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A Streptomyces sp. NEAU-HV9: Isolation, Identification, and Potential as a Biocontrol Agent against Ralstonia Solanacearum of Tomato Plants.

ABSTRACT: Ralstonia solanacearum is an important soil-borne bacterial plant pathogen. In this study, an actinomycete strain named NEAU-HV9 that showed strong antibacterial activity against Ralstonia solanacearum was isolated from soil using an in vitro screening technique. Based on physiological and morphological characteristics and 98.90% of 16S rRNA gene sequence similarity with Streptomyces panaciradicis 1MR-8T, the strain was identified as a member of the genus Streptomyces. Tomato seedling and pot culture experiments showed that after pre-inoculation with the strain NEAU-HV9, the disease occurrence of tomato seedlings was effectively prevented for R. solanacearum. Then, a bioactivity-guided approach was employed to isolate and determine the chemical identity of bioactive constituents with antibacterial activity from strain NEAU-HV9. The structure of the antibacterial metabolite was determined as actinomycin D on the basis of extensive spectroscopic analysis. To our knowledge, this is the first report that actinomycin D has strong antibacterial activity against R. solanacearum with a MIC (minimum inhibitory concentration) of 0.6 mg L-1 (0.48 μmol L-1). The in vivo antibacterial activity experiment showed that actinomycin D possessed significant preventive efficacy against R. solanacearum in tomato seedlings. Thus, strain NEAU-HV9 could be used as BCA (biological control agent) against R. solanacearum, and actinomycin D might be a promising candidate for a new antibacterial agent against R. solanacearum.

SUBMITTER: Ling L

PROVIDER: S-EPMC7142955 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ralstonia solanacearum (biovar2, race3) is the causal agent of bacterial wilt and this quarantine phytopathogen is responsible for massive losses in several commercially important crops. Biological control of this pathogen might become a suitable plant protection measure in areas where R. solanacearum is endemic. Two bacterial strains, Bacillus velezensis (B63) and Pseudomonas fluorescens (P142) with in vitro antagonistic activity toward R. solanacearum (B3B) were tested for rhizosphere competence, efficient biological control of wilt symptoms on greenhouse-grown tomato, and effects on the indigenous rhizosphere prokaryotic communities. The population densities of B3B and the antagonists were estimated in rhizosphere community DNA by selective plating, real-time quantitative PCR, and R. solanacearum-specific fliC PCR-Southern blot hybridization. Moreover, we investigated how the pathogen and/or the antagonists altered the composition of the tomato rhizosphere prokaryotic community by 16S rRNA gene amplicon sequencing. B. velezensis (B63) and P. fluorescens (P142)-inoculated plants showed drastically reduced wilt disease symptoms, accompanied by significantly lower abundance of the B3B population compared to the non-inoculated pathogen control. Pronounced shifts in prokaryotic community compositions were observed in response to the inoculation of B63 or P142 in the presence or absence of the pathogen B3B and numerous dynamic taxa were identified. Confocal laser scanning microscopy (CLSM) visualization of the gfp-tagged antagonist P142 revealed heterogeneous colonization patterns and P142 was detected in lateral roots, root hairs, epidermal cells, and within xylem vessels. Although competitive niche exclusion cannot be excluded, it is more likely that the inoculation of P142 or B63 and the corresponding microbiome shifts primed the plant defense against the pathogen B3B. Both inoculants are promising biological agents for efficient control of R. solanacearum under field conditions.

Project description:Ralstonia solanacearum, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found that R. solanacearum is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the R. solanacearum flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (fliC), and for the flagellar motor switch protein (fliM) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted R. solanacearum FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lacking fliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphA cassette was used to make a nonpolar fliM mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The fliC mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the fliC mutant lacked a 30-kDa band corresponding to flagellin. The fliM mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the fliC mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.

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A Streptomyces sp. NEAU-HV9: Isolation, Identification, and Potential as a Biocontrol Agent against Ralstonia Solanacearum of Tomato Plants.

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