Background Ralstonia solanacearum species complex (RSSC) strains are destructive plant pathogenic bacteria and the causative agents of bacterial wilt disease, infecting over 200 plant species worldwide. In addition to chromosomal genes, their virulence is mediated by mobile genetic elements including integrated DNA of bacteriophages, i.e., prophages, which may carry fitness-associated auxiliary genes or modulate host gene expression. Although experimental studies have characterised several prophages that shape RSSC virulence, the global diversity, distribution, and wider functional gene content of RSSC prophages are unknown. In this study, prophages were identified in a diverse collection of 192 RSSC draft genome assemblies originating from six continents. Results Prophages were identified bioinformatically and their diversity investigated using genetic distance measures, gene content, GC, and total length. Prophage distributions were characterised using metadata on RSSC strain geographic origin and lineage classification (phylotypes), and their functional gene content was assessed by identifying putative prophage-encoded auxiliary genes. In total, 313 intact prophages were identified, forming ten genetically distinct clusters. These included six prophage clusters with similarity to the Inoviridae, Myoviridae, and Siphoviridae phage families, and four uncharacterised clusters, possibly representing novel, previously undescribed phages. The prophages had broad geographical distributions, being present across multiple continents. However, they were generally host phylogenetic lineage-specific, and overall, prophage diversity was proportional to the genetic diversity of their hosts. The prophages contained many auxiliary genes involved in metabolism and virulence of both phage and bacteria. Conclusions Our results show that while RSSC prophages are highly diverse globally, they make lineage-specific contributions to the RSSC accessory genome, which could have resulted from shared coevolutionary history.
Volatile organic compounds (VOCs) produced by soil bacteria have been shown to exert plant pathogen biocontrol potential owing to their strong antimicrobial activity. While the impact of VOCs on soil microbial ecology is well established, their effect on plant pathogen evolution is yet poorly understood. Here we experimentally investigated how plant-pathogenic Ralstonia solanacearum bacterium adapts to VOC-mixture produced by a biocontrol Bacillus amyloliquefaciens T-5 bacterium and how these adaptations might affect its virulence. We found that VOC selection led to a clear increase in VOC-tolerance, which was accompanied with cross-tolerance to several antibiotics commonly produced by soil bacteria. The increasing VOC-tolerance led to trade-offs with R. solanacearum virulence, resulting in almost complete loss of pathogenicity in planta. At the genetic level, these phenotypic changes were associated with parallel mutations in genes encoding lipopolysaccharide O-antigen (wecA) and type-4 pilus biosynthesis (pilM), which both have been linked with outer membrane permeability to antimicrobials and plant pathogen virulence. Reverse genetic engineering revealed that both mutations were important, with pilM having a relatively larger negative effect on the virulence, while wecA having a relatively larger effect on increased antimicrobial tolerance. Together, our results suggest that microbial VOCs are important drivers of bacterial evolution and could potentially be used in biocontrol to select for less virulent pathogens via evolutionary trade-offs.
Ralstonia solanacearum is a destructive plant pathogenic bacterium and the causative agent of bacterial wilt disease, infecting over 200 plant species worldwide. In addition to chromosomal genes, its virulence is mediated by mobile genetic elements including integrated DNA of bacteriophages, i.e. prophages, which may carry fitness-associated auxiliary genes or modulate host gene expression. Although experimental studies have characterised several prophages that shape R. solanacearum virulence, the global diversity, distribution, and wider functional gene content of R. solanacearum prophages is unknown. In this study, prophages were identified in a diverse collection of 192 R. solanacearum draft genome assemblies originating from six continents. Prophages were identified bioinformatically and their diversity investigated using genetic distance measures, gene content, GC, and total length. Prophage distribution was characterised using metadata on R. solanacearum geographic origin and lineage classification (phylotypes), and their functional gene content was assessed by identifying putative prophage-encoded auxiliary genes. In total, 343 intact prophages were identified, forming ten genetically distinct clusters. These included five prophage clusters belonging to the Inoviridae, Myoviridae, and Siphoviridae phage families, and five uncharacterised clusters, possibly representing novel, previously undescribed phages. The prophages had broad geographical distribution being present across multiple continents. However, they were generally host phylogenetic lineage-specific, and overall, prophage diversity was proportional to the genetic diversity of their hosts. The prophages contained a myriad of auxiliary genes involved in metabolism and virulence of both phage and bacteria. Our results show that while R. solanacearum prophages are highly diverse globally, they make lineage-specific contributions to the R. solanacearum accessory genome, which could result from shared coevolutionary history.
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Crop losses to plant pathogens are a growing threat to global food security and more effective control strategies are urgently required. Biofumigation, an agricultural technique where Brassica plant tissues are mulched into soils to release antimicrobial plant allelochemicals called isothiocyanates (ITCs), has been proposed as an environmentally friendly alternative to agrochemicals. Whilst biofumigation has been shown to suppress a range of plant pathogens, its effects on plant pathogenic bacteria remain largely unexplored. Here, we used a laboratory model system to compare the efficacy of different types of ITCs against Ralstonia solanacearum plant bacterial pathogen. Additionally, we evaluated the potential for ITC‐tolerance evolution under high, intermediate, and low transfer frequency ITC exposure treatments. We found that allyl‐ITC was the most efficient compound at suppressing R. solanacearum growth, and its efficacy was not improved when combined with other types of ITCs. Despite consistent pathogen growth suppression, ITC tolerance evolution was observed in the low transfer frequency exposure treatment, leading to cross‐tolerance to ampicillin beta‐lactam antibiotic. Mechanistically, tolerance was linked to insertion sequence movement at four positions in genes that were potentially associated with stress responses (H‐NS histone like protein), cell growth and competitiveness (acyltransferase), iron storage ([2‐Fe‐2S]‐binding protein) and calcium ion sequestration (calcium‐binding protein). Interestingly, pathogen adaptation to the growth media also indirectly selected for increased ITC tolerance through potential adaptations linked with metabolism and antibiotic resistance (dehydrogenase‐like protein) and transmembrane protein movement (Tat pathway signal protein). Together, our results suggest that R. solanacearum can rapidly evolve tolerance to allyl‐ITC plant allelochemical which could constrain the long‐term efficiency of biofumigation biocontrol and potentially shape pathogen evolution with plants.
Ralstonia solanacearum species complex (RSSC) is a destructive group of plant pathogenic bacteria and the causative agent of bacterial wilt disease. Experimental studies have attributed RSSC virulence to insertion sequences (IS), transposable genetic elements which can both disrupt and activate host genes. Yet, the global diversity and distribution of RSSC IS are unknown. In this study, IS were bioinformatically identified in a diverse collection of 356 RSSC isolates representing five phylogenetic lineages and their diversity investigated based on genetic distance measures and comparisons with the ISFinder database. IS phylogenetic associations were determined based on their distribution across the RSSC phylogeny. Moreover, IS positions within genomes were characterised and their potential gene disruptions determined based on IS proximity to coding sequences. In total, we found 24732 IS belonging to eleven IS families and 26 IS subgroups with over half of the IS found in the megaplasmid. While IS families were generally widespread across the RSSC phylogeny, IS subgroups showed strong lineage-specific distributions and genetically similar bacterial isolates had similar IS contents. Similar associations with bacterial host genetic background were also observed with IS insertion positions which were highly conserved in closely related bacterial isolates. Finally, IS were found to disrupt genes with predicted functions in virulence, stress tolerance, and metabolism suggesting that they might be adaptive. This study highlights that RSSC insertion sequences track the evolution of their bacterial hosts potentially contributing to both intra- and inter-lineage genetic diversity.
Ralstonia solanacearum is a destructive plant pathogenic bacterium which harbours a wide variety of virulence genes allowing it to infect over 200 plant species worldwide. Its virulence is also affected by the presence of integrated bacteriophages, termed prophages. While several such prophages have been identified, the global distribution and diversity of R. solanacearum prophages is unknown. To study this, we first identified prophages present in a diverse collection of 192 assembled R. solanacearum genomes. Prophage diversity was explored by calculating prophage genetic distances and clustering with characterised prophages in a neighbour-joining tree. Prophage clusters were further verified by assessing gene content, GC content, and prophage length. Prophage identities were determined using the NCBI Virus database, and prophage-encoded virulence genes identified by analysing pangenome content. 343 intact prophages were identified, forming ten prophage clusters with distinct gene content, GC content, and length profiles. Five prophage clusters, containing 159 prophages, belonged to the Inoviridae, Myoviridae, and Siphoviridae phage families. The remaining 184 prophages were uncharacterised and may therefore represent novel prophages. Transcriptional regulators with potential virulence effects were identified in three prophage clusters, including one uncharacterised cluster. These prophage clusters were unequally distributed throughout the R. solanacearum population being host genotype specific. This research demonstrates that R. solanacearum contains a high level of uncharacterised prophage diversity and highlights novel prophages that could contribute to pathogen virulence. Given their potential host-genotype-specific virulence effects, R. solanacearumprophages could be co-evolving with their hosts, and may contribute to global variation in R. solanacearum virulence.
Ralstonia solanacearum species complex (RSSC) is a destructive group of plant pathogenic bacteria and the causative agent of bacterial wilt disease. Experimental studies have attributed RSSC virulence to insertion sequences (IS), transposable genetic elements which can both disrupt and activate host genes. Yet, the global diversity and distribution of RSSC IS are unknown. In this study, IS were bioinformatically identified in a diverse collection of 356 RSSC strains representing four phylogenetic lineages, and their diversity investigated based on genetic distance measures and comparisons with the ISFinder database. IS distributions were characterised using metadata on RSSC lineage classification and potential gene disruptions by IS were determined based on their proximity to coding sequences. In total, we found 24,732 IS belonging to eleven IS families and 26 IS subgroups, with over half of the IS found in the megaplasmid. While IS families were generally widespread across the RSSC phylogeny, IS subgroups showed strong lineage-specific distributions and genetically similar bacterial strains had similar IS contents. Further, IS present in multiple lineages were generally found in different genomic regions suggesting potential recent horizontal transfer. Finally, IS were found to disrupt many genes with predicted functions in virulence, stress tolerance, and metabolism, suggesting that they might be adaptive. This study highlights that RSSC insertion sequences track the evolution of their bacterial hosts, potentially contributing to both intra- and inter-lineage genetic diversity.
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