xanthomonas spp. XVT12 and Xac as bioassay targets. Adjustment of pH of culture broth of both tested isolates appeared to have differential effect on the strength of activity regarding to different targets. The adjustment of pH to 3-6 appeared to favor the activity on Xac, while the adjustment to 7-9 favors that on XVT12. Moreover, the addition of acetone appeared to annihilate the antagonisity of BS001 culture broth against Xac, whereas that against XVT12 remained not much changed. By Sep-Pak C18 cartridge clean up treatment, the Xac active antibiotic constituent contained in the Millipore filtered culture filtrate of both isolates was consistently eluted by 10 to 100% series of methanol, while that of XVT12 active ones was eluted only by methanol approximately at 70%. The cell free culture filtrate when added to suspension culture of XVT12, appeared to be lethal; the viable count of XVT12 at 1 X 109 cfu/ml was reduced about 100 folds 24 hrs after treatment. The accumulated evidence suggests clearly the involvement of certain chemically related peptide antibiotics in the observed antagonisity of tested bacilli against tested xanthomonads. For improving the antibiotic production, effect of 19 amino acids and 8 inorganic nitrogen was investigated using modified Czapek's Hoagland as a basal medium. With the exception like cysteine for both test isolates and aspartic acid for WG6-14, provision of amino nitrogen apparently supported better growth and endospore formation as comparing to that by inorganic nitrogen. Among the amino nitrogen tested, the addition of alanine, arginine, citrulline, glutamate, glycine, methionine and valine were good in supporting the bacterial growth (up to 108 cfu/ml). The endospore formation in these cultures, however, was rather poor (only up to 107 cfu/ml). The provision of inorganic nitrogen was considerable less effective in promoting the bacterial growth. NH4H2PO4 and KNO3 were among them the best for supporting the growth; whereas for endospore formation, Ca(NO3)2 takes the lead. As for the antibiotic production, the best efficacy in regarding to antagonisity against both Xac and XVT12 was consistently detected from ornithine, citrulline, and histidine amended medium. The addition of KNO3 and NaNO3 also supported well the production of antibiotic against Xac, but that for XVT12 was not as good. The effect of 12 kinds of sugars including mono- and disaccharides on growth, sporulation and antagonisity was also tested. All the tested sugars appeared to support well the growth of both BS001 and WG6-14. Glucose, mannose, fructose, raffinose, maltose, mannitol, and sucrose were among them the best for antagonisity expression and mannose was among them the best for supporting sporulation. In SYM medium wherein most of the amino nitrogen were well supplied by natural substrate contained, extra addition of amino nitrogen was found not necessary as regard to promotion of the growth and sporulation of both tested isolates. The addition of histidine at 6-14 hrs after culturing, however, significantly increased the spore yield of BS001.
As for the potential of disease control application, some preliminary greenhouse and field trials were conducted. A monthly spray application of BS001 at 200 and 500X in dilution was shown to be effective in reducing Xac infection on naval orange in the field. In both experiments, what worth to mention is that in addition to efficacy of disease control, significant promotion of plant growth and improved vigor was generally observed. The plant growth promoting effect was even more prominent when the culture broth was applied by drenching application. In an attempt to control basal rot of Zizania latifolia presumably caused by certain saprophytic bacteria to be identified, drenching application of WG6-14 at 500 X in dilution, and 250 L/ha rate of application, was found effective in reducing the disease incidence in the field (data not shown). And it was especially worth noting that in accompany to that was the significantly increased tiller number and total yield of the smut gall production. Another attempt of disease control was for Sclerotium rot on Stachys sieboldii in field at Puli. The application of 500X diluted BS001 broth culture was also found effective in increasing the survival stand and the yield of tuberose root. Although the effectiveness of disease control of the both tested isolates need further works for a final documentation proof, the accumulated evidence has clearly indicated their possibility and great value in agricultural application.
|Appears in Collections:||植物病理學系|
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