評估納豆菌K1菌株抑制稻熱病以及促進植物生長之潛力

Abstract

納豆菌因可分泌納豆激酶而被廣泛應用為人類食用益生菌。納豆菌屬枯草桿菌群，具分泌多種抗生物質的能力，因此具有作為植物保護劑的潛力。在本研究中，我們使用一株具分泌多量納豆激酶的納豆菌K1菌株，評估其生物防治以及促進植物生長的潛力。我們根據其在LB培養基上的菌落型態，挑選出四株彼此具有差異性的納豆菌，分別命名為WA， WB， WC及K1。根據16S rRNA與gyrB序列繪製的親緣演化樹以及碳源代謝活性的結果判斷，四種菌落皆屬相同菌株。在對峙培養實驗發現BsK1相較於其他三種株菌落具有更佳的抗稻熱病真菌（MoGuy11）之能力。此外，我們使用枯草桿菌標準菌株Bs168及一株台灣本土商用枯草桿菌菌株BY1336作為對照比較。BsK1及BY1336處理組相較於Bs168能更為有效地抑制稻熱病菌絲生長，而且觀察到菌絲會有異常膨大現象。我們使用對葉稻熱病具不同抗性的水稻品種，包括感病等級的台梗9號以及極感等級的台農67號來評估上述菌株的抗病能力。 若菌液是施用在根部時，BsK1或是BY1336皆可降低台農67號的葉稻熱病罹病指數; 若施用在葉面則只有Bs168具抗病效果。此外我們發現BsK1及Bs168僅有活細胞才具有抑制稻熱病菌絲的作用，而BY1336 不論是活細胞或是分子量大於3kDa的上清液皆具有抑制能力。利用影像質譜分析上述三株菌株所分泌的酯肽類抗生素發現，BY1336具分泌 iturins, surfactins 以及fengycins的能力，Bs168則完全偵測不到這些抗生物質，而BsK1只能偵測到surfactins。我們發現BsK1與稻熱病菌共同培養下所生成的surfactins的量較單獨培養多，因此推斷BsK1會受到病原菌的誘導而生合成抗生物質。另外，我們也比較了三株菌促進植物生長的能力。三株菌培養在LB時所釋放的揮發性氣體可促進阿拉伯芥及菸草小苗的生長。然而若直接將上述菌株施加到小苗根部，BY1336反而會抑制菸草及阿拉伯芥的生長，BsK1則不影響菸草生長。綜合上述結果， 因BsK1具有與商用菌株相似的抗病及促進植物生長能力，且對植物傷害更小，研判具有發展成為植保製劑的潛力。

Bacillus subtilis natto has been regarded as a kind of human probiotics due to its production of fibrinolytic enzyme (nattokinase). In this study, an elite Bacillus subtilis natto strain K1 with high productivity of nattokinase was evaluated for its biocontrol and plant growth-promoting properties. This bacterium displayed at least four distinct colony morphologies, and designated as WA, WB, WC and K1, respectively. Based on 16S rRNA/ gyrB constructed phylogenetic trees and carbon utilization traits, these four kinds of colonies belong to the same strain. However, in dual culture assay, BsK1 showed highest inhibition on the mycelia growth of a rice blast fungus, Magnaporthe oryzae Guy11 (MoGuy11). In comparison with a commercial strain (Bacillus sp. strain Y1336, BY1336) and a model strain of Bacillus subtilis (B. subtilis 168, Bs168), BsK1 displayed comparable biocontrol activity as that of BY1336. In dual culture plate assay, both BY1336 and BsK1 caused swollen hyphae and growth inhibition on MoGuy11 mycelia. Inoculation of either BsK1 or BY1336 on roots of rice cultivar TNG67 decreased the disease incidence of leaf blast. In contrast, leaf spray of these strains on cultivar TNG67 did not display any biocontrol ability. I found the broth filtrate of BY1336 could inhibit the growth of MoGuy11, however, that of BsK1 could not. Imaging mass analysis was conducted to confirm the synthesis of lipopeptide antibiotics. BY1336 could produce three kinds of lipopeptides (surfactins, fengycins, and iturins) in the presence or absence of MoGuy11. In contrast, BsK1 could only synthesize surfactins in the presence of MoGuy11. It suggests that the antibiotics of BsK1 are only synthesized in response to its antagonist. I also found both BsK1 and BY1336 were able to promote the growth of Arabidopsis thaliana Col-0 and Nicotiana tabacum W38 through volatile compounds emission when they were grown in Luria Bertani agar (LBA). In direct inoculation of BY1336 and BsK1 on both seedlings, growth of AtCol-0 were inhibited by both strains, but only BY1336 suppressed the growth of NtbW38. Taken together, BsK1 just like the commercial strain BY1336, has the potential to serve as elite biological control agent as well as plant growth stimulator.