拮抗棉花枯萎病菌的地黄内生细菌筛选、鉴定和促生潜能
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国家自然科学基金(U1204301);河南省自然科学基金(182300410069);河南省重点研发与推广专项(科技攻关)(212102310223);国家级大学生创新创业训练计划(202010483004)


Screening, identification and growth-promoting of antagonistic endophytic bacteria associated with Rehmannia glutinosa against Fusarium oxysporum f. sp. vasinfectum
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    摘要:

    [目的] 为了筛选棉花枯萎病菌拮抗性菌株资源,从药用植物地黄根块中分离内生细菌,分析优良菌株的促植物生长特性和耐盐碱特性,发掘优良菌株资源,为研发棉花枯萎病生防菌剂提供参考价值。[方法] 采用平板对峙法对分离内生细菌进行棉花枯萎病菌拮抗性试验,荧光显微镜观察法研究病原菌菌丝的变化、分光光度计法测定吲哚乙酸(IAA)含量和1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性、平板培养法测定溶磷特性、分隔培养法测定产生挥发性物质抑菌性、比浊法测定内生菌的耐盐碱特性、通过测定代表菌株理化特性、16S rDNA序列并分析系统发育地位、盆栽接种试验验证防病效果。[结果] 地黄内生细菌对棉花枯萎病菌具有拮抗性,其中菌株DH9、DH66、DH92拮抗作用较强。与对照组菌丝对比,处理组菌丝出现打结、弯曲和断裂现象,菌丝末端分枝明显增多,多数边缘菌丝呈珊瑚状分枝,存在明显被菌体包埋等现象。菌株DH83、DH66、DH92、DH9、DH56产生挥发性物质对棉花枯萎病菌均有抑制作用,但抑制效果不明显。产生IAA含量均大于1.32 mg/L的有7株(DH92、DH30、DH71、DH83、DH93、DH9、DH56),其中DH92产量最高,为34.696 mg/L。菌株DH92和DH30产生ACC脱氨酶活力分别为118.612μmol/(mg·h)和103.795 μmol/(mg·h)。菌株DH92无机磷溶磷能力最强,溶磷圈直径/菌落直径(D/d)为1.51;菌株DH71溶解有机磷能力最强,D/d为4.50。菌株DH9和DH56分别能够耐受在7%、3% NaCl盐浓度,在pH 8–10均能生长,具有一定耐盐碱性。结合菌株培养特征、生理生化特性和16S rDNA测序及系统发育分析,结果表明DH30最相似菌株为沙福芽孢杆菌(Bacillus safensis),DH9最相似菌株为枯草芽孢杆菌(Bacillus subtilis),DH92最相似菌株为菠萝泛菌(Pantoea ananatis)。DH92处理组防治效果达77.29%,其他防效在63%以上,可作为棉花枯萎病的生防菌株资源。[结论] 药用植物地黄根块中存在棉花枯萎病菌拮抗性菌株资源,其中菠萝泛菌DH92从地黄根块中分离未曾见报道。优良地黄内生细菌具有促进植物生长特性和一定耐盐碱性,为防治棉花枯萎病和研发生物防治菌剂提供了基础。

    Abstract:

    [Objective] To screen antagonistic strain resources of Cotton Fusarium Wilt (Fusarium oxysporum f. sp. vasinfectum), endophytic bacteria were isolated from the root of Rehmannia glutinosa, a medical herb, and the excellent strains’ characteristics of plant growth promotion and salt and alkali tolerance were analyzed, so as to explore the excellent strain resources and provide reference value for the research and development of biological control agents of F. oxysporum f. sp. vasinfectum. [Methods] The antagonistic test of F. oxysporum f. sp. vasinfectum was performed by plate confrontation method, and the changes in mycelia were studied through fluorescence microscopy. Such indexes as indoleacetic acid (IAA) production and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity were determined by spectrophotometry. Phosphate solubilization was assayed by plate culture method. Bacteriostasis of volatile substances was examined by separation culture method. Salt and alkali tolerance was measured by turbidimetric method. The physiological and biochemical characteristics of the strains were determined, 16S rRNA gene were sequenced and analyzed for phylogenetic status analysis, and disease-control effects were demonstrated by greenhouse inoculation test. [Results] The endophytic bacteria of R. glutinosa exerted antagonistic effects on F. oxysporum f. sp. vasinfectum, among which the strains DH9, DH66 and DH92 exhibited obvious antagonistic effects. Compared with the control group, the hyphae in the treatment group showed knotting, bending and breaking, the end branches of hyphae increased significantly, and most of the edges were coral branches, and were obviously embedded by the bacteria. The volatile substances produced by strains DH83, DH66, DH92, DH9 and DH56 exerted inhibitory effects on F. oxysporum f. sp. vasinfectum, but such effects were not salient. IAA contents of seven strains (DH92, DH30, DH71, DH83, DH93, DH9 and DH56) were higher than 1.32mg/L.DH92 had the highest yield, which amounted to 34.696 mg/L. The activities of ACC deaminase produced by DH92 and DH30 were 118.612 μmol/(mg·h) and 103.795μmol/(mg·h), respectively. DH92 had the highest ability to dissolve inorganic phosphorus, with diameter of dissolving circle/colony (D/d) being 1.51, DH71 had the highest ability to dissolve organic phosphorus, and D/d was 4.50. The strains DH9 and DH56 could tolerate 7% and 3% NaCl concentration respectively, and could grow at pH 8–10, with certain resistance. Combined with the culture characteristics, physiological and biochemical characteristics and 16S rDNA sequencing and phylogenetic analysis, results showed that DH30 exhibited the highest similarity with Bacillus safensis, DH9 showed the highest similarity with Bacillus subtilis, and DH92 had the highest similarity with pantoa ananatis. Control effect of treatment group with DH92 was 77.29%, and those of others were above 63%. Therefore, these strains can be used as biocontrol resources against F. oxysporum f. sp. vasinfectum.[Conclusion] There are antagonistic strains of F. oxysporum f. sp. vasinfectum in the root of R. glutinosa, among them. The isolation of Pantoa ananatis DH92 from R. glutinosa root has not been reported before. The excellent endophytic bacteria of R. glutinosa have the characteristics of plant growth promotion and certain salt and alkali tolerance, which provide theoretical basis for the control of F. oxysporum f. sp. vasinfectum and the development of biological control agents.

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赵龙飞,徐亚军,邓振山,赖心河,周金源,马素珍,符可,周璞,朱艳芳. 拮抗棉花枯萎病菌的地黄内生细菌筛选、鉴定和促生潜能[J]. 微生物学报, 2021, 61(8): 2338-2357

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  • 收稿日期:2020-08-02
  • 最后修改日期:2020-11-01
  • 在线发布日期: 2021-08-04
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