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Biocontrol of Ralstonia solanacearum by treatment with lytic bacteriophages.

ABSTRACT: Ralstonia solanacearum is a Gram-negative bacterium and the causative agent of bacterial wilt in many important crops. We treated R. solanacearum with three lytic phages: ?RSA1, ?RSB1, and ?RSL1. Infection with ?RSA1 and ?RSB1, either alone or in combination with the other phages, resulted in a rapid decrease in the host bacterial cell density. Cells that were resistant to infection by these phages became evident approximately 30 h after phage addition to the culture. On the other hand, cells infected solely with ?RSL1 in a batch culture were maintained at a lower cell density (1/3 of control) over a long period. Pretreatment of tomato seedlings with ?RSL1 drastically limited penetration, growth, and movement of root-inoculated bacterial cells. All ?RSL1-treated tomato plants showed no symptoms of wilting during the experimental period, whereas all untreated plants had wilted by 18 days postinfection. ?RSL1 was shown to be relatively stable in soil, especially at higher temperatures (37 to 50°C). Active ?RSL1 particles were recovered from the roots of treated plants and from soil 4 months postinfection. Based on these observations, we propose an alternative biocontrol method using a unique phage, such as ?RSL1, instead of a phage cocktail with highly virulent phages. Using this method, ?RSL1 killed some but not all bacterial cells. The coexistence of bacterial cells and the phage resulted in effective prevention of wilting.

SUBMITTER: Fujiwara A

PROVIDER: S-EPMC3131639 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Biocontrol of Ralstonia solanacearum by treatment with lytic bacteriophages.

Fujiwara Akiko A Fujisawa Mariko M Hamasaki Ryosuke R Kawasaki Takeru T Fujie Makoto M Yamada Takashi T

Applied and environmental microbiology 20110415 12

Ralstonia solanacearum is a Gram-negative bacterium and the causative agent of bacterial wilt in many important crops. We treated R. solanacearum with three lytic phages: ϕRSA1, ϕRSB1, and ϕRSL1. Infection with ϕRSA1 and ϕRSB1, either alone or in combination with the other phages, resulted in a rapid decrease in the host bacterial cell density. Cells that were resistant to infection by these phages became evident approximately 30 h after phage addition to the culture. On the other hand, cells in ...[more]

PMID: 21498752

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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 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.

Dataset Information

Biocontrol of Ralstonia solanacearum by treatment with lytic bacteriophages.

Publications

Biocontrol of Ralstonia solanacearum by treatment with lytic bacteriophages.

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