Plant-Pathogenic Ralstonia Phylotypes Evolved Divergent Respiratory Strategies and Behaviors To Thrive in Xylem.

Bacterial pathogens in the Ralstonia solanacearum species complex (RSSC) infect the water-transporting xylem vessels of plants, causing bacterial wilt disease. Strains in RSSC phylotypes I and III can reduce nitrate to dinitrogen via complete denitrification. The four-step denitrification pathway enables bacteria to use inorganic nitrogen species as terminal electron acceptors, supporting their growth in oxygen-limited environments such as biofilms or plant xylem. Reduction of nitrate, nitrite, and nitric oxide all contribute to the virulence of a model phylotype I strain. However, little is known about the physiological role of the last denitrification step, the reduction of nitrous oxide to dinitrogen by NosZ. We found that phylotypes I and III need NosZ for full virulence. However, strains in phylotypes II and IV are highly virulent despite lacking NosZ. The ability to respire by reducing nitrate to nitrous oxide does not greatly enhance the growth of phylotype II and IV strains. These partial denitrifying strains reach high cell densities during plant infection and cause typical wilt disease. However, unlike phylotype I and III strains, partial denitrifiers cannot grow well under anaerobic conditions or form thick biofilms in culture or in tomato xylem vessels. Furthermore, aerotaxis assays show that strains from different phylotypes have different oxygen and nitrate preferences. Together, these results indicate that the RSSC contains two subgroups that occupy the same habitat but have evolved divergent energy metabolism strategies to exploit distinct metabolic niches in the xylem. IMPORTANCE Plant-pathogenic Ralstonia spp. are a heterogeneous globally distributed group of bacteria that colonize plant xylem vessels. Ralstonia cells multiply rapidly in plants and obstruct water transport, causing fatal wilting and serious economic losses of many key food security crops. The virulence of these pathogens depends on their ability to grow to high cell densities in the low-oxygen xylem environment. Plant-pathogenic Ralstonia can use denitrifying respiration to generate ATP. The last denitrification step, nitrous oxide reduction by NosZ, contributes to energy production and virulence for only one of the three phytopathogenic Ralstonia species. These complete denitrifiers form thicker biofilms in culture and in tomato xylem, suggesting they are better adapted to hypoxic niches. Strains with partial denitrification physiology form less biofilm and are more often planktonic. They are nonetheless highly virulent. Thus, these closely related bacteria have adapted their core metabolic functions to exploit distinct microniches in the same habitat.

Bacteriocin Production Correlates with Epidemiological Prevalence of Phylotype I Sequevar 18 Ralstonia pseudosolanacearum in Madagascar.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is a major threat to vegetable crops in Madagascar. For more effective disease management, surveys were carried out in the main vegetable production areas of the country, leading to the collection of 401 new RSSC isolates. Phylogenetic assignment of the isolates revealed a high prevalence of phylotype I sequevar 18. This result contrasts sharply with the epidemiological pattern of RSSC in neighboring islands, including Reunion Island, Comoros, Mayotte, Mauritius, Rodrigues, and the Seychelles, where phylotype I sequevar 31 is widespread. Molecular typing characterization of the Malagasy isolates allowed the identification of 96 haplotypes. Some are found in various plots located in different provinces, which suggests that they were probably disseminated via infected plant material. To find out a potential explanation for the observed epidemiological pattern, we examined the capacity of the Malagasy strains to produce bacteriocin. Interestingly, the highly prevalent genetic lineages I-18 produce bacteriocins that are active against all the genetic lineages present in the country. This work sheds light on the potential impact of bacteriocins in the epidemiology of Malagasy RSSC. IMPORTANCE Knowledge of the epidemiology of a plant pathogen is essential to develop effective control strategies. This study focuses on the epidemiological pattern of Ralstonia pseudosolanacearum phylotype I populations responsible for bacterial wilt in Madagascar. We identified, with the newly collected isolates in three provinces, four genetic lineages probably propagated via infected plant material in Madagascar. We revealed that the epidemiological situation in Madagascar contrasts with that of neighboring Indian Ocean islands. Interestingly, our study on the bacteriocin-producing capacity of Malagasy isolates revealed a correlation between the inhibitory activity of the producing strains and the observed epidemiology. These results suggested that the epidemiology of plant pathogens may be impacted by bacteriocin production.

Molecular Epidemiology of Ralstonia pseudosolanacearum Phylotype I Strains in the Southwest Indian Ocean Region and Their Relatedness to African Strains.

The increasing requirement for developing tools enabling fine strain traceability responsible for epidemics is tightly linked with the need to understand factors shaping pathogen populations and their environmental interactions. Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is one of the most important plant diseases in tropical and subtropical regions. Sadly, little, outdated, or no information on its epidemiology is reported in the literature, although alarming outbreaks are regularly reported as disasters. A large set of phylotype I isolates (n = 2,608) was retrieved from diseased plants in fields across the Southwest Indian Ocean (SWIO) and Africa. This collection enabled further assessment of the epidemiological discriminating power of the previously published RS1-MLVA14 scheme. Thirteen markers were validated and characterized as not equally informative. Most had little infra-sequevar polymorphism, and their performance depended on the sequevar. Strong correlation was found with a previous multilocus sequence typing scheme. However, 2 to 3% of sequevars were not correctly assigned through endoglucanase gene sequence. Discriminant analysis of principal components (DAPC) revealed four groups with strong phylogenetic relatedness to sequevars 31, 33, and 18. Phylotype I-31 isolates were highly prevalent in the SWIO and Africa, but their dissemination pathways remain unclear. Tanzania and Mauritius showed the greatest diversity of RSSC strains, as the four DAPC groups were retrieved. Mauritius was the sole territory harboring a vast phylogenetic diversity and all DAPC groups. More research is still needed to understand the high prevalence of phylotype I-31 at such a large geographic scale.

Contrasting genetic diversity and structure among Malagasy Ralstonia pseudosolanacearum phylotype I populations inferred from an optimized Multilocus Variable Number of Tandem Repeat Analysis scheme.

The Ralstonia solanacearum species complex (RSSC), composed of three species and four phylotypes, are globally distributed soil-borne bacteria with a very broad host range. In 2009, a devastating potato bacterial wilt outbreak was declared in the central highlands of Madagascar, which reduced the production of vegetable crops including potato, eggplant, tomato and pepper. A molecular epidemiology study of Malagasy RSSC strains carried out between 2013 and 2017 identified R. pseudosolanacearum (phylotypes I and III) and R. solanacearum (phylotype II). A previously published population biology analysis of phylotypes II and III using two MultiLocus Variable Number of Tandem Repeats Analysis (MLVA) schemes revealed an emergent epidemic phylotype II (sequevar 1) group and endemic phylotype III isolates. We developed an optimized MLVA scheme (RS1-MLVA14) to characterize phylotype I strains in Madagascar to understand their genetic diversity and structure. The collection included isolates from 16 fields of different Solanaceae species sampled in Analamanga and Itasy regions (highlands) in 2013 (123 strains) and in Atsinanana region (lowlands) in 2006 (25 strains). Thirty-one haplotypes were identified, two of them being particularly prevalent: MT007 (30.14%) and MT004 (16.44%) (sequevar 18). Genetic diversity analysis revealed a significant contrasting level of diversity according to elevation and sampling region. More diverse at low altitude than at high altitude, the Malagasy phylotype I isolates were structured in two clusters, probably resulting from different historical introductions. Interestingly, the most prevalent Malagasy phylotype I isolates were genetically distant from regional and worldwide isolates. In this work, we demonstrated that the RS1-MLVA14 scheme can resolve differences from regional to field scales and is thus suited for deciphering the epidemiology of phylotype I populations.

Spontaneous mutations in a regulatory gene induce phenotypic heterogeneity and adaptation of Ralstonia solanacearum to changing environments.

An evolution experiment with the bacterial plant pathogen Ralstonia solanacearum revealed that several adaptive mutations conferring enhanced fitness in plants arose in the efpR gene encoding a regulator of virulence and metabolic functions. In this study, we found that an efpR mutant systematically displays colonies with two morphotypes: the type S ('smooth', similar to the wild type) and the type EV ('efpR variant'). We demonstrated that the efpH gene, a homologue of efpR, plays a key role in the control of phenotypic heterogeneity, the ΔefpR-ΔefpH double mutant being stably locked into the EV type. Using mixed infection assays, we demonstrated that the type EV is metabolically more proficient than the type S and displays fitness gain in specific environments, whereas the type S has a better fitness into the plant environment. We provide evidence that this efpR-dependent phenotypic heterogeneity is a general feature of strains of the R. solanacearum species complex and could occur in natural conditions. This study highlights the potential role of phenotypic heterogeneity in this plant pathogen as an adaptive trait to changing environments.

Bacterial Diseases of Bananas and Enset: Current State of Knowledge and Integrated Approaches Toward Sustainable Management.

Bacterial diseases of bananas and enset have not received, until recently, an equal amount of attention compared to other major threats to banana production such as the fungal diseases black leaf streak (Mycosphaerella fijiensis) and Fusarium wilt (Fusarium oxysporum f. sp. cubense). However, bacteria cause significant impacts on bananas globally and management practices are not always well known or adopted by farmers. Bacterial diseases in bananas and enset can be divided into three groups: (1) Ralstonia-associated diseases (Moko/Bugtok disease caused by Ralstonia solanacearum and banana blood disease caused by R. syzygii subsp. celebesensis); (2) Xanthomonas wilt of banana and enset, caused by Xanthomonas campestris pv. musacearum and (3) Erwinia-associated diseases (bacterial head rot or tip-over disease Erwinia carotovora ssp. carotovora and E. chrysanthemi), bacterial rhizome and pseudostem wet rot (Dickeya paradisiaca formerly E. chrysanthemi pv. paradisiaca). Other bacterial diseases of less widespread importance include: bacterial wilt of abaca, Javanese vascular wilt and bacterial fingertip rot (probably caused by Ralstonia spp., unconfirmed). This review describes global distribution, symptoms, pathogenic diversity, epidemiology and the state of the art for sustainable disease management of the major bacterial wilts currently affecting banana and enset.

Tube-Wise Diagnostic Microarray for the Multiplex Characterization of the Complex Plant Pathogen Ralstonia solanacearum.

Ralstonia solanacearum is a well-known agricultural and ecological threat worldwide. The complexity of the R. solanacearum species complex (Rssc) represents a challenge for the accurate characterization of epidemiological strains by official services and research laboratories. The majority of protocols only focus on a narrow range of strains; however, this species complex includes strains that represent major constraints and are under strict regulation. The main drawback associated with the current methods of detecting and characterizing Rssc strains is their reliance on combining different protocols to properly characterize the strains at the ecotype level, which require time and money. Therefore, we used microarray technology (ArrayTube) to develop a standard protocol, which characterizes 17 major groups of interest in the Rssc, in a single multiplex reaction. These 17 majors groups are linked with a phylogenetic assignation (phylotypes, sequevars), but also with an ecotype assignation associated with a range of hosts (e.g., brown rot, Moko). Probes were designed with a 50-mer length constraint and thoroughly evaluated for any flaws or secondary structures. The strains are characterized based on a DNA extraction from pure culture. Validation data showed strong intra-repeatability, inter-repeatability, and reproducibility as well as good specificity. A hierarchical analysis of the probe groups is suitable for an accurate characterization. Compared with single marker detection tests, the method described in this paper addresses efficiently the issue of combining several tests by testing a large number of phylogenetic markers in a single reaction assay. This custom microarray (RsscAT) represents a significant improvement in the epidemiological monitoring of Rssc strains worldwide, and it has the potential to provide insights for phylogenetic incongruence of Rssc strains based on the host of isolation and may be used to indicate potentially emergent strains.

Sequevar Diversity and Virulence of Ralstonia solanacearum Phylotype I on Mayotte Island (Indian Ocean).

The genetic and phenotypic diversity of the Ralstonia solanacearum species complex, which causes bacterial wilt to Solanacae, was assessed in 140 strains sampled from the main vegetable production areas of the Mayotte island. Only phylotype I strains were identified in the five surveyed areas. The strains were distributed into the following 4 sequevars: I-31 (85.7%), I-18 (5.0%), I-15 (5.7%), and I-46 (3.6%). The central area of Mayotte was the most diverse region, harboring 4 sequevars representing 47.1% of the collected strains. Virulence tests were performed under field and controlled conditions on a set of 10 tomato breeding line accessions and two commercial hybrid tomato cultivars. The strains belonging to sequevar I-31 showed the highest virulence on the tomatoes (pathotypes T-2 and T-3), whereas sequevars I-18, I-15, and I-46 were grouped into the weakly T-1 pathotype. When the tomato accessions were challenged in the field and growth chambers, the highest level of resistance were observed from the genetically related accessions Hawaii 7996, R3034, TML46, and CLN1463. These accessions were considered moderately to highly resistant to representative strains of the most virulent and prevalent sequevar (I-31). Interestingly, the Platinum F1 cultivar, which was recently commercialized in Mayotte for bacterial wilt resistance, was highly or moderately resistant to all strains. This study represents the first step in the rationalization of resistance deployment strategies against bacterial wilt-causing strains in Mayotte.

Genetic Diversity of the Ralstonia solanacearum Species Complex in the Southwest Indian Ocean Islands.

Epidemiological surveillance of plant pathogens based on genotyping methods is mandatory to improve disease management strategies. In the Southwest Indian Ocean (SWIO) islands, bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC) is hampering the production of many sustainable and cash crops. To thoroughly analyze the genetic diversity of the RSSC in the SWIO, we performed a wide sampling survey (in Comoros, Mauritius, Reunion, Rodrigues, and Seychelles) that yielded 1,704 isolates from 129 plots, mainly from solanaceous crops. Classification of the isolates to the four major RSSC phylogenetic groups, named phylotypes, showed that 87% were phylotype I, representing the most prevalent strain in each of the SWIO islands. Additionally, 9.7% were phylotype II, and 3.3% were phylotype III; however, these isolates were found only in Reunion. Phylotype IV (2 isolates), known to be restricted to Indonesia-Australia-Japan, was reported in Mauritius, representing the first report of this group in the SWIO. Partial endoglucanase (egl) sequencing, based on the selection of 145 isolates covering the geographic and host diversity in the SWIO (also including strains from Mayotte and Madagascar), revealed 14 sequevars with Reunion and Mauritius displaying the highest sequevar diversity. Through a multilocus sequence analysis (MLSA) scheme based on the partial sequencing of 6 housekeeping genes (gdhA, gyrB, rplB, leuS, adk, and mutS) and 1 virulence-associated gene (egl), we inferred the phylogenetic relationships between these 145 SWIO isolates and 90 worldwide RSSC reference strains. Phylotype I was the most recombinogenic, although recombination events were detected among all phylotypes. A multilocus sequence typing (MLST) scheme identified 29 sequence types (STs) with variable geographic distributions in the SWIO. The outstanding epidemiologic feature was STI-13 (sequevar I-31), which was overrepresented in the SWIO and obviously reflected a lineage strongly adapted to the SWIO environment. A goeBURST analysis identified eight clonal complexes (CCs) including SWIO isolates, four CCs being geographically restricted to the SWIO, and four CCs being widespread beyond the SWIO. This work, which highlights notable genetic links between African and SWIO strains, provides a basis for the epidemiological surveillance of RSSC and will contribute to BW management in the SWIO.

Molecular Epidemiology of Bacterial Wilt in the Madagascar Highlands Caused by Andean (Phylotype IIB-1) and African (Phylotype III) Brown Rot Strains of the Ralstonia solanacearum Species Complex.

The Ralstonia solanacearum species complex (RSSC) is a highly diverse cluster of bacterial strains found worldwide, many of which are destructive and cause bacterial wilt (BW) in a wide range of host plants. In 2009, potato production in Madagascar was dramatically affected by several BW epidemics. Controlling this disease is critical for Malagasy potato producers. The first important step toward control is the characterization of strains and their putative origins. The genetic diversity and population structure of the RSSC were investigated in the major potato production areas of the Highlands. A large collection of strains (n = 1224) was assigned to RSSC phylotypes based on multiplex polymerase chain reaction (PCR). Phylotypes I and III have been present in Madagascar for a long time but rarely associated with major potato BW outbreaks. The marked increase of BW prevalence was found associated with phylotype IIB sequevar 1 (IIB-1) strains (n = 879). This is the first report of phylotype IIB-1 strains in Madagascar. In addition to reference strains, epidemic IIB-1 strains (n = 255) were genotyped using the existing MultiLocus Variable-Number Tandem Repeat Analysis (MLVA) scheme RS2-MLVA9, producing 31 haplotypes separated into two related clonal complexes (CCs). One major CC included most of the worldwide haplotypes distributed across wide areas. A regional-scale investigation suggested that phylotype IIB-1 strains were introduced and massively spread via latently infected potato seed tubers. Additionally, the genetic structure of phylotype IIB-1 likely resulted from a bottleneck/founder effect. The population structure of phylotype III, described here for the first time in Madagascar, exhibited a different pattern. Phylotype III strains (n = 217) were genotyped using the highly discriminatory MLVA scheme RS3-MLVA16. High genetic diversity was uncovered, with 117 haplotypes grouped into 11 CCs. Malagasy phylotype III strains were highly differentiated from continental African strains, suggesting no recent migration from the continent. Overall, population structure of phylotype III involves individual small CCs that correlate to restricted geographic areas in Madagascar. The evidence suggests, if at all, that African phylotype III strains are not efficiently transmitted through latently infected potato seed tubers.

TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity.

Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47-91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other's EBEs. Investigation of sequence divergence between RipTAL repeats allows for a reconstruction of repeat array biogenesis, for example through slipped strand mispairing or gene conversion. Using these studies we show how RipTALs of broad host range strains evolved convergently toward a shared target sequence. Finally, we discuss the differences between TALE-likes of plant pathogens in the context of disease ecology.

Degradation of the Plant Defense Signal Salicylic Acid Protects Ralstonia solanacearum from Toxicity and Enhances Virulence on Tobacco.

UNLABELLED: Plants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule. IMPORTANCE: Plant pathogens such as the bacterial wilt agent Ralstonia solanacearum threaten food and economic security by causing significant losses for small- and large-scale growers of tomato, tobacco, banana, potato, and ornamentals. Like most plants, these crop hosts use salicylic acid (SA) both indirectly as a signal to activate defenses and directly as an antimicrobial chemical. We found that SA inhibits growth of R. solanacearum and induces a general stress response that includes repression of multiple bacterial wilt virulence factors. The ability to degrade SA reduces the pathogen's sensitivity to SA toxicity and increases its virulence on tobacco.

A novel multilocus variable number tandem repeat analysis typing scheme for African phylotype III strains of the Ralstonia solanacearum species complex.

Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used. Results and Discussion. Sixteen polymorphic TR loci, which included seven microsatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more discriminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC phylotype III.

Genomic and proteomic evidence supporting the division of the plant pathogen Ralstonia solanacearum into three species.

BACKGROUND: The increased availability of genome sequences has advanced the development of genomic distance methods to describe bacterial diversity. Results of these fast-evolving methods are highly correlated with those of the historically standard DNA-DNA hybridization technique. However, these genomic-based methods can be done more rapidly and less expensively and are less prone to technical and human error. They are thus a technically accessible replacement for species delineation. Here, we use several genomic comparison methods, supported by our own proteomic analyses and metabolic characterization as well as previously published DNA-DNA hybridization analyses, to differentiate members of the Ralstonia solanacearum species complex into three species. This pathogen group consists of diverse and widespread strains that cause bacterial wilt disease on many different plants. RESULTS: We used three different methods to compare the complete genomes of 29 strains from the R. solanacearum species complex. In parallel we profiled the proteomes of 73 strains using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). Proteomic profiles together with genomic sequence comparisons consistently and comprehensively described the diversity of the R. solanacearum species complex. In addition, genome-driven functional phenotypic assays excitingly supported an old hypothesis (Hayward et al. (J Appl Bacteriol 69:269-80, 1990)), that closely related members of the R. solanacearum could be identified through a simple assay of anaerobic nitrate metabolism. This assay allowed us to clearly and easily differentiate phylotype II and IV strains from phylotype I and III strains. Further, genomic dissection of the pathway distinguished between proposed subspecies within the current phylotype IV. The assay revealed large scale differences in energy production within the R. solanacearum species complex, indicating coarse evolutionary distance and further supporting a repartitioning of this group into separate species. CONCLUSIONS: Together, the results of these studies support the proposed division of the R. solanacearum species complex into three species, consistent with recent literature, and demonstrate the utility of proteomic and genomic approaches to delineate bacterial species.

In planta comparative transcriptomics of host-adapted strains of Ralstonia solanacearum.

Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana) and NPB (not pathogenic to banana) strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions. Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem. Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes.

Comparative genomic analysis of Ralstonia solanacearum reveals candidate genes for host specificity.

BACKGROUND: Ralstonia solanacearum is a vascular soil-borne plant pathogen with an unusually broad host range. This economically destructive and globally distributed bacterium has thousands of distinct lineages within a heterogeneous and taxonomically disputed species complex. Some lineages include highly host-adapted strains (ecotypes), such as the banana Moko disease-causing strains, the cold-tolerant potato brown rot strains (also known as R3bv2) and the recently emerged Not Pathogenic to Banana (NPB) strains. RESULTS: These distinct ecotypes offer a robust model to study host adaptation and the emergence of ecotypes because the polyphyletic Moko strains include lineages that are phylogenetically close to the monophyletic brown rot and NPB strains. Draft genomes of eight new strains belonging to these three model ecotypes were produced to complement the eleven publicly available R. solanacearum genomes. Using a suite of bioinformatics methods, we searched for genetic and evolutionary features that distinguish ecotypes and propose specific hypotheses concerning mechanisms of host adaptation in the R. solanacearum species complex. Genome-wide, few differences were identified, but gene loss events, non-synonymous polymorphisms, and horizontal gene transfer were identified among type III effectors and were associated with host range differences. CONCLUSIONS: This extensive comparative genomics analysis uncovered relatively few divergent features among closely related strains with contrasting biological characteristics; however, several virulence factors were associated with the emergence of Moko, NPB and brown rot and could explain host adaptation.

A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

Moko Disease-Causing Strains of Ralstonia solanacearum from Brazil Extend Known Diversity in Paraphyletic Phylotype II.

The epidemic situation of Moko disease-causing strains in Latin America and Brazil is unclear. Thirty-seven Ralstonia solanacearum strains from Brazil that cause the Moko disease on banana and heliconia plants were sampled and phylogenetically typed using the endoglucanase (egl) and DNA repair (mutS) genes according to the phylotype and sequevar classification. All of the strains belonged to phylotype II and a portion of the strains was typed as the Moko disease-related sequevars IIA-6 and IIA-24. Nevertheless, two unsuspected sequevars also harbored the Moko disease-causing strains IIA-41 and IIB-25, and a new sequevar was described and named IIA-53. All of the strains were pathogenic to banana and some of the strains of sequevars IIA-6, IIA-24, and IIA-41 were also pathogenic to tomato. The Moko disease-causing strains from sequevar IIB-25 were pathogenic to potato but not to tomato. These results highlight the high diversity of strains of Moko in Brazil, reinforce the efficiency of the egl gene to reveal relationships among these strains, and contribute to a better understanding of the diversity of paraphyletic Moko disease-causing strains of the R. solanacearum species complex, where the following seven distinct genetic clusters have been described: IIA-6, IIA-24, IIA-41, IIA-53, IIB-3, IIB-4, and IIB-25.

Ralstonia solanacearum requires PopS, an ancient AvrE-family effector, for virulence and To overcome salicylic acid-mediated defenses during tomato pathogenesis.

UNLABELLED: During bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression of popS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in the R. solanacearum species complex. The phylogeny of popS mirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. A popS mutant of R. solanacearum UW551 had reduced virulence on agriculturally important Solanum spp., including potato and tomato plants. However, the popS mutant had wild-type virulence on a weed host, Solanum dulcamara, suggesting that some species can avoid the effects of PopS. The popS mutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, the popS mutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated the popS mutant virulence defect. Finally, the popS mutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which are R. solanacearum's natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed in Nicotiana leaf tissue, unlike the AvrE homolog DspEPcc from the necrotroph Pectobacterium carotovorum subsp. carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophic R. solanacearum. IMPORTANCE: The type III-secreted AvrE effector family is widely distributed in high-impact plant-pathogenic bacteria and is known to suppress plant defenses for virulence. We characterized the biology of PopS, the only AvrE homolog made by the bacterial wilt pathogen Ralstonia solanacearum. To our knowledge, this is the first study of R. solanacearum effector function in roots and stems, the natural infection sites of this pathogen. Unlike the functionally redundant R. solanacearum effectors studied to date, PopS is required for full virulence and wild-type colonization of two natural crop hosts. R. solanacearum is a biotrophic pathogen that causes a nonnecrotic wilt. Consistent with this, PopS suppressed plant defenses but did not elicit cell death, unlike AvrE homologs from necrosis-causing plant pathogens. We propose that AvrE family effectors have functionally diverged to adapt to the necrotic or nonnecrotic lifestyle of their respective pathogens.

Constraints on genome dynamics revealed from gene distribution among the Ralstonia solanacearum species.

Because it is suspected that gene content may partly explain host adaptation and ecology of pathogenic bacteria, it is important to study factors affecting genome composition and its evolution. While recent genomic advances have revealed extremely large pan-genomes for some bacterial species, it remains difficult to predict to what extent gene pool is accessible within or transferable between populations. As genomes bear imprints of the history of the organisms, gene distribution pattern analyses should provide insights into the forces and factors at play in the shaping and maintaining of bacterial genomes. In this study, we revisited the data obtained from a previous CGH microarrays analysis in order to assess the genomic plasticity of the R. solanacearum species complex. Gene distribution analyses demonstrated the remarkably dispersed genome of R. solanacearum with more than half of the genes being accessory. From the reconstruction of the ancestral genomes compositions, we were able to infer the number of gene gain and loss events along the phylogeny. Analyses of gene movement patterns reveal that factors associated with gene function, genomic localization and ecology delineate gene flow patterns. While the chromosome displayed lower rates of movement, the megaplasmid was clearly associated with hot-spots of gene gain and loss. Gene function was also confirmed to be an essential factor in gene gain and loss dynamics with significant differences in movement patterns between different COG categories. Finally, analyses of gene distribution highlighted possible highways of horizontal gene transfer. Due to sampling and design bias, we can only speculate on factors at play in this gene movement dynamic. Further studies examining precise conditions that favor gene transfer would provide invaluable insights in the fate of bacteria, species delineation and the emergence of successful pathogens.