Honour C. McCann

Outbreak sequencing provides insight into the origin and evolutionary processes acting on emerging pathogens. Sequencing a historic multihost outbreak of Ralstonia spp. in Martinique shows the outbreak was caused by two lineages that diverged at separate times from mainland populations. One lineage (Ralstonia pseudosolanacearum I-18) was originally introduced from Asia to South America, where it became well established prior to its dissemination to Martinique, where it retains a signature of specialization on solanaceous hosts. The novel lineage first identified during the outbreak (Ralstonia solanacearum IIB-4NPB) arose from a mainland population endemic to the Americas prior to its arrival in Martinique, where host-range expansion was observed. In contrast to minor changes in secreted effector protein repertoires, the emergent R. solanacearum IIB-4NPB acquired a novel integrative and conjugative element (ICERsoRUN1145). After identifying all Ralstonia spp. ICEs and mapping their spatial and phylogenetic distribution among Ralstonia spp. sampled during the outbreak, we found closely related ICEs circulating in mainland populations of R. pseudosolanacearum, indicating likely exchange between introduced and endemic Ralstonia spp. The family of ICEs in Ralstonia (ICERs) has a conserved bipartite structure and display a striking pattern of functional specialization in each cargo gene insertion hotspot: the first hotspot is a target for metabolic gene acquisition, and the second is a target for defense element acquisition. This work provides unparalleled phylogenetic and spatial resolution of an unusual outbreak and highlights the role of horizontal transfer in shaping the ecological success of an emerging pathogen.

Long term chronic infections of plants by bacterial pathogens are largely unknown. Understanding how pathogens adapt during chronic infection provides a key insight to pathogen evolutionary strategy both for persistence and survival, but also for potential future outbreaks. Pseudomonas savastanoi pv. fraxini ( Psf) , a member of phylogroup 3 within the Pseudomonas syringae species complex, causes canker disease in European ash ( Fraxinus excelsior ). Infections persist for years within the bark parenchyma, where bacteria are enclosed in cavities that contribute to the gradual expansion of host periderm. This pathosystem therefore provides an opportunity to examine pathogen evolution in a long-lived, largely unmanaged host. We combined population genomics and phenotypic analysis of 124 Psf strains collected from six sites across the UK. Phylogenetic analysis revealed a highly clonal population, with only 833 core genome SNPs across a 5.3 Mb genome, and a relatively small accessory genome largely shaped by gain and loss of large mobile genetic elements. Despite this limited genomic diversity, mutations were enriched in regulatory genes, including two-component systems, chemotaxis proteins, and cell envelope-associated loci. Notably, the global regulator gacA/S was independently mutated multiple times within the same clonal lineage. These mutations, typically small deletions, were associated with changes in motility, nutrient utilisation, stress tolerance, and virulence across genetic backgrounds. As a result, phenotypic heterogeneity was observed within otherwise clonal populations, including within individual lesions. These findings indicate that repeated mutation of regulatory systems represents a key mechanism of adaptation in this chronic plant–pathogen interaction, enabling phenotypic diversification despite limited sequence divergence. This study provides a microevolutionary perspective on P. syringae populations in the phyllosphere and highlights the role of regulatory variation in the evolution of low-virulence, ecologically restricted pathogens.

SUMMARY: Wild plant species are threatened by diverse pathogens, but disease symptoms are rarely observed in nature. This suggests that wild plants harbor valuable sources of resistance. In this study, we show that the model bacterial pathogen Pseudomonas syringae pv. tomato ( Pto ) DC3000 triggered defense responses in all tested accessions of a wild Solanaceae species, Solanum americanum . Pto DC3000‐triggered immunity in S. americanum required a type III secretion system. We show that seven Pto DC3000 effectors (AvrPto, HopAD1, HopAM1, HopC1, HopAA1‐1, HopM1, and AvrE1) triggered hypersensitive responses (HR) in S. americanum accession SP2273. Significantly, sequential deletion of the HR‐triggering effectors from Pto DC3000 resulted in enhanced virulence in S. americanum . However, the well‐conserved effectors, HopM1 and AvrE1, were indispensable for virulence. We conclude that the immunity triggered by multiple effectors contributes to nonhost resistance in S. americanum against P. syringae . We propose that the identification of the corresponding disease resistance genes for HopM1 and AvrE1 in S. americanum would accelerate the development of durable immunity to P. syringae pathogens in Solanaceae crops.

The evolutionary processes underlying disease outbreaks remain unknown for most bacterial plant pathogens. We sequenced an outbreak of lethal wilt disease in Martinique caused by two distantly related lineages in the Ralstonia solanacearum species complex. One lineage ( R. solanacearum IIB-4NPB) exhibited the broadest host range ever documented for a single lineage in the field, while the other ( R. pseudosolanacearum I-18) expanded in parallel but retained specialisation on solanaceaous hosts. Phylogenomic analysis of 407 outbreak isolates shows both lineages independently disseminated from mainland populations into Martinique. We resolved fine-scale geographic patterns of genomic diversity and identified spatial hotspots of interspecies mobile element exchange, resulting in the identification of a new family of Ralstonia integrative conjugative elements (ICEs) associated with the outbreak. ICE accessory gene integration sites display striking functional specialization and differentiation despite variable gene content: each site acquires only metabolism-associated or defence-related genes, respectively. This work provides insight into the origin and genomic changes associated with an outbreak of plant disease, and highlights the role of mobile elements in driving pathogen emergence.

Research initiatives undertaken in response to disease outbreaks accelerate our understanding of microbial evolution, mechanisms of virulence and resistance, and plant–pathogen coevolutionary interactions. The emergence and global spread of Pseudomonas syringae pv. actinidiae (Psa) on kiwifruit ( Actinidia chinensis ) showed that there are parallel paths to host adaptation and antimicrobial resistance evolution, accelerated by the movement of mobile elements. Significant progress has been made in identifying type 3 effectors required for virulence and recognition in A. chinensis and Actinidia arguta , broadening our understanding of how host-mediated selection shapes virulence. The rapid development of Actinidia genomics after the Psa3 pandemic began has also generated new insight into molecular mechanisms of immunity and resistance gene evolution in this recently domesticated, nonmodel host. These findings include the presence of close homologs of known resistance genes RPM1 and RPS2 as well as the novel expansion of CC _G10 -NLRs (nucleotide-binding leucine-rich repeats) in Actinidia spp. The advances and approaches developed during the pandemic response can be applied to new pathosystems and new outbreak events.

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa . PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn 6212 . Deletion of Tn 6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn 6212 -encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn 6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth.

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness, yet ICEs that carry accessory genes encoding functions other than antimicrobial resistance remain poorly characterized. Recent observation of the rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements among a diverse array of P. syringae . Fifty-three unique ICE types were identified across multiple phylogroups. These ICEs display distinct evolutionary histories compared to their bacterial hosts, are highly recombinogenic, exhibit a conserved structure and are punctuated by hotspots of accessory gene integration. Many carry a 16 kb transposon-like entity (Tn 6212 ) that shows little polymorphism indicating recent dissemination. Deletion of Tn 6212 did not alter pathogen growth in planta , but mutants displayed significant fitness defects when grown on TCA cycle intermediates. These were largely attributable to a single LysR regulator. RNA-seq analysis of a set of nested Tn 6212 deletions confirmed a central role of LysR in enhanced expression of more than 300 genes and down-regulation of genes controlling expression of energetically demanding loci. Together the transcriptional data indicate a major role for Tn 6212 in manipulation of bacterial metabolism with primary effects on RNA degradation, protein synthesis and potential diversion of ATP to growth.

Ralstonia solanacearum causes bacterial wilt disease in solanaceous crops. Identification of avirulence type III-secreted effectors recognized by specific disease resistance proteins in host plant species is an important step toward developing durable resistance in crops. In the present study, we show that R. solanacearum effector RipJ functions as an avirulence determinant in Solanum pimpinellifolium LA2093. In all, 10 candidate avirulence effectors were shortlisted based on the effector repertoire comparison between avirulent Pe_9 and virulent Pe_1 strains. Infection assays with transgenic strain Pe_1 individually carrying a candidate avirulence effector from Pe_9 revealed that only RipJ elicits strong bacterial wilt resistance in S. pimpinellifolium LA2093. Furthermore, we identified that several RipJ natural variants do not induce bacterial wilt resistance in S. pimpinellifolium LA2093. RipJ belongs to the YopJ family of acetyltransferases. Our sequence analysis indicated the presence of partially conserved putative catalytic residues. Interestingly, the conserved amino acid residues in the acetyltransferase catalytic triad are not required for effector-triggered immunity. In addition, we show that RipJ does not autoacetylate its lysine residues. Our study reports the identification of the first R. solanacearum avirulence protein that triggers bacterial wilt resistance in tomato. We expect that our discovery of RipJ as an avirulence protein will accelerate the development of bacterial wilt-resistant tomato varieties in the future. [ ] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Population genomics is transforming our understanding of pathogen biology and evolution, and contributing to the prevention and management of disease in diverse crops. We provide an overview of key methods in bacterial population genomics and describe recent work focusing on three topics of critical importance to plant pathology: (i) resolving pathogen origins and transmission pathways during outbreak events, (ii) identifying the genetic basis of host specificity and virulence, and (iii) understanding how pathogens evolve in response to changing agricultural practices. [ ] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) threatens the cultivation of important crops worldwide. We sequenced 30 RSSC phylotype I ( R. pseudosolanacearum ) strains isolated from pepper (Capsicum annuum) and tomato (Solanum lycopersicum) across the Republic of Korea. These isolates span the diversity of phylotype I, have extensive effector repertoires and are subject to frequent recombination. Recombination hotspots among South Korean phylotype I isolates include multiple predicted contact-dependent inhibition loci, suggesting that microbial competition plays a significant role in Ralstonia evolution. Rapid diversification of secreted effectors presents challenges for the development of disease-resistant plant varieties. We identified potential targets for disease resistance breeding by testing for allele-specific host recognition of T3Es present among South Korean phyloype I isolates. The integration of pathogen population genomics and molecular plant pathology contributes to the development of location-specific disease control and development of plant cultivars with durable resistance to relevant threats.