Isolation of Ralstonia solanacearum -infecting bacteriophages from tomato fields in Chiang Mai, Thailand, and their experimental use as biocontrol agents

Abstract

Aims: To isolate and characterize novel bacteriophages infecting the phytopathogen, Ralstonia solanacearum, and to evaluate them as resources with potential uses in the biocontrol of bacterial wilt. Methods and Results: Fourteen phages infecting R. solanacearum were isolated from soil samples collected in Chiang Mai, Thailand. The phages showed different host ranges when tested against 59 R. solanacearum strains isolated from Thailand and Japan. These phages were characterized as nine podoviruses and five myoviruses based on their morphology. Podovirus J2 in combination with another podovirus (φRSB2) lysed host cells very efficiently in contaminated soil. J2 treatment prevented wilting of tomato plants infected with a highly virulent R. solanacearum strain. Conclusions: Treatment with J2 effectively reduced the amount of the bacterial wilt pathogen in contaminated soil and prevented bacterial wilt of tomato in pot experiments. Myovirus J6 possessed jumbo phage features, giving a unique opportunity to study its utilization as a biocontrol agent. Significance and Impact of the Study: As exemplified by J2, the phages isolated in this study represent valuable resources with potential uses in biocontrol of bacterial wilt. A rare jumbo phage J6 served as a valuable subject to understand and utilize this new group of phages.

Replications of Two Closely Related Groups of Jumbo Phages Show Different Level of Dependence on Host-encoded RNA Polymerase

“…Bacteria were cultured in CPG medium containing 0.1% (w/v) casamino acids, 1.0% (w/v) peptone, and 0.5% (w/v) glucose (Horita and Tsuchiya, 2002) at 28°C with shaking at 200–300 rpm. Bacteriophages ΦRP12 and ΦRP31 were isolated from tomato fields in Chiang Mai, Thailand as described previously (Bhunchoth et al, 2015). Each phage was routinely propagated using R. solanacearum strain MAFF 730138 as the host.…”

Jumbo Bacteriophages: An Overview

“…More recently, larger viruses have been isolated, and their discovery has greatly enriched our understanding of biological entity diversity and evolution (Bhunchoth et al, 2015; Sharma et al, 2015). Jumbo phages have been isolated from diverse niches and exhibit extremely high genetic diversity.…”

Bacteriophage-Based Bacterial Wilt Biocontrol for an Environmentally Sustainable Agriculture

“…Subsequently, the characterization of 14 bacteriophages isolated from soil in Thailand, belonging to the Podoviridae and Myoviridae families, revealed that the combination of two podoviruses (J2 and φRSB2) efficiently lysed R. solanacearum cells in contaminated soil but, only J2 treatment prevented disease development in tomato plants (Bhunchoth et al, 2015) ( Table 1 ). The application of mixtures of other six lytic bacteriophages, isolated from soil in Sri Lanka, to the rhizosphere of tomato plants as a soil drench reduced the incidence of bacterial wilt about 10–20%, either by applying bacteriophage mixtures immediately before inoculation with the pathogen, or watering the soil several times with such mixtures (Kalpage and De Costa, 2014) ( Table 1 ).…”

“…Japanese patent with publication number JP2005278513 describes three types of R. solanacearum bacteriophages with lytic activity against the pathogen, which were isolated from soil in Japan. The phages were initially characterized by their genome size and host range, which was assayed against six Japanese strains isolated from tobacco plants, all of them of race 1: M4S, Ps29, and Ps65 of biovar 3, and C319, Ps72, and Ps74 of biovar 4 (Bhunchoth et al, 2015, Supplementary Table S1) and, therefore, all of them belonging to phylotypes I or IV of the former species complex, and presently not R. solanacearum ( Figure 1 ). Biocontrol assays were performed against strain C319 by spraying a phage of type 1 or 2 onto tobacco plants or as a soil conditioner (Yamada and Yamamoto, 2005).…”

“…Thus, bacterial infectious diseases can be treated by the action of phages, a biocontrol method also known as phage therapy. This method can be considered as a promising strategy against bacterial wilt disease (Fujiwara et al, 2011; Bae et al, 2012; Bhunchoth et al, 2015), since it has been successfully applied for controlling some important plant diseases caused by phytopathogenic bacteria (Jones et al, 2007; Balogh et al, 2010; Doffkay et al, 2015; Buttimer et al, 2017). …”