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Genomic Analysis of the First European Bacteriophages with Depolymerase Activity and Biocontrol Efficacy against the Phytopathogen Ralstonia solanacearum.

ABSTRACT: Ralstonia solanacearum is the causative agent of bacterial wilt, one of the most destructive plant diseases. While chemical control has an environmental impact, biological control strategies can allow sustainable agrosystems. Three lytic bacteriophages (phages) of R. solanacearum with biocontrol capacity in environmental water and plants were isolated from river water in Europe but not fully analysed, their genomic characterization being fundamental to understand their biology. In this work, the phage genomes were sequenced and subjected to bioinformatic analysis. The morphology was also observed by electron microscopy. Phylogenetic analyses were performed with a selection of phages able to infect R. solanacearum and the closely related phytopathogenic species R. pseudosolanacearum. The results indicated that the genomes of vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 range from 40,688 to 41,158 bp with almost 59% GC-contents, 52 ORFs in vRsoP-WF2 and vRsoP-WM2, and 53 in vRsoP-WR2 but, with only 22 or 23 predicted proteins with functional homologs in databases. Among them, two lysins and one exopolysaccharide (EPS) depolymerase, this type of depolymerase being identified in R. solanacearum phages for the first time. These three European phages belong to the same novel species within the Gyeongsanvirus, Autographiviridae family (formerly Podoviridae). These genomic data will contribute to a better understanding of the abilities of these phages to damage host cells and, consequently, to an improvement in the biological control of R. solanacearum.

SUBMITTER: Biosca EG

PROVIDER: S-EPMC8703784 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Three new lytic bacteriophages were found to effectively control the pathogen Ralstonia solanacearum, a quarantine bacterium in many countries, and causative agent of bacterial wilt, one of the most important vascular plant diseases. Bacterial wilt management has been carried out with fluctuating effects, suggesting the need to find alternative treatments. In this work, three lytic phages were isolated from environmental water from geographically distant regions in Spain. They proved to specifically infect a collection of R. solanacearum strains, and some of the closely related pathogenic species Ralstonia pseudosolanacearum, without affecting non-target environmental bacteria, and were able to lyze the pathogen populations within a wide range of conditions comprising environmental values of water temperatures, pH, salinity, and lack of aeration found in storage tanks. The three bacteriophages displayed high efficiency in controlling R. solanacearum, with reductions of the bacterial populations of several orders of magnitude in just a few hours, and proved to be able to survive in freshwater for months at environmental temperatures keeping activity on R. solanacearum, pointing out their suitability for field application through irrigation. Concerning their biocontrol potential, they were effective in reducing high populations of the pathogen in environmental water, and bacterial wilt incidence in planta by watering with either one phage or their combinations in assays with more than 300 plants. This is the first report on effective R. solanacearum biocontrol by applying single or combined bacteriophages through irrigation water in conditions mimicking those of the natural settings. The three phages belong to the Podoviridae family and are members of the T7likevirus genus. They are the first isolated phages from river water with activity against R. solanacearum, showing the longest persistence in natural water reported until now for phages with biocontrol potential, and consistently being able to control the disease in the host plant under environmental conditions. Consequently, the use of these bacteriophages for the prevention and/or biocontrol of the bacterial wilt disease caused by R. solanacearum has been patented. Evidence provided reveals the suitability of these waterborne phages to be effectively considered as a valuable strategy within the frame of sustainable integrated management programs.

Project description:Ralstonia solanaceraum is the quarantine plant pathogenic bacterium that causes bacterial wilt in over 200 host plants, which include economically important crops such as potato, tomato, tobacco, banana, and ginger. Alternative biological methods of disease control that can be used in integrated pest management are extensively studied. In search of new proteins with antibacterial activity against R. solanacearum, we identified L-amino acid oxidases (LAOs) from fruiting bodies of Amanita phalloides (ApLAO) and Infundibulicybe geotropa (CgLAO). We describe an optimized isolation procedure for their biochemical characterization, and show that they are dimeric proteins with estimated monomer molecular masses of 72 and 66 kDa, respectively, with isoelectric point of pH 6.5. They have broad substrate specificities for hydrophobic and charged amino acids, with highest Km for L-Leu, and broad pH optima at pH 5 and pH 6, respectively. An enzyme with similar properties is also characterized from the mycelia of I. geotropa (CgmycLAO). Fractionated aqueous extracts of 15 species of mushrooms show that LAO activity against L-Leu correlates with antibacterial activity. We confirm that the LAO activities mediate the antibacterial actions of ApLAO, CgLAO, and CgmycLAO. Their antibacterial activities are greater against Gram-negative versus Gram-positive bacteria, with inhibition of growth rate, prolongation of lag-phase, and decreased endpoint biomass. In Gram-positive bacteria, they mainly prolong the lag phase. These in vitro antibacterial activities of CgLAO and CgmycLAO are confirmed in vivo in tomato plants, while ApLAO has no effect on disease progression in planta. Transmission electron microscopy shows morphological changes of R. solanacearum upon LAO treatments. Finally, broad specificity of the antibacterial activities of these purified LAOs were seen for in vitro screening against 14 phytopathogenic bacteria. Therefore, these fungal LAOs show great potential as new biological phytoprotective agents and show the fruiting bodies of higher fungi to be a valuable source of antimicrobials with unique features.

Dataset Information

Genomic Analysis of the First European Bacteriophages with Depolymerase Activity and Biocontrol Efficacy against the Phytopathogen Ralstonia solanacearum.

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