Bacillus mycoides的鑑定與其防治番茄萎凋病之效果評估

Identification and evaluation of Bacillus mycoides as a biocontrol agent for controlling tomato Fusarium wilt

Fusarium oxysporum f. sp. lycopersici（Fol）引起的番茄萎凋病，是台灣夏季番茄生產的主要限制因子之一。從植株體內及根圈土壤分離到140株菌株，分別導入栽培介質中培育番茄幼苗，17％的菌株可促進植株生長；15％的菌株可以有效防治番茄萎凋病。其中CHT2401、CHT2402與CHR001三菌株均可產生類似根狀的菌落（rhizoidal colony），依生理、生化、分子測試及培養形態之結果，鑑定為Bacillus mycoides。此三菌株適合生長溫度為28-36℃，在pH 4-8均生長良好，但對測試16種碳、32種氮素源之需求則有所差異。再者，三菌株培養在不同基質的培養基上，對Fol的拮抗作用亦有所不同。試驗發現：三菌株培養於nutrient agar（NB）及soy agar（SA）上生長良好，並表現有較佳的拮抗作用。菌株若培養在potato dextrose agar（PDA）、corn meal agar及malt extract agar等平板上則對Fol的拮抗效果不佳。B. mycoides菌株培養於黃豆粉與玉米粉所調製之營養液，混入帶Fol菌源之泥土中，可明顯降低番茄萎凋病之發生，且其效果與使用之黃豆粉與玉米粉濃度成正相關。番茄幼苗根經浸泡於B. mycoides菌株培養液，發現BM細菌培養於NB、5％玉米粉及5%黃豆粉，分別可降低萎凋病罹病度達36.3 - 72.67％。番茄幼苗根浸泡於Potato dextrose broth（PDB）培養的菌液卻有加速病害發生的現象。綜合結果顯示：營養液會影響B. mycoides的防病功效。

"Fusarium wilt caused by Fusarium oxysporum f. s p. lycopersici (Fol) is one of the limiting factors for the production of tomato in summer in Taiwan. The objectives of this study are to identify growth-promoting bacteria from tomato roots and rhizosphere and to evaluate their ability to control Fusarium wilt. Of 140 bacterial isolates tested, 17% were able to promote the growth of tomato seedlings and 15% reduced the incidence of Fusarium wilt. Three isolates designated CHT2401, CHT2402 and CHR001 formed rhizoidal colonies resembling Bacillus mycoides. Their identities were further confirmed by physiological and biochemical tests according to Biolog system and by sequence comparison of gyrB gene and ITS region with other known Bacillus spp. whose sequences are available in database. The optimum temperature for bacterial growth ranged from 28 to 36°C and the optimum pH ranged from 4 to 8. Among 16 carbohydrates and 32 nitrogen sources tested for growth, three B. mycoides isolates had very different preferences. The ability of B. mycoides isolates to suppress Fusarium wilt was markedly affected by the composition of the media. Bacteria grown in nutrient agar (NA) or soy powder agar were more effective in reducing Fusarium wilt than those grown on potato dextrose agar, corn meal agar, and malt extract agar. Soils mixed with bacteria suspensions that were grown in soybean meal or corn meal provided excellent protection for tomato seedlings from the damage of Fusarium wilt in a concentration-dependent manner. Tomato roots dipping in bacterial suspensions grown in nutrient broth, soybean meal or corn meal and planted in Fol-infested soils also developed fewer Fusarium wilt and powdery mildew compared to controls. However, plants dipping in bacterial suspensions grown in potato dextrose broth developed severe Fusarium wilt. Overall, our results demonstrate the importance of medium composition in the biocontrol effectiveness of B. mycoides against tomato Fusarium wilt. The identified B. mycoide isolates may be useful in developing effective biocontrol agents and providing an alternative for diseases management."