Biocontrol of bacterial wilt in tomato with a cocktail of lytic bacteriophages.

Bacteriophages (phages) have been proposed as promising alternative pesticides against various bacterial diseases of crops. However, the efficacy of phages in managing plant bacterial diseases is variable and poorly understood in natural settings. In this study, two lytic phages, RpT1 and RpY2, were investigated for their biocontrol potential against bacterial wilt by Ralstonia pseudosolanacearum invasion in tomato plants. The two phages possess similar morphology and genome organization to those of the Autographiviridae family with a broad host range. Treatment with the two phages (alone or in combination) resulted in a significant reduction in bacterial wilt incidence. Three days post-treatment with phages, which was performed after R. pseudosolanacearum inoculation with a specified density of 108 PFU (plaque forming units)/g of soil, led to the most effective biocontrol activity compared to other treatments and a lower density of phage. A phage cocktail containing both RpT1 and RpY2 suppressed disease symptoms in agricultural soils, mimicking their ability to control diseases in natural settings. Furthermore, supplementation with specific adjuvants enhanced the biocontrol potential of both phages. The persistence of the two phages under various environmental conditions indicates their stable activity in soils. Consequently, the consistent biocontrol activity of these phages provides insights into the proper application, timing, and density of phages for effective phage therapy in bacterial wilt control in tomato. KEY POINTS: • Biocontrol potential of phages in natural settings individually and as a cocktail. • Apparent long-term persistence of phages in natural soils, various temperatures, and pH. • An effective approach for developing phages for biocontrol.

Complete Genome Sequence of a Novel Bacteriophage RpY1 Infecting Ralstonia solanacearum Strains.

Ralstonia solanacearum species complex is deleterious plant pathogenic bacteria causing bacterial wilt in the members of solanaceous crops and the bacterial wilt is difficult to control. Bacteriophages-based biocontrol is an environmentally friendly and promising strategy to control bacterial plant diseases. In this study, we isolated 72 phages from the various crop cultivated soils in Korea using five different strains of R. solanacearum. Among 72 phages, phage RpY1 was selected for further study based on the specificity of the targeted host. This phage was identified as a member of Podoviridae with a head measuring 60-70 nm in length and short tail according to the morphology of transmission electron microscopy images. The genome size of phage RpY1 is 43,284 bp with G + C content of 61.4% and 53 open reading frames (ORFs), including 18 annotated ORFs and 35 hypothetical proteins. This phage genome showed no homology to the genome of known phages except for the DU_RP_II phage infecting R. solanacearum; however, the host range of phage RpY1 is much narrower than that of DU_RP_II.

Biochemical and Structural Basis of Triclosan Resistance in a Novel Enoyl-Acyl Carrier Protein Reductase.

Enoyl-acyl carrier protein reductases (ENR), such as FabI, FabL, FabK, and FabV, catalyze the last reduction step in bacterial type II fatty acid biosynthesis. Previously, we reported metagenome-derived ENR homologs resistant to triclosan (TCL) and highly similar to 7-α hydroxysteroid dehydrogenase (7-AHSDH). These homologs are commonly found in Epsilonproteobacteria, a class that contains several human-pathogenic bacteria, including the genera Helicobacter and Campylobacter Here we report the biochemical and predicted structural basis of TCL resistance in a novel 7-AHSDH-like ENR. The purified protein exhibited NADPH-dependent ENR activity but no 7-AHSDH activity, despite its high homology with 7-AHSDH (69% to 96%). Because this ENR was similar to FabL (41%), we propose that this metagenome-derived ENR be referred to as FabL2. Homology modeling, molecular docking, and molecular dynamic simulation analyses revealed the presence of an extrapolated six-amino-acid loop specific to FabL2 ENR, which prevented the entry of TCL into the active site of FabL2 and was likely responsible for TCL resistance. Elimination of this extrapolated loop via site-directed mutagenesis resulted in the complete loss of TCL resistance but not enzyme activity. Phylogenetic analysis suggested that FabL, FabL2, and 7-AHSDH diverged from a common short-chain dehydrogenase reductase family. This study is the first to report the role of the extrapolated loop of FabL2-type ENRs in conferring TCL resistance. Thus, the FabL2 ENR represents a new drug target specific for pathogenic Epsilonproteobacteria.

Rhizobacterial syntrophy between a helper and a beneficiary promotes tomato plant health.

Microbial interactions impact the functioning of microbial communities. However, microbial interactions within host-associated communities remain poorly understood. Here, we report that the beneficiary rhizobacterium Niallia sp. RD1 requires the helper Pseudomonas putida H3 for bacterial growth and beneficial interactions with the plant host. In the absence of the helper H3 strain, the Niallia sp. RD1 strain exhibited weak respiration and elongated cell morphology without forming bacterial colonies. A transposon mutant of H3 in a gene encoding succinate-semialdehyde dehydrogenase displayed much attenuated support of RD1 colony formation. Through the subsequent addition of succinate to the media, we found that succinate serves as a public good that supports RD1 growth. Comparative genome analysis highlighted that RD1 lacked the gene for sufficient succinate, suggesting its evolution as a beneficiary of succinate biosynthesis. The syntrophic interaction between RD1 and H3 efficiently protected tomato plants from bacterial wilt and promoted tomato growth. The addition of succinate to the medium restored complex II-dependent respiration in RD1 and facilitated the cultivation of various bacterial isolates from the rhizosphere. Taken together, we delineate energy auxotrophic beneficiaries ubiquitous in the microbial community, and these beneficiaries could benefit host plants with the aid of helpers in the rhizosphere.