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.

Fire blight, caused by the plant-pathogenic bacterium Erwinia amylovora, is a highly contagious and difficult-to-control disease due to its efficient dissemination and survival and the scarcity of effective control methods. Copper and antibiotics are the most used treatments but pose environmental and human health risks. Bacteriophages (phages) constitute an ecological, safe, and sustainable fire blight control alternative. The goal of this study was to search for specific E. amylovora phages from plant material, soil, and water samples in Mediterranean environments. A collection of phages able to specifically infect and lyse E. amylovora strains was generated from former fire blight-affected orchards in Eastern Spain. Following in vitro characterization, assays in immature fruit revealed that preventively applying some of the phages or their combinations delayed the onset of fire blight symptoms and reduced the disease’s severity, suggesting their biocontrol potential in Spain and other countries. The morphological and molecular characterization of the selected E. amylovora phages classified them as members of the class Caudoviricetes (former Myoviridae family) and genus Kolesnikvirus. This study reveals Mediterranean settings as plausible sources of E. amylovora-specific bacteriophages and provides the first effective European phage cocktails in plant material for the development of sustainable fire blight management measures.

Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most destructive diseases of solanaceous plants, affecting staple crops worldwide. The bacterium survives in water, soil, and other reservoirs, and is difficult to control. In this sense, the use of three specific lytic R. solanacearum bacteriophages was recently patented for bacterial wilt biocontrol in environmental water and in plants. To optimize their applications, the phages and the bacterium need to be accurately monitored and quantified, which is laborious and time-consuming with biological methods. In this work, primers and TaqMan probes were designed, and duplex and multiplex real-time quantitative PCR (qPCR) protocols were developed and optimized for the simultaneous quantification of R. solanacearum and their phages. The quantification range was established from 10⁸ to 10 PFU/mL for the phages and from 10⁸ to 10² CFU/mL for R. solanacearum. Additionally, the multiplex qPCR protocol was validated for the detection and quantification of the phages with a limit ranging from 10² targets/mL in water and plant extracts to 10³ targets/g in soil, and the target bacterium with a limit ranging from 10³ targets/mL in water and plant extracts to 10⁴ targets/g in soil, using direct methods of sample preparation.

Ralstonia solanacearum is a pathogen that causes bacterial wilt producing severe damage in staple solanaceous crops. Traditional control has low efficacy and/or environmental impact. Recently, the bases of a new biotechnological method by lytic bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 with specific activity against R. solanacearum were established. However, some aspects remain unknown, such as the survival and maintenance of the lytic activity after submission to a preservation method as the lyophilization. To this end, viability and stability of lyophilized vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 and their capacity for bacterial wilt biocontrol have been determined against one pathogenic Spanish reference strain of R. solanacearum in susceptible tomato plants in different conditions and making use of various cryoprotectants. The assays carried out have shown satisfactory results with respect to the viability and stability of the bacteriophages after the lyophilization process, maintaining high titers throughout the experimental period, and with respect to the capacity of the bacteriophages for the biological control of bacterial wilt, controlling this disease in more than 50% of the plants. The results offer good prospects for the use of lyophilization as a conservation method for the lytic bacteriophages of R. solanacearum in view of their commercialization as biocontrol agents.

Fire blight, caused by the plant-pathogenic bacterium Erwinia amylovora, is a highly contagious and difficult-to-control disease due to the efficient dissemination, survival, and scarcity of effective control methods. Copper and antibiotics are the most used treatments but pose environmental and human health risks. Bacteriophages (phages) constitute an ecological, safe, and sustainable fire blight control alternative. The goal of this study was to search for specific E. amylovora phages from plant material, soil, and water samples in Mediterranean environments. A collection of phages able to specifically infect and lyse E. amylovora strains was generated from former fire blight-affected orchards in Eastern Spain. Following in vitro characterization, assays on immature fruit revealed that preventively applying some of the phages or their combinations delayed the onset of fire blight symptoms and reduced the disease severity, suggesting their biocontrol potential in Spain and other countries. Morphological and molecular characterization of the selected E. amylovora phages classified them as members of the class Caudoviricetes (former Myoviridae family) and genus Kolesnikvirus. This study reveals Mediterranean settings as plausible sources of E. amylovora-specific bacteriophages, and provides the first effective European phage cocktails in plant material for the development of sustainable fire blight management measures.