比較探討兩株芽孢桿菌分離株特性及其與可食菊科植物萃取物綜合配方之生物防治效果

Abstract

芽孢桿菌屬具有促進植物生長以及防治病害之功用，又因其可產生耐抗逆境之內生孢子，故產品耐儲藏，現今已有許多商品上市。芽孢桿菌 EN01 於2018年從新北市永和區農田間之青蔥葉上分離而得，此菌株經生化鑑定及分子鑑定後，將其命名為貝萊斯芽孢桿菌 EN01。實驗室分離之液化澱粉芽孢桿菌 WF02以及兩株分離自市售產品之芽孢桿菌 (枯草桿菌Y1336, 臺灣寶®; 液化澱粉芽孢桿菌 BPD1, 活力磷寶®) 用於後續實驗之比較分析。WF02 在先前研究已證實可減緩番茄青枯病及瓜類果斑病之發病率及嚴重度。此外，亦發現芽孢桿菌屬與菊科植物萃取物進行綜合配方時，可提升其生物膜形成以及細胞活力。根據對峙培養結果發現EN01 及 WF02分別對於抑制植物病原性真菌及植物病原性細菌有較佳之功效。值得注意的是在供試菌株中，除了BPD1外，在未添加誘導物之MOLP培養基中所培養得到之發酵上清液，雖不具有抑制細菌卻具有抑制真菌之功效。此外，芽孢桿菌產生之揮發性物質還具有抑制真菌生長及影響細菌病原性之功能。在盆栽試驗中，單獨施用EN01 與WF02或是分別與菊科植物萃取物進行綜合配方後可有效減緩瓜類幼苗之果斑病嚴重度，以及減少病原菌在寄主植物體內之族群量。經實驗發現此效果歸因於兩菌株可誘導植物之過氧化氫及次級代謝物之生成，如: 苯丙氨酸氨裂合酶、過氧化酶及木質素。綜合上述，兩芽孢桿菌 (EN01 及WF02) 在與適當濃度之菊科植物萃取物進行綜合配方時，具有作為生物防治製劑之潛力。

Bacillus species are well known for their beneficial effects on plant growth promotion and biocontrol of plant diseases. Since they are able to produce endospores, allow them to resist adverse environments, and make them easily be formulated as a stable dry powder with a long shelf life of the commercial products. Bacillus sp. strain EN01 was newly isolated from green onion in a vegetable field located at New Taipei City, Taiwan in 2018. This strain was classified as B. velezensis by biochemical and phylogenetic analyses. A laboratory-isolated B. amyloliquefaciens strain WF02 and two commercial strains isolated from commercial products, B. subtilis strain Y1336 and B. amyloliquefaciens strain BPD1, were used for comparative analyses. WF02 has presented potential biocontrol effects of bacterial wilt in tomato and bacterial fruit blotch in cucurbit plants. It has been found that there were synergistic effects of the formulation containing Bacillus strains with edible Asteraceae plant extract (AP) on the biofilm formation ability as well as cellular vitality of individual Bacillus spp. strains under limited nutrient environment. According to the results of dual culture assays, both EN01 and WF02 showed excellent antifungal and antibacterial activities compared with those of commercial strains, respectively. Notably, the supernatant of the respective bacterial culture grown in the MOLP medium without additives showed no antibacterial activity, except that of BPD1. On the other hand, either broth or supernatant of the tested strains showed inhibition of the pathogenic fungi growth. Otherwise, the volatile organic compounds (VOCs) of individual Bacillus spp. strains showed in vitro antifungal activities and affected the pathogenesis properties of plant pathogenic bacteria. In pot experiments, either single-strain inoculant (EN01 or WF02) or formulated with AP displayed biocontrol activities of bacterial fruit blotch on muskmelon seedlings. Both the disease severity and pathogen population were declined compared with those of the control (Acidovorax citrulli, Aac). According to the results of enzymatic and histochemical staining assays, these effects might derive from the induction of H2O2 as well as the production of secondary metabolite-related compounds, such as phenylalanine ammonia-lyase and lignin. To sum up, these two Bacillus spp. strains, EN01 and WF02, while formulating with appropriate concentrations of AP, can be potential microbial biological control agents.