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Characterization of a Novel Endophytic Actinomycete, Streptomyces physcomitrii sp. nov., and Its Biocontrol Potential Against Ralstonia solanacearum on Tomato.

ABSTRACT: Bacterial wilt of tomato is a destructive disease caused by Ralstonia solanacearum throughout the world. An endophytic actinomycete with antagonistic activity, designated strain LD120^T, was isolated from moss (Physcomitrium sphaericum (Ludw) Fuernr). The biocontrol test demonstrated that co-inoculation by the isolate and the pathogen gave the greatest biocontrol efficiency of 63.6%. Strain LD120^T had morphological characteristics and chemotaxonomic properties identical to those of members of the genus Streptomyces. The diamino acid present in the cell wall was LL-diaminopimelic acid. Arabinose, glucose, rhamnose, and ribose occured in whole cell hydrolysates. The menaquinones detected were MK-9(H₄), MK-9(H₆), MK-9(H₈), and MK-9(H₂). The polar lipid profile was found to contain diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylinositol. The major cellular fatty acids were found to be iso-C_16:0, iso-C_17:0, anteiso-C_15:0, and C_16:1 ω7c. The DNA G+C content of the draft genome sequence, consisting of 7.6 Mbp, was 73.1%. Analysis of the 16S rRNA gene sequence showed that strain LD120^T belongs to the genus Streptomyces, with the highest sequence similarity to Streptomyces azureus NRRL B-2655^T (98.97%), but phylogenetically clustered with Streptomyces anandii NRRL B-3590^T (98.62%). Multilocus sequence analysis based on five other house-keeping genes (atpD, gyrB, rpoB, recA, and trpB) and the low level of DNA-DNA relatedness, as well as phenotypic differences, allowed strain LD120^T to be differentiated from its closely related strains. Therefore, the strain was concluded to represent a novel species of the genus Streptomyces, for which the name Streptomycesphyscomitrii sp. nov. was proposed. The type strain was LD120^T (=CCTCC AA 2018049^T = DSM 110638^T).

SUBMITTER: Zhuang X

PROVIDER: S-EPMC7765990 | 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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Characterization of a Novel Endophytic Actinomycete, Streptomyces physcomitrii sp. nov., and Its Biocontrol Potential Against Ralstonia solanacearum on Tomato.

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