Calcium modulation of bacterial wilt disease on potato.

Ralstonia solanacearum species complex (RSSC) is a phytopathogenic bacterial group that causes bacterial wilt in several crops, being potato (Solanum tuberosum) one of the most important hosts. The relationship between the potato plant ionome (mineral and trace elements composition) and the resistance levels to this pathogen has not been addressed until now. Mineral content of xylem sap, roots, stems and leaves of potato genotypes with different levels of resistance to bacterial wilt was assessed in this work, revealing a positive correlation between divalent calcium (Ca) cation concentrations and genotype resistance. The aim of this study was to investigate the effect of Ca on bacterial wilt resistance, and on the growth and virulence of RSSC. Ca supplementation significantly decreased the growth rate of Ralstonia pseudosolanacearum GMI1000 in minimal medium and affected several virulence traits such as biofilm formation and twitching motility. We also incorporate for the first time the use of microfluidic chambers to follow the pathogen growth and biofilm formation in conditions mimicking the plant vascular system. By using this approach, a reduction in biofilm formation was observed when both, rich and minimal media, were supplemented with Ca. Assessment of the effect of Ca amendments on bacterial wilt progress in potato genotypes revealed a significant delay in disease progress, or a complete absence of wilting symptoms in the case of partially resistant genotypes. This work contributes to the understanding of Ca effect on virulence of this important pathogen and provides new strategies for an integrated control of bacterial wilt on potato. IMPORTANCE: Ralstonia solanacearum species complex (RSSC) includes a diverse group of bacterial strains that cause bacterial wilt. This disease is difficult to control due to pathogen aggressiveness, persistence, wide range of hosts, and wide geographic distribution in tropical, subtropical, and temperate regions. RSSC causes considerable losses depending on the pathogen strain, host, soil type, environmental conditions, and cultural practices. In potato, losses of $19 billion per year have been estimated for this pathogen worldwide. In this study, we report for the first time the mineral composition found in xylem sap and plant tissues of potato germplasm with different levels of resistance to bacterial wilt. This study underscores the crucial role of calcium (Ca) concentration in the xylem sap and stem in relation to the resistance of different genotypes. Our in vitro experiments provide evidence of Ca's inhibitory effect on the growth, biofilm formation, and twitching movement of the model RSSC strain R. pseudosolanacearum GMI1000. This study introduces a novel element, the Ca concentration, which should be included into the integrated disease control management strategies for bacterial wilt in potatoes.

The Micacocidin Production-Related RSc1806 Deletion Alters the Quorum Sensing-Dependent Gene Regulation of Ralstonia pseudosolanacearum Strain OE1-1.

The soil-borne phytopathogenic gram-negative bacterium Ralstonia solanacearum species complex (RSSC) produces staphyloferrin B and micacocidin as siderophores that scavenge for trivalent iron (Fe3+) in the environment, depending on the intracellular divalent iron (Fe2+) concentration. The staphyloferrin B-deficient mutant reportedly retains its virulence, but the relationship between micacocidin and virulence remains unconfirmed. To elucidate the effect of micacocidin on RSSC virulence, we generated the micacocidin productivity-deficient mutant (ΔRSc1806) that lacks RSc1806, which encodes a putative polyketide synthase/non-ribosomal peptide synthetase, using the RSSC phylotype I Ralstonia pseudosolanacearum strain OE1-1. When incubated in the condition without Fe2+, ΔRSc1806 showed significantly lower Fe3+-scavenging activity, compared with OE1-1. Until 8 days after inoculation on tomato plants, ΔRSc1806 was not virulent, similar to the mutant (ΔphcA) missing phcA, which encodes the LysR-type transcriptional regulator PhcA that regulates the expression of the genes responsible for quorum sensing (QS)-dependent phenotypes including virulence. The transcriptome analysis revealed that RSc1806 deletion significantly altered the expression of more than 80% of the PhcA-regulated genes in the mutant grown in medium with or without Fe2+. Among the PhcA-regulated genes, the transcript levels of the genes whose expression was affected by the deletion of RSc1806 were strongly and positively correlated between the ΔRSc1806 and the phcA-deletion mutant. Furthermore, the deletion of RSc1806 significantly modified QS-dependent phenotypes, similar to the effects of the deletion of phcA. Collectively, our findings suggest that the deletion of micacocidin production-related RSc1806 alters the regulation of PhcA-regulated genes responsible for QS-dependent phenotypes including virulence as well as Fe3+-scavenging activity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Novel Ralstonia species from human infections: improved matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification and analysis of antimicrobial resistance patterns.

A collection of 161 Ralstonia isolates, including 90 isolates from persons with cystic fibrosis, 27 isolates from other human clinical samples, 8 isolates from the hospital environment, 7 isolates from industrial samples, and 19 environmental isolates, was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification and yielded confident species level identification scores for only 62 (39%) of the isolates, including four that proved misidentified subsequently. Whole-genome sequence analysis of 32 representative isolates for which no confident MALDI-TOF MS species level identification was obtained revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively, and provided the basis for updating an in-house MALDI-TOF MS database. A reanalysis of all mass spectra with the updated MALDI-TOF MS database increased the percentage of isolates with confident species level identification up to 77%. The antimicrobial susceptibility of 30 isolates mainly representing novel human clinical and environmental Ralstonia species was tested toward 17 antimicrobial agents and demonstrated that the novel Ralstonia species were generally multi-resistant, yet susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline. An analysis of genomic antimicrobial resistance genes in 32 novel and publicly available genome sequences revealed broadly distributed beta-lactam resistance determinants.IMPORTANCEThe present study demonstrated that a commercial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification database can be tailored to improve the identification of Ralstonia species. It also revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively. An analysis of minimum inhibitory concentration values demonstrated that the novel Ralstonia species were generally multi-resistant but susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline.

Characterization of Ralstonia insidiosa C1 isolated from Alpine regions: Capability in polyhydroxyalkanoates degradation and production.

This study ventures into the exploration of potential poly-3-hydroxybutyrate (PHB) degradation in alpine environments. PHB-degrading bacteria were identified in both campus soil, representing a residential area, and Mt. Kurodake soil, an alpine region in Hokkaido, Japan. Next-generation sequencing analysis indicated that the campus soil exhibited higher microbial diversity, while Ralstonia insidiosa C1, isolated from Mt. Kurodake soil, displayed the highest proficiency in PHB degradation. R. insidiosa C1 efficiently degraded up to 3% (w/v) of PHB and various films composed of other biopolymers at 14 °C. This bacterium synthesized homopolymers using substrates such as 3-hydroxybutyric acid, sugars, and acetic acid, while also produced copolymers using a mixture of fatty acids. The analysis results confirmed that the biopolymer synthesized by strain C1 using glucose was PHB, with physical properties comparable to commercial products. The unique capabilities of R. insidiosa C1, encompassing both the production and degradation of bioplastics, highlight its potential to establish a novel material circulation model.

Comparative genomics and host range analysis of four Ralstonia pseudosolanacearum strains isolated from sunflower reveals genomic and phenotypic differences.

BACKGROUND: Bacterial wilt caused by Ralstonia solanacearum species complex (RSSC) is one of the devastating diseases in crop production, seriously reducing the yield of crops. R. pseudosolanacearum, is known for its broad infrasubspecific diversity and comprises 36 sequevars that are currently known. Previous studies found that R. pseudosolanacearum contained four sequevars (13, 14, 17 and 54) isolated from sunflowers sown in the same field. RESULTS: Here, we provided the complete genomes and the results of genome comparison of the four sequevars strains (RS639, RS642, RS647, and RS650). Four strains showed different pathogenicities to the same cultivars and different host ranges. Their genome sizes were about 5.84 ~ 5.94 Mb, encoding 5002 ~ 5079 genes and the average G + C content of 66.85% ~ 67%. Among the coding genes, 146 ~ 159 specific gene families (contained 150 ~ 160 genes) were found in the chromosomes and 34 ~ 77 specific gene families (contained 34 ~ 78 genes) in the megaplasmids from four strains. The average nucleotide identify (ANI) values between any two strains ranged from 99.05% ~ 99.71%, and the proportion of the total base length of collinear blocks accounts for the total gene length of corresponding genome was all more than 93.82%. Then, we performed a search for genomic islands, prophage sequences, the gene clusters macromolecular secretion systems, type III secreted effectors and other virulence factors in these strains, which provided detailed comparison results of their presence and distinctive features compared to the reference strain GMI1000. Among them, the number and types of T2SS gene clusters were different in the four strains, among which RS650 included all five types. T4SS gene cluster of RS639 and RS647 were missed. In the T6SS gene cluster, several genes were inserted in the RS639, RS647, and RS650, and gene deletion was also detected in the RS642. A total of 78 kinds of type III secreted effectors were found, which included 52 core and 9 specific effectors in four strains. CONCLUSION: This study not only provided the complete genomes of multiple R. pseudosolanacearum strains isolated from a new host, but also revealed the differences in their genomic levels through comparative genomics. Furthermore, these findings expand human knowledge about the range of hosts that Ralstonia can infect, and potentially contribute to exploring rules and factors of the genetic evolution and analyzing its pathogenic mechanism.

Molecular basis for the interference of the Arabidopsis WRKY54-mediated immune response by two sequence-unrelated bacterial effectors.

Arabidopsis thaliana WRKY proteins are potential targets of pathogen-secreted effectors. RESISTANT TO RALSTONIA SOLANACEARUM 1 (RRS1; AtWRKY52) is a well-studied Arabidopsis nucleotide-binding and leucine-rich repeat (NLR) immune receptor carrying a C-terminal WRKY domain that functions as an integrated decoy. RRS1-R recognizes the effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia pseudosolanacearum by direct interaction through its WRKY domain. AvrRps4 and PopP2 were previously shown to interact with several AtWRKYs. However, how these effectors selectively interact with their virulence targets remains unknown. Here, we show that several members of subgroup IIIb of the AtWRKY family are targeted by AvrRps4 and PopP2. We demonstrate that several AtWRKYs induce cell death when transiently expressed in Nicotiana benthamiana, indicating the activation of immune responses. AtWRKY54 was the only cell death-inducing AtWRKY that interacted with both AvrRps4 and PopP2. We found that AvrRps4 and PopP2 specifically suppress AtWRKY54-induced cell death. We also demonstrate that the amino acid residues required for the avirulence function of AvrRps4 and PopP2 are critical for suppressing AtWRKY54-induced cell death. AtWRKY54 residues predicted to form a binding interface with AvrRps4 were predominantly located in the DNA binding domain and necessary for inducing cell death. Notably, one AtWRKY54 residue, E164, contributes to affinity with AvrRps4 and is exclusively present among subgroup IIIb AtWRKYs, yet is located outside of the DNA-binding domain. Surprisingly, AtWRKY54 mutated at E164 evaded AvrRps4-mediated cell death suppression. Taking our observations together, we propose that AvrRp4 and PopP2 specifically target AtWRKY54 to suppress plant immune responses.

Transcriptomic profiling reveals host-specific evolutionary pathways promoting enhanced fitness in the plant pathogen Ralstonia pseudosolanacearum.

The impact of host diversity on the genotypic and phenotypic evolution of broad-spectrum pathogens is an open issue. Here, we used populations of the plant pathogen Ralstonia pseudosolanacearum that were experimentally evolved on five types of host plants, either belonging to different botanical families or differing in their susceptibility or resistance to the pathogen. We investigated whether changes in transcriptomic profiles, associated with or independent of genetic changes, could occur during the process of host adaptation, and whether transcriptomic reprogramming was dependent on host type. Genomic and transcriptomic variations were established for 31 evolved clones that showed better fitness in their experimental host than the ancestral clone. Few genomic polymorphisms were detected in these clones, but significant transcriptomic variations were observed, with a large number of differentially expressed genes (DEGs). In a very clear way, a group of genes belonging to the network of regulation of the bacterial virulence such as efpR, efpH or hrpB, among others, were deregulated in several independent evolutionary lineages and appeared to play a key role in the transcriptomic rewiring observed in evolved clones. A double hierarchical clustering based on the 400 top DEGs for each clone revealed 2 major patterns of gene deregulation that depend on host genotype, but not on host susceptibility or resistance to the pathogen. This work therefore highlights the existence of two major evolutionary paths that result in a significant reorganization of gene expression during adaptive evolution and underscore clusters of co-regulated genes associated with bacterial adaptation on different host lines.

Identification, genetic diversity, and pathogenicity of Ralstonia pseudosolanacearum causing cigar tobacco bacterial wilt in China.

Ralstonia pseudosolanacearum, previously known as R. solanacearum species complex (RSSC) phylotypes I and III, is a plant pathogenic bacterium causing significant yield losses in economical crops. In the May of 2020 and 2021, cigar tobacco bacterial wilt was first observed in fields in Danzhou, Hainan Province, China. A total of eight bacterial isolates were isolated and identified as R. pseudosolanacearum with race 1, biovar III by 16S rRNA gene sequencing, Biolog, and host identification. The amino acid sequence showed that Hainan strains and 15 R. pseudosolanacearum reference strains from flue-cured tobacco in Shandong and Guizhou Provinces, all belonged to RS1000 type containing the avrA gene, only Guizhou strains also had the popP1 gene. On the basis of phylotype-specific multiplex PCR amplification, mismatch repair gene and endoglucanase gene-base tree, Hainan strains were identified as phylotype I sequevar 70, and showed stronger pathogenic capabilities on three different varieties than those reference strains. This is the first report of cigar tobacco bacterial wilt caused by R. pseudosolanacearum sequevar 70. The results revealed the diversity of RSSC in Nicotiana tabacum in China and provided useful information regarding the epidemiology of cigar tobacco wilt disease, as well as the breeding for disease resistance in local cigar tobacco.

Whole genome resequencing and complementation tests reveal candidate loci contributing to bacterial wilt (Ralstonia sp.) resistance in tomato.

Tomato (Solanum lycopersicum) is one of the most economically important vegetable crops worldwide. Bacterial wilt (BW), caused by the Ralstonia solanacearum species complex, has been reported as the second most important plant pathogenic bacteria worldwide, and likely the most destructive. Extensive research has identified two major loci, Bwr-6 and Bwr-12, that contribute to resistance to BW in tomato; however, these loci do not completely explain resistance. Segregation of resistance in two populations that were homozygous dominant or heterozygous for all Bwr-6 and Bwr-12 associated molecular markers suggested the action of one or two resistance loci in addition to these two major QTLs. We utilized whole genome sequence data analysis and pairwise comparison of six BW resistant and nine BW susceptible tomato lines to identify candidate genes that, in addition to Bwr-6 and Bwr-12, contributed to resistance. Through this approach we found 27,046 SNPs and 5975 indels specific to the six resistant lines, affecting 385 genes. One sequence variant on chromosome 3 captured by marker Bwr3.2dCAPS located in the Asc (Solyc03g114600.4.1) gene had significant association with resistance, but it did not completely explain the resistance phenotype. The SNP associated with Bwr3.2dCAPS was located within the resistance gene Asc which was inside the previously identified Bwr-3 locus. This study provides a foundation for further investigations into new loci distributed throughout the tomato genome that could contribute to BW resistance and into the role of resistance genes that may act against multiple pathogens.

Genomic and Biological Characterization of Ralstonia solanacearum Inovirus Brazil 1, an Inovirus that Alters the Pathogenicity of the Phytopathogen Ralstonia pseudosolanacearum.

Filamentous bacteriophages contain a single-stranded DNA genome and have a peculiar lifestyle, since they do not cause host cell lysis, but establish a persistent association with the host, often causing behavioral changes, with effects on bacterial ecology. Over the years, a gradual reduction in the incidence of bacterial wilt has been observed in some fields from Brazil. This event, which has been associated with the loss of pathogenicity of Rasltonia spp. isolates due to infection by filamentous viruses of the inovirus group, is widely reported for Ralstonia spp. Asian isolates infected by inoviruses. In an attempt to elucidate which factors are associated with the phenomenon reported in Brazil, we investigated one isolate of R. solanacearum (UB-2014), with unusual characteristics for R. solanacearum, obtained from eggplant with mild wilt symptoms. To verify if the presence of filamentous bacteriophage was related to this phenotype, we performed viral purification and nucleic acid extraction. The phage genome was sequenced, and phylogenetic analyses demonstrated that the virus belongs to the family Inoviridae and was named as Ralstonia solanacerarum inovirus Brazil 1 (RSIBR1). RSIBR1 was transmitted to R. pseudosolanacearum GMI1000, and the virus-infected GMI1000 (GMI1000 VI) isolate showed alterations in phenotypic characteristics, as well as loss of pathogenicity, similarly to that observed in R. solanacearum isolate UB-2014. The presence of virus-infected UB-2014 and GMI1000 VI plants without symptoms, after 3 months, confirms that the infected isolates can colonize the plant without causing disease, which demonstrates that the phage infection changed the behavior of these pathogens.

Ralstonia wenshanensis sp. nov., a novel bacterium isolated from a tobacco field in Yunnan, China.

A Gram-negative, aerobic, motile with paired polar flagella and rod-shaped bacterium strain (56D2T) was isolated from tobacco planting soil in Yunnan, PR China. Major fatty acids were C16  :  1 ω7c (summed feature 3), C16  :  0 and C18  :  1 ω7c (summed feature 8). The polar lipid profile of strain 56D2T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminophospholipid and one unidentified glycolipid. Moreover, strain 56D2T contained ubiquinone Q-8 as the sole respiratory quinone. 16S rRNA gene sequence analysis showed that strain 56D2T was closely related to members of the genus Ralstonia and the two type strains with the highest sequence identities were R. mannitolilytica LMG 6866T (98.36 %) and R. pickettii K-288T (98.22 %). The 16S rRNA gene sequence identities between strain 56D2T and other members of the genus Ralstonia were below 98.00 %. Genome sequencing revealed a genome size of 5.87 Mb and a G+C content of 63.7 mol%. The average nucleotide identity values between strain 56D2T and R. pickettii K-288T, R. mannitolilytica LMG 6866 T and R. insidiosa CCUG 46789T were less than 95 %, and the in silico DNA-DNA hybridization values (yielded by formula 2) were less than 70 %. Based on these data, we conclude that strain 56D2T represents a novel species of the genus Ralstonia, for which the name Ralstonia wenshanensis sp. nov. is proposed. The type strain of Ralstonia wenshanensis sp. nov. is 56D2T (=CCTCC AB 2021466T=GDMCC 1.2886T=JCM 35178T).

Characterization of clinical Ralstonia strains and their taxonomic position.

To improve understanding of the role of Ralstonia in cystic fibrosis (CF), whole genomes of 18 strains from clinical samples were sequenced using Illumina technology. Sequences were analysed by core genome Multi-Locus Sequence Typing, Average Nucleotide Identity based on BLAST (ANIb), RAST annotation, and by ResFinder. Phylogenetic analysis was performed for the 16S rRNA gene, and the OXA-22 and OXA-60 ß-lactamase families. The minimal inhibitory concentrations (MICs) were determined using broth microdilution. ANIb data for the 18 isolates and 54 strains from GenBank, supported by phylogenetic analysis, showed that 8 groups of clusters (A-H), as well as subgroups that should be considered as species or subspecies. Groups A-C contain strains previously identified as Ralstonia solanacearum and Ralstonia pseudosolanacearum. We propose that group A is a novel species. Group B and C are Ralstonia syzygii, Ralstonia solanacearum, respectively. Group D is composed of Ralstonia mannitolilytica and Group E of Ralstonia pickettii. Group F and G should be considered novel species. Group H strains belong to R. insidiosa. OXA-22 and OXA-60 family ß-lactamases were encoded by all strains. Co-trimoxazole generally showed high activity with low MICs (≤1 mg/l) as did ciprofloxacin (≤0.12 mg/l). MICs against the other antibiotics were more variable, but generally high. RAST annotation revealed limited differences between the strains, and virulence factors were not identified. The taxonomy of the genus Ralstonia is in need of revision, but sequencing additional isolates is needed. Antibiotic resistance levels are high. Annotation did not identify potential virulence factors.

Modulating the active site lid of an alcohol dehydrogenase from Ralstonia sp. enabled efficient stereospecific synthesis of 17ß-hydroxysteroids.

Enzymatic stereospecific reduction of 17-oxosteroids offers an attractive approach to access 17ß-hydroxysteroids of pharmaceutical importance. In this study, by adjusting the flexibility of α6-helix at the substrate entrance of the alcohol dehydrogenase from Ralstonia sp. (RasADH), the catalytic activity toward the stereospecific 17ß-reduction of androstenedione was improved without sacrifice of the enantioselectivity. Among the mutants, F205I and F205A exhibited up to 623- and 523-fold improvement in catalytic efficiency, respectively, towards a range of different 17-oxosteroids compared to the wild-type enzyme. The corresponding 17ß-hydroxysteroids were prepared in optically pure form with high space-time productivity and isolated yields using F205I as the biocatalyst, indicating that these mutants are promising biocatalysts for this useful transformation. These results suggest that modulating the flexibility of the active site lid offers an effective approach to engineer alcohol dehydrogenase for accommodating bulky steroidal substrates.

Bacteremia due to Ralstonia mannitolilytica: A report of the first case in Peru. / Bacteremia por Ralstonia mannitolilytica: primer reporte de caso en Perú.

The first report of Ralstonia mannitolilytica bacteremia in Peru is presented. The patient was a pediatric cancer patient with a long-term central venous access device. For the diagnosis, the MicroScan Walk Away 96 automated system was used. 16S rDNA was amplified by conventional PCR, and the bacterial genus and species were identified by genetic sequencing. In addition, the bacterial resistance profile to major antimicrobials was determined. The article discusses the need to actively monitor Ralstonia mannitolilytica, especially in hospital areas of immunocompromised patients.

Se presenta el primer reporte de una bacteriemia por Ralstonia mannitolilytica en Perú. Se trata de un paciente pediátrico con cáncer que porta un dispositivo de acceso venoso central de larga duración. Para establecer el diagnóstico, se utilizó el sistema automático MicroScan Walk Away 96. Se amplificó el rADN 16S mediante PCR convencional y se identificó el género y la especie bacteriana mediante secuenciación genética. Además, se determinó el perfil de resistencia bacteriana a los principales antimicrobianos. El artículo discute la necesidad de monitorizar activamente la presencia de Ralstonia mannitolilytica, especialmente en áreas hospitalarias de pacientes inmunodeprimidos.

A high-throughput virulence screening method for the Ralstonia solanacearum species complex.

Ralstonia solanacearum species complex strains are the causative agents for wilting diseases of many plants, including the economically important brown rot of potato. We developed a high-throughput virulence screen that is implemented in 96-well microtiter plates using seedlings grown in soft water agar to save space, effort, and resources. Nicotiana glutinosa was determined to be the most effective host for this assay, and we confirmed bacterial growth and systemic spread in inoculated seedlings. In our assay, N. glutinosa seeds were sown quickly and easily on top of individual water agar wells of a 96-well plate by pipetting out desired number of seeds in an aqueous suspension. They were inoculated on the same day by first touching a bacterial colony with an autoclaved toothpick and then stabbing the toothpick into the center of the water agar well. Such inoculation method resulted in inocula above a threshold of 2 × 104 CFU per well achieving consistent virulence results and enabling reduction of inoculum preparation efforts to facilitate high-throughput screening. Our assay is suitable for forward genetic screening of a large number of strains, isolates or mutants for disease symptoms under both cool (20 °C) and warm (28 °C) temperature conditions before detailed studies can be narrowed down to a manageable number of desired candidates. Our virulence screen method provides a valuable tool for future work in understanding genetics of virulence of Rssc, especially cool virulence of the highly regulated race 3 biovar 2 group of R. solanacearum, leading toward development of effective control strategies.

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.

Host adaptation and microbial competition drive Ralstonia solanacearum phylotype I evolution in the Republic of Korea.

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.

Transposable elements contribute to the genome plasticity of Ralstonia solanacearum species complex.

The extensive genetic diversity of Ralstonia solanacearum, a serious soil-borne phytopathogen, has led to the concept that R. solanacearum encompasses a species complex [R. solanacearum species complex (RSSC)]. Insertion sequences (ISs) are suggested to play an important role in the genome evolution of this pathogen. Here, we identified and analysed transposable elements (TEs), ISs and transposons, in 106 RSSC genomes and 15 Ralstonia spp. We mapped 10 259 IS elements in the complete genome of 62 representative RSSC strains and closely related Ralstonia spp. A unique set of 20 IS families was widespread across the strains, IS5 and IS3 being the most abundant. Our results showed six novel transposon sequences belonging to the Tn3 family carrying passenger genes encoding antibiotic resistance and avirulence proteins. In addition, internal rearrangement events associated with ISs were demonstrated in Ralstonia pseudosolanacearum strains. We also mapped IS elements interrupting avirulence genes, which provided evidence that ISs plays an important role in virulence evolution of RSSC. Additionally, the activity of ISs was demonstrated by transcriptome analysis and DNA hybridization in R. solanacearum isolates. Altogether, we have provided collective data of TEs in RSSC genomes, opening a new path for understanding their evolutionary impact on the genome evolution and diversity of this important plant pathogen.

Ptr1 evolved convergently with RPS2 and Mr5 to mediate recognition of AvrRpt2 in diverse solanaceous species.

The Ptr1 (Pseudomonas tomato race 1) locus in Solanum lycopersicoides confers resistance to strains of Pseudomonas syringae pv. tomato expressing AvrRpt2 and Ralstonia pseudosolanacearum expressing RipBN. Here we describe the identification and phylogenetic analysis of the Ptr1 gene. A single recombinant among 585 F2 plants segregating for the Ptr1 locus was discovered that narrowed the Ptr1 candidates to eight nucleotide-binding leucine-rich repeat protein (NLR)-encoding genes. From analysis of the gene models in the S. lycopersicoides genome sequence and RNA-Seq data, two of the eight genes emerged as the strongest candidates for Ptr1. One of these two candidates was found to encode Ptr1 based on its ability to mediate recognition of AvrRpt2 and RipBN when it was transiently expressed with these effectors in leaves of Nicotiana glutinosa. The ortholog of Ptr1 in tomato and in Solanum pennellii is a pseudogene. However, a functional Ptr1 ortholog exists in Nicotiana benthamiana and potato, and both mediate recognition of AvrRpt2 and RipBN. In apple and Arabidopsis, recognition of AvrRpt2 is mediated by the Mr5 and RPS2 proteins, respectively. Phylogenetic analysis places Ptr1 in a distinct clade compared with Mr5 and RPS2, and it therefore appears to have arisen by convergent evolution for recognition of AvrRpt2.

Truncating the Structure of Lipopolysaccharide in Escherichia coli Can Effectively Improve Poly-3-hydroxybutyrate Production.

Poly-3-hydroxybutyrate is an environmentally friendly polymer with many promising applications and can be produced in Escherichia coli cells after overexpressing the heterologous gene cluster phaCAB. In this study, we found that truncating the structure of lipopolysaccharide in E. coli can effectively enhance poly-3-hydroxybutyrate production. E. coli mutant strains WJW00, WJD00, and WJJ00 were constructed by deleting rfaD from E. coli strain W3110, DH5α, and JM109, respectively. Compared to the controls W3110/pDXW-8-phaCAB, DH5a/pDXW-8-phaCAB, and JM109/pDXW-8-phaCAB, the yield of poly-3-hydroxybutyrate in WJW00/pDXW-8-phaCAB, WJD00/pDXW-8-phaCAB, and WJJ00/pDXW-8-phaCAB cells increased by 200%, 81.5%, and 75.6%, respectively, and the conversion rate of glucose to poly-3-hydroxybutyrate was increased by ∼250%. Further analysis revealed that LPS truncation in E. coli rebalanced carbon and nitrogen metabolism, increased the levels of acetyl-CoA, γ-aminobutyric acid, NADPH, NADH, and ATP, and decreased the levels of organic acids and flagella, resulting in the high ratio of carbon to nitrogen. These metabolic changes in these E. coli mutants led to the significant increase of poly-3-hydroxybutyrate production.