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

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.