Ralstonia Pseudosolanacearum Research Articles

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

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. 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 ~ 5.94Mb, 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 ~ 77specific 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. 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.

Current Status of Bacterial Diseases of Vegetable Crops

Emerging bacterial plant diseases became a major threat for the vegetable production system. In the recent past, with changes in farming practices, abrupt weather climate, and the introduction of new varieties and hybrids are considered major factors for the emergence and outbreak of new bacterial pathogens (diseases). The bacterial wilt is an emerging threat for the cultivation of almost all the solanaceous vegetables caused by Ralstonia pseudosolanacearum in India. Incidence of more emerging bacterial diseases in vegetable crops viz tomato leaf blight (Xanthomonas axenopodis pv vesictoria) up to 20% and fruit canker- (Corneybacterium michiganens) up to 5%, bacterial speck (Pseudomonas syringae); brinjal little leaf of brinjal (phytoplasma) up to 40%, cabbage and cauliflower black rot (X. compestris pv compestris) up to 10% and summer squash soft rot and wilting (Erwinia spp.) up to 20% on summer sqash and 19.5% on cauliflower has been recorded. It clearly revealed the cross-infectivity of the bacterial pathogen, indicating the emergence of these pathogens as a broad spectrum covering a wide host range. Future research in this field will include a better understanding of the molecular basis of variability in the pathogen, pathogenesis, accurate and reliable diagnostic of the disease and to engineer novel and durable protection strategies against devastating post-harvest diseases of vegetable crops.

Antibacterial Activity and Mechanism of Three Root Exudates from Mulberry Seedlings against Ralstonia pseudosolanacearum.

Bacterial wilt is a significant soil-borne disease that poses a threat to mulberry production yield and quality of agricultural production worldwide. However, the disease resistance mechanisms dependent on root exudates are not well understood. In this present study, we investigated the antibacterial mechanisms of the main active substances (erucamide, oleamide, and camphor bromide) present in mulberry root exudates (MRE) against Ralstonia pseudosolanacearum (Rp), the causal agent of bacterial wilt. Our findings revealed that these three active substances inhibited the growth activity of Rp by affecting the cell morphology and extracellular polysaccharide content, as well as triggering a burst of reactive oxygen species. The active substances induced oxidative stress, leading to a decrease in Rp growth. Additionally, the expression levels of key genes in the hrp gene cluster (hrpB, hrpX, and hrpF) and other virulence-related genes (such as ripAW, ripAE, Rs5-4819, Rs5-4374, ace, egl3, and pehB) were significantly reduced upon treatment with the active substances. Further pathogenicity experiments demonstrated that root exudates (at a concentration of 1.5 mg·mL-1) delayed or slowed down the occurrence of bacterial wilt in mulberry. These findings provide valuable insight into the antimicrobial mechanisms of MRE against Rp and lay a theoretical foundation for the development and application of biocontrol agents to control mulberry bacterial wilt.

Draft genomes of two phylotype I and II Ralstonia solanacearum species complex (RSSC) isolates causing bacterial wilt in tomato plants from Costa Rica.

Bacteria from RSSC hold agricultural significance as they are the causal agents of bacterial wilt. Here, we report the draft genomes of two bacteria extracted from vascular tissues of infected tomato plants. Isolate RALF-MA was classified as Ralstonia pseudosolanacearum (phylotype I) and RALSA-MA as Ralstonia solanacearum (phylotype II).

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.

Development of antibody to virulence factor flagellin and its evaluation in screening Ralstonia pseudosolanacearum.

The bacterial wilt disease caused by Ralstonia pseudosolanacearum presents a notable economic risk to a variety of crucial crops worldwide. During preliminary isolation of this phytopathogen, several colonies of other saprophytic bacteria may be mistaken with it. So, the present study aims to address this issue by proposing the application of immunogenic proteins, particularly flagellin (FliC), to enable a rapid and early identification of bacterial wilt. In this study, a novel approach is unveiled for the early detection of R. pseudosolanacearum. The study exploits the immunogenic attributes of flagellin (FliC), by generating polyclonal antibodies against recombinant FliC within model organisms-rabbits and mice. The efficacy of these antibodies is meticulously assessed throughdiscerning techniques, including DAS-ELISA and Western blot analyses, which elucidate their remarkable specificity in identifying various R. pseudosolanacearum strains. Furthermore, the introduction of antibody-coated latex agglutinating reagents offers an additional layer of confirmation, substantiating the feasibility of establishing a laboratory-based toolkit for swift screening and unambiguous identification of the bacterial wilt pathogen. This study presents a significant stride toward enhancing early diagnostic capabilities, potentially revolutionizing agricultural practices by safeguarding crop yield and quality through proactive pathogen detection and mitigation strategies.

Commodity risk assessment of Petunia spp. and Calibrachoa spp. unrooted cuttings from Guatemala.

The European Commission requested the EFSA Panel on Plant Health to evaluate the probability of entry of pests (likelihood of pest freedom at entry), including both, regulated and non-regulated pests, associated with unrooted cuttings of the genera Petunia and Calibrachoa produced under physical isolation in Guatemala. The relevance of any pest for this opinion was based on evidence following defined criteria, based on the methodology used for high-risk plants adapted for the specificity of this assessment. Nineteen EU regulated pests (Bemisia tabaci, pepper golden mosaic virus, pepper huasteco yellow vein virus, tomato severe leaf curl virus, tomato yellow leaf curl virus, tomato spotted wilt virus, Liriomyza huidobrensis, Liriomyza sativae, Liriomyza trifolii, Bactericera cockerelli, Eotetranichus lewisi, Epitrix subcrinita, Epitrix cucumeris, Helicoverpa zea, Chloridea virescens, Spodoptera ornithogalli, Ralstonia solanacearum, Ralstonia pseudosolanacearum, Xanthomonas vesicatoria) and one EU non-regulated (Phenacoccus solenopsis) pest fulfilled all relevant criteria and were selected for further evaluation. For these pests, the risk mitigation measures proposed in the technical dossier from Guatemala were evaluated taking into account the possible limiting factors, and an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The limited and partially conflicting information provided in the dossier contributes to the wide estimates of pest freedom. The estimated degree of pest freedom varies among the pests evaluated, with Ralstonia spp. (R. solanacearum and R. pseudosolanacearum) being the pest most frequently expected on the imported cuttings. The expert knowledge elicitation indicated, with 95% certainty, that between 9916 and 10,000 bags containing unrooted cuttings per 10,000 would be free of Ralstonia spp.

Exploring the competitive potential of Ralstonia pseudosolanacearum and Ralstonia solanacearum: Insights from a comparative adaptability study

AbstractBacterial wilt, caused by Ralstonia pseudosolanacearum (Rpsol) and R. solanacearum (Rsol), poses a significant challenge to solanaceous plant cultivation worldwide, particularly in tropical and subtropical regions. Even though Brazil is recognized as one of the centres of origin and diversity of Rsol, in certain regions of this large country there is an emerging prevalence of Rpsol in production fields. Therefore, this study aimed to comprehensively investigate the adaptive traits of Rpsol and Rsol using a polyphasic approach. A diverse collection of isolates from both species was assessed for their physiological, biochemical, ecological and pathogenic traits. Rsol isolates demonstrated greater adaptability to a broader range of temperature, salinity and pH. They also exhibited enhanced abilities in biofilm formation and bacteriocin production. Conversely, Rpsol isolates exhibited a broader utilization of carbon sources and displayed a wider spectrum of resistance to inhibitory substances. Moreover, they demonstrated higher infectivity towards different solanaceous hosts, showing a faster invasion and colonization process in the roots and stems of tomato plants compared to Rsol isolates. Based on our findings, we concluded that Rsol exhibited greater physiological and ecological adaptability, while Rpsol showed greater pathogenic and biochemical adaptability. These results suggest that the coexistence of both species is maintained through a balance of distinct traits within each species.

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.

Virulence of Novel Ralstonia pseudosolanacearum (Phylotype I) Strains from Rose, Blueberry, and Mandevilla on Seed Potato.

Potato (Solanum tuberosum L.) ranks fourth among the most important staple food in the world. Ralstonia solanacearum (phylotype [phy] IIB, sequevar [seq] 1 and 2), also known as R3B2, the causal agent of brown rot disease on potato, is extremely damaging, causing great economical losses to potato in temperate regions. It is thought that members of Ralstonia pseudosolanacearum (phy I) are not pathogenic at low temperatures and are usually found in warmer climates. R. pseudosolanacearum strain PD 7123 (seq 33) isolated from roses in the Netherlands, strain P824 (seq 13) isolated from blueberry, and strain P781 (seq 14) from mandevilla in Florida are phylogenetically closely related and could share the same host. The virulence and ability of these novel strains to multiply latently in potato in temperate regions is unknown. The objective of this work was to assess the virulence and presence of latent infections of the mentioned R. pseudosolanacearum strains on three commercial seed potato cultivars under warmer (28°C) and temperate (20°C) temperatures. At 28°C, all three R. pseudosolanacearum strains caused severe symptoms on all potato cultivars. Overall disease severity on potato was lower at 20°C than 28°C, but major differences in virulence of the three strains were observed at 42 days postinoculation (dpi) among potato cultivars. All asymptomatic potato plants and most of their daughter tubers had latent infections at 20°C. Altogether, these results show that the phy I strains from rose, blueberry, and mandevilla may pose a threat to potato production in temperate climates and the worldwide movement of seed potatoes.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

First specific detection and validation of tomato wilt caused by Fusarium brachygibbosum using a PCR assay.

Tomato wilt is a widespread soilborne disease of tomato that has caused significant yield losses in many tomato growing regions of the world. Previously, it was reported that tomato wilt can be caused by many pathogens, such as Fusarium oxysporum, Ralstonia solanacearum, Ralstonia pseudosolanacearum, Fusarium acuminatum, and Plectosphaerella cucumerina. In addition, we have already reported that Fusarium brachygibbosum caused symptomatic disease of tomato wilt for the first time in China. The symptoms of tomato wilt caused by these pathogens are similar, making it difficult to distinguish them in the field. However, F. brachygibbosum specific identification method has not been reported. Therefore, it is of great importance to develop a rapid and reliable diagnostic method for Fusarium brachygibbosum to establish a more effective plan to control the disease. In this study, we designed F. brachygibbosum-specific forward primers and reverse primers with a fragment size of 283bp located in the gene encoding carbamoyl phosphate synthase arginine-specific large chain by whole genome sequence comparison analysis of the genomes of eight Fusarium spp.. We then tested different dNTP, Mg2+ concentrations, and annealing temperatures to determine the optimal parameters for the PCR system. We evaluated the specificity, sensitivity and stability of the PCR system based on the optimized reaction system and conditions. The PCR system can specifically identify the target pathogens from different fungal pathogens, and the lower detection limit of the target pathogens is at concentrations of 10 pg/uL. In addition, we can accurately identify F. brachygibbosum in tomato samples using the optimized PCR method. These results prove that the PCR method developed in this study can accurately identify and diagnose F. brachygibbosum.

The Ralstonia pseudosolanacearum Type III Effector RipL Delays Flowering and Promotes Susceptibility to Pseudomonas syringae in Arabidopsis thaliana.

The plant defense responses to microbial infection are tightly regulated and integrated with the developmental program for optimal resources allocation. Notably, the defense- associated hormone salicylic acid (SA) acts as a promoter of flowering while several plant pathogens actively target the flowering signaling pathway to promote their virulence or dissemination. Ralstonia pseudosolanacearum inject tens of effectors in the host cells that collectively promote bacterial proliferation in plant tissues. Here, we characterized the function of the broadly conserved R. pseudosolanacearum effector RipL, through heterologous expression in Arabidopsis thaliana . RipL-expressing transgenic lines presented a delayed flowering, which correlated with a low expression of flowering regulator genes. Delayed flowering was also observed in Nicotiana benthamiana plants transiently expressing RipL. In parallel, RipL promoted plant susceptibility to virulent strains of Pseudomonas syringae in the effector-expressing lines or when delivered by the type III secretion system. Unexpectedly, SA accumulation and SA-dependent immune signaling were not significantly affected by RipL expression. Rather, the RNA-seq analysis of infected RipL-expressing lines revealed that the overall amplitude of the transcriptional response was dampened, suggesting that RipL could promote plant susceptibility in an SA-independent manner. Further elucidation of the molecular mechanisms underpinning RipL effect on flowering and immunity may reveal novel effector functions in host cells.

A Mutation of a Putative NDP-Sugar Epimerase Gene in Ralstonia pseudosolanacearum Attenuates Exopolysaccharide Production and Bacterial Virulence in Tomato Plant.

Ralstonia solanacearum species complex (RSSC) is a soil borne plant pathogen causing bacterial wilt on various important crops, including Solanaceae plants. The bacterial pathogens within the RSSC produce exopolysaccharide (EPS), a highly complicated nitrogen-containing heteropolymeric polysaccharide, as a major virulence factor. However, the biosynthetic pathway of the EPS in the RSSC has not been fully characterized. To identify genes in EPS production beyond the EPS biosynthetic gene operon, we selected the EPS-defective mutants of R. pseudosolanacearum strain SL341 from Tn5-inserted mutant pool. Among several EPS-defective mutants, we identified a mutant, SL341P4, with a Tn5-insertion in a gene encoding a putative NDP-sugar epimerase, a putative membrane protein with sugar-modifying moiety, in a reverse orientation to EPS biosynthesis gene cluster. This protein showed similar to other NDP-sugar epimerases involved in EPS biosynthesis in many phytopathogens. Mutation of the NDP-sugar epimerase gene reduced EPS production and biofilm formation in R. pseudosolanacearum. Additionally, the SL341P4 mutant exhibited reduced disease severity and incidence of bacterial wilt in tomato plants compared to the wild-type SL341 without alteration of bacterial multiplication. These results indicate that the NDP-sugar epimerase gene is required for EPS production and bacterial virulence in R. pseudosolanacearum.

Prediction of gene clusters associated with host specificity of Ralstonia pseudosolanacearum race 4 based on comparative genomic analysis

Prediction of gene clusters associated with host specificity of Ralstonia pseudosolanacearum race 4 based on comparative genomic analysis

First Report of Bacterial Wilt of Eggplant (Solanum melongena) Caused by Ralstonia pseudosolanacearum in Mexico.

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is a major disease of solanaceous crops worldwide. In May 2022, symptoms of wilting, yellowing, and reduced growth were observed on eggplant (Solanum melongena) cv. Barcelona in a commercial greenhouse located in Culiacán, Sinaloa, Mexico. The disease incidence was recorded up to 30%. Sections of stems from diseased plants showed discoloration of the vascular tissue and the pith. Colonies with typical RSSC morphology were isolated from five eggplant stems on Petri plates containing casamino acid-peptone-glucose (CPG) medium supplemented with 1% 2,3,5-triphenyltetrazolium chloride (TZC), and incubated at 25°C for 48-h (Schaad et al. 2001; Garcia et al. 2019). On CPG medium + TZC, white and irregular colonies with pinkish centers were observed. On King's B medium, mucoid and white colonies were produced. The strains were Gram-negative in the KOH test and were nonfluorescent on King's B medium. Strains were positive using commercial Rs ImmunoStrip® (Agdia, USA). For molecular identification, DNA was extracted, and the partial endoglucanase gene (egl) was amplified by PCR and sequenced using the primer pair Endo-F/Endo-R (Fegan and Prior 2005). BLASTn searches showed 100% identity with available sequences of R. pseudosolanacearum from Musa sp. in Colombia (MW016967) and from Eucalyptus pellita in Indonesia (MW748363, MW748376, MW748377, MW748379, MW748380, MW748382). To confirm the bacterial identity, DNA was amplified with the primers 759/760 (Opina et al. 1997) and Nmult21:1F/Nmult22:RR (Fegan and Prior 2005) to generate 280 and 144-bp amplicons for RSSC and phylotype I (= R. pseudosolanacearum), respectively. A phylogenetic analysis was performed using the Maximum Likelihood method and the strain was distinguished as R. pseudosolanacearum sequevar 14. The strain (CCLF369) is currently preserved in the Culture Collection of the Research Center for Food and Development (Culiacán, Sinaloa, Mexico) and the sequence was deposited in GenBank (accession number OQ559102). Pathogenicity tests were performed by injection of 20-μl of a bacterial suspension (108 CFU/ml) at the base of the stem of five eggplants cv. Barcelona. Five plants inoculated with sterile distilled water were used as control. Plants were kept in a greenhouse at 28/37°C (night/day) for 12 days. All inoculated plants exhibited wilting, chlorosis, and necrosis of leaves between 8 and 11 days after inoculation, whereas control plants remained asymptomatic. The bacterial strain was only isolated from symptomatic plants and confirmed to be R. pseudosolanacearum using the molecular techniques mentioned above, fulfilling Koch´s postulates. Ralstonia pseudosolanacearum has been previously reported to cause bacterial wilt of tomato in Sinaloa, Mexico (García-Estrada et al. 2023); however, to our knowledge, this is the first report of R. pseudosolanacearum infecting eggplant in Mexico. Further studies on epidemiology and management strategies for this disease are required on vegetable crops in Mexico.

Presence of Ralstonia pseudosolanacearum (Phylotype I) in Aquatic Environments in the Netherlands.

Ralstonia pseudosolanacearum, a European Union quarantine organism, was until recently absent in the aquatic environments and outdoor cultivation systems of the region. This bacterium was only sporadically reported in restricted greenhouse cultivation systems in some EU countries. In this paper, we report the first findings of R. pseudosolanacearum (phylotype I) in surface water in two distinct geographic locations in the Netherlands in 2020. In 2021, the population of R. pseudosolanacearum in surface water ranged from 104 to 106 CFU/liter. An inoculum reservoir for R. pseudosolanacearum in these aquatic environments was the wild bittersweet plant where population densities ranged from 105 to 107 CFU/ml concentrated bittersweet extract. The virulence of the R. pseudosolanacearum isolates from surface water and bittersweet was confirmed by a pathogenicity test on Solanum lycopersicum cv. Moneymaker plants, resulting in wilting and necrosis of the plants. Sequence analysis of the egl locus of R. pseudosolanacearum isolates from surface water and bittersweet revealed that these isolates are closely related to R. pseudosolanacearum (phylotype I) isolates found previously in the Netherlands on rose. R. pseudosolanacearum (phylotype I) has a very broad host plant range, including potato, many ornamentals, and other economically important crops. This highlights the risk for various host plants grown in the vicinity of the geographic locations where R. pseudosolanacearum has been found and shows the importance of unraveling the epidemiological parameters of the survival, establishment, and spread of R. pseudosolanacearum in temperate climates.

An NLR Integrated Domain toolkit to identify plant pathogen effector targets.

Plant immune receptors, known as Nod-like Receptors (NLRs), possess unique integrated decoy domains that enable plants to attract pathogen effectors and initiate a specific immune response. The aim of this study was to create a library of these integrated domains (IDs) and screen them with pathogen effectors to identify new targets for effector virulence and NLR-effector interactions. This works compiles IDs found in NLRs from seven different plant species and produced a library of 78 plasmid clones containing a total of 104 IDs, representing 43 distinct Interpro domains. They conducted a yeast-two-hybrid assay, followed by an in planta interaction test, using 32 conserved effectors from Ralstonia pseudosolanacearum type III. Through these screenings, they discovered three interactions involving different IDs (kinase, DUF3542, WRKY) interacting with two unrelated type III effectors (RipAE and PopP2). Of particular interest was the interaction between PopP2 and ID#85, an atypical WRKY domain integrated into a soybean NLR gene (GmNLR-ID#85). Using a FRET-FLIM technique to detect protein-protein interactions in living plant cells, we demonstrated that PopP2 physically associates with ID#85 in the nucleus. However, unlike the known WRKY-containing Arabidopsis RRS1-R NLR receptor, GmNLR-ID#85 could not be acetylated by PopP2 and failed to activate RPS4-dependent immunity when introduced into the RRS1-R immune receptor. The generated library of 78 plasmid clones, encompassing these screenable IDs, is publicly available through Addgene. This resource is expected to be valuable for the scientific community in discovering new targets for effectors and potentially engineering novel plant immune receptors.

The transcription regulator ChpA affects the global transcriptome including quorum sensing-dependent genes in Ralstonia pseudosolanacearum strain OE1-1.

The gram-negative plant-pathogenic β-proteobacterium Ralstonia pseudosolanacearum strain OE1-1 produces methyl 3-hydroxymyristate as a quorum sensing (QS) signal through methyltransferase PhcB and senses the chemical via the sensor histidine kinase PhcS. This leads to activation of the LysR family transcription regulator PhcA, which regulates the genes (QS-dependent genes) responsible for QS-dependent phenotypes, including virulence. The transcription regulator ChpA, which possesses a response regulator receiver domain and also a hybrid sensor histidine kinase/response regulator phosphore-acceptor domain but lacks a DNA-binding domain, is reportedly involved in QS-dependent biofilm formation and virulence of R. pseudosolanacearum strain GMI1000. To explore the function of ChpA in QS of OE1-1, we generated a chpA-deletion mutant (ΔchpA) and revealed that the chpA deletion leads to significantly altered QS-dependent phenotypes. Furthermore, ΔchpA exhibited a loss in its infectivity in xylem vessels of tomato plant roots, losing virulence on tomato plants, similar to the phcA-deletion mutant (ΔphcA). Transcriptome analysis showed that the transcript levels of phcB, phcQ, phcR, and phcA in ΔchpA were comparable to those in OE1-1. However, the transcript levels of 89.9% and 88.9% of positively and negatively QS-dependent genes, respectively, were significantly altered in ΔchpA compared with OE1-1. Furthermore, the transcript levels of these genes in ΔchpA were positively correlated with those in ΔphcA. Together, our results suggest that ChpA is involved in the regulation of these QS-dependent genes, thereby contributing to the behaviour in host plant roots and virulence of OE1-1.

Comprehensive Control System for Ginger Bacterial Wilt Disease Based on Anaerobic Soil Disinfestation

Bacterial wilt is a soil-borne disease that causes severe damage in ginger-growing regions of Japan (eight prefectures in the Shikoku, Kyushu, and Honshu regions). Because the pathogen Ralstonia pseudosolanacearum usually lives in deeper soil and infects host plants via the roots, it is not easy to eliminate even with chemical pesticides (such as soil fumigants). In our previous study, we found that anaerobic soil disinfestation with diluted ethanol (Et-ASD) effectively eliminated this pathogen. We conducted field experiments to confirm the effectiveness of Et-ASD in a ginger monoculture system. Eight trials were conducted in seven different ginger fields from spring to autumn. Excluding one trial in early spring, seven trials in summer successfully eliminated the pathogen from the field soil (below the detection limit by the developed sensitive bio-PCR method), and no disease recurrence was observed after ASD treatment. In addition, other useful methods for controlling the disease were explored, including proper field management after the disease outbreak and the disinfection of seed rhizomes. Based on these results, a comprehensive control system for bacterial wilt disease in ginger was developed.

First Report of Ralstonia pseudosolanacearum Causing Wilt Disease in Tomato (Solanum lycopersicum L.) Plants from Mexico.

Mexico produces more than four million tons of tomato fruits and ranks tenth worldwide. In February 2022, tomato plants in a greenhouse in Culiacan, Sinaloa State, were affected by wilt diseases with an incidence of 20% and irreversible wilt and death of the infected plants (severity up 70%). When cut stems from affected plants, a reddish to brown discoloration of the vascular system was observed and these were disinfected with 1% NaClO for 5 min and then placed in a humid chamber. Characteristic milky-white exudate was obtained. From that exudate, irregular, mucoid, and white colonies with pink centres were obtained on casamino peptone glucose (CPG) plates supplemented with 1% 2,3,5-triphenyl 15 tetrazolium chloride (TZC); these characteristics are typical of the Ralstonia solanacearum species complex (RSSC) (Garcia et al., 2019). Identification of the pathogen was done by PCR using specific primer pairs reported by Paudel et al. (2022), RssC-wF3 (5'-TATATATCCTCGACTTTTCCATGAAGCTGTG-3') - RssCwR3 (5'-CTATATATATACCCCACTTGTTGAGGAACTG-3') and Rpseu-wF5 (5'-TTTTATTTTTTTGGTGTCCGGGCCAAGATAG-3') - Rpseu-wR5 (5'- TTATATTACTCGAACGTGCTGCAAAACCACT-3'), which amplified fragments of 162 and 251 bp for RSSC and Ralstonia pseudosolanacearum, respectively. Additionally, 759 (5'-GTCGCCGTCAACTCACTTTCC-3') - 760 (5'-GTCGCCGTCAGCAATGCGGAATCG-3') (Opina, et al., 1997) and Nmult21:1F (5'-CGTTGATGAGGCGCGCAATTT-3') - Nmult22:RR (5'- TCGCTTGACCCTATAACGAGTA-3') (Fegan and Prior, 2005) were used to generate 282 and 144 bp amplicons for RSSC and phylotype I, respectively. Subsequen to making the specific detection, the representative strain ClnMx was used to generate a sequence for the endoglucanase (egl) gene for separation into sequevars by using the primers Endo-F (5'- ATGCATGCCGCTGGTCGCCGC-3') and Endo-R (5'-GCGTTGCCCGGCACGAACACC-3'), which amplified a fragment of 750 bp (Fegan et al., 1998). The egl sequence (GenBank Access ON542479) showed 100% identity with the well-defined R. pseudosolanacearum sequevar 14, which was isolated from tomato plants from Senegal (UW763, I-14 GenBank Access CP051174) (Steidl et al., 2021), as well as, the strain MAFF 301070 (GenBank Access AB508612) from Japanese tomato. For pathogenicity tests, four 1-month-old tomato plants were infected using an insulin syringe that contained a pure bacterial suspension with approximately 2x108 CFU/mL. For each plant, 20 µL was infiltrated into the axil of the third upper leaf, and for untreated controls, tomato plants were infiltrated with sterile water. All plants were kept at 28°C under greenhouse conditions. Symptoms resembling those observed in the field were observed in inoculated plants six days after inoculation, and the plant pathogen was recovered on TZC medium. To confirm the bacteria identification a PCR using the specific primer pairs mentioned early was carried out. In contrast, water-treated control plants remained healthy. Koch's postulates were carried out twice with similar results. Ralstonia solanacearum species complex (RSSC) causes severe economic losses in many countries of the world because of their capability to infect a wide range of host plants, including potato, tomato, eggplant, tobacco, and, banana, among others. Ralstonia pseudosolanacearum has been reported to cause tomato wilt disease mainly on the Afro-Eurasian continent in areas such as Senegal, Cambodia, and Japan (Klass et al., 2019). To our knowledge, this is the first report of R. pseudosolanacearum causing bacterial wilt diseases in tomato plants from Mexico and because, the control of this bacteria is a challenge by the long survival time in soil, water, and infected plant tissues, the identification of this important pathogen could provide relevant information for developing management strategies.