Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (8): 272-282.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0038

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Isolation, Identification and Growth-promoting Characteristics of Endohyphal Bacterium 7-2H from Endophytic Fungi of Spiranthes sinensis

FANG Lan1(), LI Yan-yan2, JIANG Jian-wei1, CHENG Sheng1, SUN Zheng-xiang1(), ZHOU Yi1()   

  1. 1. College of Agriculture, Yangtze University, Jingzhou 434025
    2. Tobacco Research Institute of Hubei Province, Wuhan 430000
  • Received:2023-01-17 Online:2023-08-26 Published:2023-09-05
  • Contact: SUN Zheng-xiang, ZHOU Yi E-mail:1605867934@qq.com;sunzhengxiang9904@126.com;zhouyi@yangtzeu.edu.cn

Abstract:

Endohyphal bacteria are a special class of bacteria that play an essential role in host fungi and ecosystems. To explore the endohyphal bacteria resources with growth-promoting functions in endophytic fungi of Spiranthes sinensis and explore their biological functions,the endohyphal bacteria were isolated by mycelial tissue grinding and identified by colony morphology, physiological and biochemical properties and molecular characteristics, after detecting the presence of endohyphal bacteria in the mycelium of Epicoccum sorghium by fluorescence in situ hybridization(FISH). Then the endogeneity of the bacteria was verified by specific primers, and the growth-promoting characteristics were determined by Salkowski colorimetry, CAS colorimetry and Mo-Sb colorimetry. The growth-promoting ability of endohyphal bacteria was preliminarily verified by rice seed growth-promoting experiments, and the whole genome sequencing and growth-promoting functional gene analysis of endohyphal bacteria were performed. The presence of bacteria within the mycelium of the fungus E. sorghinum was observed after hybridization with the fluorescein-labeled single-stranded nucleic acid probe, which might confirm the mycelium contains bacteria. An endohyphal Rhizobium oryzihabitans was isolated from the mycelium and named as 7-2H, which had the ability of producing indole-3-acetic(IAA)and siderophore as well as solublizing phosphate, and significantly increased the shoot length, root length, fresh weight and dry weight of rice seedlings. The average nucleotide identity(ANI)value between the genome of strain 7-2H and the standard strain Rhizobium oryzihabitans M15 was 96.98%. In the genome of strain 7-2H, genes related to IAA production, siderophore and phosphate solubilization were revealed. An endohyphal Rhizobium oryzihabitans is isolated, which has fine plant growth-promoting property and has a potential to be developed to be microbial fertilizer.

Key words: Spiranthes sinensis, endophytic fungus, Epicoccum sorghinum, endohyphal bacteria, Rhizobium oryzihabitans, growth-promoting characteristics, whole genome sequencing