一株番茄青枯病生防菌的鉴定与防病、定殖能力初探

Identification, colonization and disease prevention capacity of a antagonistic bacteria against Ralstonia Solanacearum

摘要:

摘要：【目的】采用根系分泌物培养基筛选到一株番茄根际优势细菌YPP-9。本文分析测定该菌株对植物青枯病菌茄科雷尔氏菌的拮抗作用和控病能力，及其在番茄根际的定殖能力，并系统分析该菌株的分类学地位。【方法】以平板双重培养法和温室盆栽试验分别测定菌株对病原菌的拮抗能力和对番茄青枯病的控病能力；利用变性梯度凝胶电泳技术分析菌株在番茄根际的定殖能力；以形态学和生理生化特性以及16S rRNA基因序列分析确定菌株的分类地位。【结果】菌株YPP-9对茄科雷尔氏菌SSF-4的平板抑菌带宽为5 mm，其盆栽控制番茄青枯病的效果达63.7%。菌株YPP-9在番茄根际具有较好的定殖能力。该菌株培养24 h后菌落呈奶酪色，革兰氏染色阳性，菌体杆状、大小1.8-4.1 μm×0.9-1.1 μm，形成芽孢，芽孢中生或偏端生且为近似柱形，孢囊不膨大，无伴孢晶体，侧生鞭毛。菌株生长pH范围为pH 5.5-8.5且最适生长pH为6.0，生长温度范围为20℃-45℃且最适生长温度为30℃。The BIOLOG GP2结果显示该菌为芽孢杆菌属。16S rRNA基因序列分析显示该菌株与Bacillus fumarioli的亲缘关系最近且序列相似性为97%，且其序列号为FJ231500。该菌株的G+C含量为41.9%，甲基萘醌主要类型为MK-7，细胞壁脂肪酸的主要种类为C14:0 iso、C15:0 iso 和C16:0 iso以及C16 : 1ω7c alcohol且含量分别为28.27%、19.59%、12.93%和10.88%。【结论】菌株YPP-9对茄科雷尔氏菌具有良好的拮抗作用和盆栽控病能力，且能良好的定殖于番茄根际。分类学上，该菌株归入芽胞杆菌属（Bacillus），并可能是一个新的种。

Abstract:

Abstract: [Objective] To isolated a bacterial strain YPP-9, dominantly colonizing the rhizosphere of tomato using root exudate medium. In this study, we investigated the antagnism and disease-controling effect against Ralstonia solanacearum, evaluated the ability to colonize the rhizosphere of tomato, and further analyzed the phylogeny of YPP-9. [Methods] To evaluate the antagnism against R. solanacearum and the biocontrol on tomato bacterial wilt by YPP-9 respectively employing plate culture method and pot experiment in green house. We analyzed the rhizosphere colonization of YPP-9 by PCR-denaturing gradient gel electrophoresis, and also identified the taxonomic position of YPP-9 using morphological and chemotaxonomic characteristics together with 16S rRNA gene phylogenetic analysis. [Results] YPP-9 suppressed the growth of R. solanacearum (strains SSF-4) in vitro with the inhibition zone of 5 mm. The disease-control efficiency against tomato bacterial wilt in pot was 63.4%. YPP-9 also colonized the rhizosphere of tomato well. The colonies were cream in colour after 24 h culture. Cells were gram-positive, rods (1.8-4.1 μm × 0.9-1.1 μm) and formed endospores. Endospores were mainly ellipsoidal to cylindrical and lied in subterminal, and occasionally paracentral, positions in no swollen sporangia. No crystal protein. The pH range for YPP-9 growth was 5.5-8.5 with the optimum at pH 6.0, and the temperature for YPP-9 growth was 20 to 45 degrees with the optimum at 30 degrees. The results of BIOLOG GP2 showed that YPP-9 was Bacillus. Phylogenetic analysis of the 16S rRNA gene sequence revealed that YPP-9 was the most closely related to Bacillus fumarioli, with the sequence similarity of 97.7%. The sequence number was FJ231500. The DNA G+C content was 41.9%. The major menaquinone was MK-7. The dominant fatty acids in cell wall were C14:0 iso, C15:0 iso, C16:0 iso and C16:1ω7c alcohol, with the contents of 28.27%, 19.59%, 12.93% and 10.88%, respectively. [Conclusion] Bacterium YPP-9 strongly inhibited R. Solanacearum in vitro and efficientlyly suppressed the disease development in pot experiment. YPP-9 also colonized the tomato rhizosphere well. Taxonomically, YPP-9 is affiliated to Bacillus, and probably a novel species.