Viability, Stability and Biocontrol Activity in Planta of Specific Ralstonia solanacearum Bacteriophages after Their Conservation Prior to Commercialization and Use.

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.

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

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.

High Resolution Structure of the Mature Capsid of Ralstonia solanacearum Bacteriophage ϕRSA1 by Cryo-Electron Microscopy.

The ϕRSA1 bacteriophage has been isolated from Ralstonia solanacearum, a gram negative bacteria having a significant economic impact on many important crops. We solved the three-dimensional structure of the ϕRSA1 mature capsid to 3.9 Šresolution by cryo-electron microscopy. The capsid shell, that contains the 39 kbp of dsDNA genome, has an icosahedral symmetry characterized by an unusual triangulation number of T = 7, dextro. The ϕRSA1 capsid is composed solely of the polymerization of the major capsid protein, gp8, which exhibits the typical "Johnson" fold first characterized in E. coli bacteriophage HK97. As opposed to the latter, the ϕRSA1 mature capsid is not stabilized by covalent crosslinking between its subunits, nor by the addition of a decoration protein. We further describe the molecular interactions occurring between the subunits of the ϕRSA1 capsid and their relationships with the other known bacteriophages.

High genomic diversity of novel phages infecting the plant pathogen Ralstonia solanacearum, isolated in Mauritius and Reunion islands.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is among the most important plant diseases worldwide, severely affecting a high number of crops and ornamental plants in tropical regions. Only a limited number of phages infecting R. solanacearum have been isolated over the years, despite the importance of this bacterium and the associated plant disease. The antibacterial effect or morphological traits of these R. solanacearum viruses have been well studied, but not their genomic features, which need deeper consideration. This study reports the full genome of 23 new phages infecting RSSC isolated from agricultural samples collected in Mauritius and Reunion islands, particularly affected by this plant bacterial pathogen and considered biodiversity hotspots in the Southwest Indian Ocean. The complete genomic information and phylogenetic classification is provided, revealing high genetic diversity between them and weak similarities with previous related phages. The results support our proposal of 13 new species and seven new genera of R. solanacearum phages. Our findings highlight the wide prevalence of phages of RSSC in infected agricultural settings and the underlying genetic diversity. Discoveries of this kind lead more insight into the diversity of phages in general and to optimizing their use as biocontrol agents of bacterial diseases of plants in agriculture.

The complete genomic sequence of the novel myovirus RP13 infecting Ralstonia solanacearum, the causative agent of bacterial wilt.

A novel lytic bacteriophage, Ralstonia phage RP13, was isolated from tomato fields in Pang Nga, Thailand. Electron microscopic observation showed it to have the features of a myovirus with a novel triangulation number (T = 21, dextro). The RP13 DNA appeared to be heavily modified. By applying RNA sequencing and RNA-sequence-mediated DNA sequencing, the whole genome of RP31 was determined to be 170,942 bp in length with a mean G+C content of 39.2%. A total of 277 ORFs were identified as structural, functional, or hypothetical genes in addition to four tRNA genes. Phylogenetic analysis suggested that RP13 is not closely related to any other known phages. Thus, we concluded that the RP13 is a novel phage infecting R. solanacearum strains and will be a useful biocontrol agent against bacterial wilt disease.

3D structure of three jumbo phage heads.

Jumbo phages are bacteriophages that carry more than 200 kbp of DNA. In this study we characterized two jumbo phages (ΦRSL2 and ΦXacN1) and one semi-jumbo phage (ΦRP13) at the structural level by cryo-electron microscopy. Focusing on their capsids, three-dimensional structures of the heads at resolutions ranging from 16 to 9 Å were calculated. Based on these structures we determined the geometrical basis on which the icosahedral capsids of these phages are constructed, which includes the accessory and decorative proteins that complement them. A triangulation number novel to Myoviridae (ΦRP13; T=21) was discovered as well as two others, which are more common for jumbo phages (T=27 and T=28). Based on one of the structures we also provide evidence that accessory or decorative proteins are not a prerequisite for maintaining the structural integrity of very large capsids.

C22 podovirus infectivity is associated with intermediate stiffness.

Bacteriophages have potential for use as biological control agents (biocontrols) of pathogenic bacteria, but their low stability is limiting for their utilization as biocontrols. Understanding of the conditions conducive to storage of phages in which infectivity is maintained over long periods will be useful for their application as biocontrols. We employed a nanomechanical approach to study how external environmental factors affect surface properties and infectivity of the podovirus C22 phage, a candidate for biocontrol of Ralstonia solanacearum, the agent of bacterial wilt in crops. We performed atomic force microscopy (AFM)-based nano-indentation on the C22 phage in buffers with varying pH and ionic strength. The infectivity data from plaque assay in the same conditions revealed that an intermediate range of stiffness was associated with phage titer that remained consistently high, even after prolonged storage up to 182 days. The data are consistent with the model that C22 phage must adopt a metastable state for maximal infectivity, and external factors that alter the stiffness of the phage capsid lead to perturbation of this infective state.

Phage combination therapies for bacterial wilt disease in tomato.

Bacteriophages have been proposed as an alternative to pesticides to kill bacterial pathogens of crops. However, the efficacy of phage biocontrol is variable and poorly understood in natural rhizosphere microbiomes. We studied biocontrol efficacy of different phage combinations on Ralstonia solanacearum infection in tomato. Increasing the number of phages in combinations decreased the incidence of disease by up to 80% in greenhouse and field experiments during a single crop season. The decreased incidence of disease was explained by a reduction in pathogen density and the selection for phage-resistant but slow-growing pathogen strains, together with enrichment for bacterial species that were antagonistic toward R. solanacearum. Phage treatment did not affect the existing rhizosphere microbiota. Specific phage combinations have potential as precision tools to control plant pathogenic bacteria.

Characterization and complete genome sequence analysis of phage GP4, a novel lytic Bcep22-like podovirus.

We isolated a novel lytic phage of Ralstonia solanacearum, GP4. The GP4 phage has a latent period of ~ 2 h at its optimal multiplicity of infection and is stable at temperatures ranging from 40-70 °C. GP4 lysed 16 strains of R. solanacearum belonging to phylotype IV. High-throughput sequencing revealed that GP4 has a linear dsDNA genome that consists of 61,129 bp, contains 83 open reading frames, and encodes a tRNA for cysteine. The GP4 genome has low similarity to other phage sequences in the GenBank database. Phylogenetic analysis indicated that GP4 can be taxonomically classified as a member of the Bcep22-like subfamily of the family Podoviridae.

Characterization of CRISPR-Cas systems in the Ralstonia solanacearum species complex.

Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of an array of short DNA repeat sequences separated by unique spacer sequences that are flanked by associated (Cas) genes. CRISPR-Cas systems are found in the genomes of several microbes and can act as an adaptive immune mechanism against invading foreign nucleic acids, such as phage genomes. Here, we studied the CRISPR-Cas systems in plant-pathogenic bacteria of the Ralstonia solanacearum species complex (RSSC). A CRISPR-Cas system was found in 31% of RSSC genomes present in public databases. Specifically, CRISPR-Cas types I-E and II-C were found, with I-E being the most common. The presence of the same CRISPR-Cas types in distinct Ralstonia phylotypes and species suggests the acquisition of the system by a common ancestor before Ralstonia species segregation. In addition, a Cas1 phylogeny (I-E type) showed a perfect geographical segregation of phylotypes, supporting an ancient acquisition. Ralstoniasolanacearum strains CFBP2957 and K60T were challenged with a virulent phage, and the CRISPR arrays of bacteriophage-insensitive mutants (BIMs) were analysed. No new spacer acquisition was detected in the analysed BIMs. The functionality of the CRISPR-Cas interference step was also tested in R. solanacearum CFBP2957 using a spacer-protospacer adjacent motif (PAM) delivery system, and no resistance was observed against phage phiAP1. Our results show that the CRISPR-Cas system in R. solanacearum CFBP2957 is not its primary antiviral strategy.

Host range and molecular characterization of a lytic Pradovirus-like Ralstonia phage RsoP1IDN isolated from Indonesia.

A lytic Ralstonia solanacearum-infecting phage designated Ralstonia phage RsoP1IDN was isolated from soil in Indonesia. The phage has a linear double-stranded DNA genome of 41,135 bp with 413-bp terminal repeats, and contains 41 annotated open reading frames. The phage is most closely related to Ralstonia phage RSB1, but different from RSB1 mainly in containing a putative HNH homing endonuclease and having a narrower host range. Our phylogenetic and genomic analyses revealed that both phages RsoP1IDN and RSB1 belong to the genus Pradovirus or a new genus, and not Phikmvvirus as previously reported for phage RSB1. RsoP1IDN is the first sequenced and characterized R. solanacearum-infecting phage isolated from Indonesia in the proposed species Ralstonia virus RsoP1IDN.

Potential use of soilborne lytic Podoviridae phage as a biocontrol agent against Ralstonia solanacearum.

A new podovirus RsPod1EGY Ralstonia phage (GenBank accession no MG711516) with a specific action against R. solanacearum phylotype IIa, sequevar I (race 3, biovar 2) was isolated from Egyptian soil. The potential efficacy of the isolated phage to be used as biocontrol agent was evaluated in vitro and under greenhouse conditions. The podovirus phage produced a plaque size of 3.0-4.0 mm in diameter and completed its infection cycle in 180 min after infection with a burst size of ∼27 virions per infected cell. On the basis of restriction endonuclease analysis, the genome size of the phage was about 41 kb of double-stranded DNA. In vitro studies showed that RsPod1EGY is stable at higher temperatures (up to 60 °C), and at a wide pH range (5-9). SDS-PAGE analysis indicated the major structural protein to be approximately 32 kDa. Bacteriolytic activity of RsPod1EGY against R. solanacearum was detected at different multiplicity of infection (MOI). RsPod1EGY proved to be effective in reduction and prevention of formation of surface polysaccharides of R. solanacearum, during the exponential growth phase of the latter. Interestingly, RsPod1EGY was effective in suppression of R. solanacearum under greenhouse conditions. All Phage-treated tomato plants showed no wilt symptoms or any latent infection during the experimental period, whereas all untreated plants have wilted by 10 days post-infection. The lytic stability of RsPod1EGY phage at higher temperature as well as its effective suppression of wilting symptoms under greenhouse conditions would contribute to biocontrol the bacterial wilt disease in Egypt under field conditions.

Sequencing, genome analysis and host range of a novel Ralstonia phage, RsoP1EGY, isolated in Egypt.

A novel Ralstonia phage was isolated from soil in Egypt. It was designated Ralstonia phage RsoP1EGY using our phage identifier naming approach to reflect the phage's bacterial host species, characteristics and origin. When tested, this phage specifically infected only race 3 biovar 2 phylotype IIB sequevar 1, and not non-race 3 biovar 2 strains of Ralstonia solanacearum. The phage has an icosahedral capsid of 60 ± 5 nm in diameter with a short tail of 15 ± 5 nm in length, typical of a podovirus. The genome of RsoP1EGY is 41,297 bp in size, containing 50 open reading frames, with no significant sequence identity to any other reported R. solanacearum or non-Ralstonia phages, except to the recently deposited but unreported and unclassified Ralstonia phage DU_RP_I. RsoP1EGY is the first sequenced and characterized R. solanacearum phage isolated in Egypt.

Genomic characterization of the novel Ralstonia phage RPSC1.

In this paper, I describe the genomic characteristics of a Ralstonia phage infecting Ralstonia solanacearum. The Ralstonia phage RPSC1 was isolated from a soil sample collected in Sichuan Province, in southwestern China. The complete genome of RPSC1 is composed of a linear double-stranded DNA 39,628 bp in length, with G+C content of 61.55%, and 43 putative protein-coding genes. All the putative protein-coding genes were on the same strand. No tRNA-encoding genes were identified. Phylogenetic and comparative genomics analyses indicate that Ralstonia phage RPSC1 should be considered a new member of the family Podoviridae. The wide host range contributes to the potential of Ralstonia phage RPSC1 as a biocontrol agent.

Complete genome sequence of DU_RP_II, a novel Ralstonia solanacearum phage of the family Podoviridae.

DU_RP_II, a bacteriophage of the family Podoviridae, which lyses Ralstonia solanacearum, was isolated from a diseased plant sample, and its genome was completely sequenced. The genome was found to be 42,091 base pairs long and to be a circular double-stranded DNA with a GC content of 62.17% and 38 predicted coding sequences. The phage showed homology to the RSK1 phage in four coding sequences, but it was concluded that the phage differed from previously reported Ralstonia phages based on the results of both morphology and bioinformatics analysis. This suggests that the phage DU_RP_II is a new member of the family Podoviridae.

Developing a bacteriophage cocktail for biocontrol of potato bacterial wilt.

Bacterial wilt is a devastating disease of potato and can cause an 80% production loss. To control wilt using bacteriophage therapy, we isolated and characterized twelve lytic bacteriophages from different water sources in Kenya and China. Based on the lytic curves of the phages with the pathogen Ralstonia solanacearum, one optimal bacteriophage cocktail, P1, containing six phage isolations was formulated and used for studying wilt prevention and treatment efficiency in potato plants growing in pots. The preliminary tests showed that the phage cocktail was very effective in preventing potato bacterial wilt by injection of the phages into the plants or decontamination of sterilized soil spiked with R. solanacearum. Eighty percent of potato plants could be protected from the bacterial wilt (caused by R. solanacearum reference strain GIM1.74 and field isolates), and the P1 cocktail could kill 98% of live bacteria spiked in the sterilized soil at one week after spraying. However, the treatment efficiencies of P1 depended on the timing of application of the phages, the susceptibility of the plants to the bacterial wilt, as well as the virulence of the bacteria infected, suggesting that it is important to apply the phage therapy as soon as possible once there are early signs of the bacterial wilt. These results provide the basis for the development of bacteriophagebased biocontrol of potato bacterial wilt as an alternative to the use of antibiotics.

Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum.

A filamentous bacteriophage, designated ϕRs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ϕRs551 is a member of the family Inoviridae, and is about 1200 nm long and 7 nm wide. ϕRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other R. solanacearum phages isolated from soil, the genome sequence of ϕRs551 is not only 100% identical to its prophage sequence in the deposited genome of R. solanacearum strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of R. solanacearum. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a R. solanacearum strain from India. When ORF13 and a core attP site of ϕRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous R. solanacearum ϕRSM phages, ϕRs551 relies on its resolvase and the core att sequence for site-directed integration into its susceptible R. solanacearum strain. The integration occurred four hours after phage infection. Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ϕRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ϕRSM phages. Study of bacteriophages of R. solanacearum would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat R. solanacearum.

Complete genome sequence of a novel lytic bacteriophage isolated from Ralstonia solanacearum.

A lytic podophage RSPI1 was isolated from tobacco field soil collected in Fujian Province, South China using host bacterium Ralstonia solanacearum Tb15-14. Whole genome sequencing of this phage was performed using the high-throughput Ion Torrent PGM Sequencer. The complete genome of RSPI1 was 43,211 bp in length with a mean DNA G + C content of 61.5%. A total of 48 open reading frames were identified with lengths ranging from 132 bp to 5,061 bp, of which, 11, 12 and 25 were identified as functional, structural and unknown genes, respectively. A BLAST analysis revealed that this phage genome had a query cover of 78-79% and a highest identity of 84% with four podophages that infect Burkholderia pseudomallei. Two neighbor-joining phylogenetic trees were constructed using phage DNA polymerase I and tail fiber protein sequences and showed that this phage is closely related to Burkholderia phage Bp-AMP1, and also related to several phages that infect Ralstonia solanacearum. These findings indicate that RSPI1 is a novel phage that infects the notorious plant pathogen Ralstonia solanacearum.

Parasites and competitors suppress bacterial pathogen synergistically due to evolutionary trade-offs.

Parasites and competitors are important for regulating pathogen densities and subsequent disease dynamics. It is, however, unclear to what extent this is driven by ecological and evolutionary processes. Here, we used experimental evolution to study the eco-evolutionary feedbacks among Ralstonia solanacearum bacterial pathogen, Ralstonia-specific phage parasite, and Bacillus amyloliquefaciens competitor bacterium in the laboratory and plant rhizosphere. We found that while the phage had a small effect on pathogen densities on its own, it considerably increased the R. solanacearum sensitivity to antibiotics produced by B. amyloliquefaciens. Instead of density effects, this synergy was due to phage-driven increase in phage resistance that led to trade-off with the resistance to B. amyloliquefaciens antibiotics. While no evidence was found for pathogen resistance evolution to B. amyloliquefaciens antibiotics, the fitness cost of adaptation (reduced growth) was highest when the pathogen had evolved in the presence of both parasite and competitor. Qualitatively similar patterns were found between laboratory and greenhouse experiments even though the evolution of phage resistance was considerably attenuated in the tomato rhizosphere. These results suggest that evolutionary trade-offs can impose strong constraints on disease dynamics and that combining phages and antibiotic-producing bacteria could be an efficient way to control agricultural pathogens.

Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages.

Jumbo phages infecting Ralstonia solanacearum were isolated in Thailand (ϕRSL2) and Japan (ϕRSF1). They were similar regarding virion morphology, genomic arrangement, and host range. Phylogenetic and proteomic tree analyses demonstrate that the ϕRSL2 and ϕRSF1 belong to a group of evolutionary related phages, including Pseudomonas phages ϕKZ, 201ϕ2-1 and all previously described ϕKZ-related phages. Despite conserved genomic co-linearity between the ϕRSL2 and ϕRSF1, they differ in protein separation patterns. A major difference was seen in the detection of virion-associated-RNA polymerase subunits. All ß- and ß'-subunits were detected in ϕRSF1, but one ß'-subunit was undetected in ϕRSL2. Furthermore, ϕRSF1 infected host cells faster (latent period: 60 and 150min for ϕRSF1 and ϕRSL2, respectively) and more efficiently than ϕRSL2. Therefore, the difference in virion-associated-RNA polymerase may affect infection efficiency. Finally, we show that ϕRSF1 is able to inhibit bacterial wilt progression in tomato plants.