The phc Quorum-Sensing System in Ralstonia solanacearum Species Complex.

Ralstonia solanacearum species complex (RSSC) strains are devastating plant pathogens distributed worldwide. The primary cell density-dependent gene expression system in RSSC strains is phc quorum sensing (QS). It regulates the expression of about 30% of all genes, including those related to cellular activity, primary and secondary metabolism, pathogenicity, and more. The phc regulatory elements encoded by the phcBSRQ operon and phcA gene play vital roles. RSSC strains use methyl 3-hydroxymyristate (3-OH MAME) or methyl 3-hydroxypalmitate (3-OH PAME) as the QS signal. Each type of RSSC strain has specificity in generating and receiving its QS signal, but their signaling pathways might not differ significantly. In this review, I describe the genetic and biochemical factors involved in QS signal input and the regulatory network and summarize control of the phc QS system, new cell-cell communications, and QS-dependent interactions with soil fungi.

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.

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.

Pathogenic and Comparative Genomic Analysis of Ralstonia pseudosolanacearum Isolated from Casuarina.

Casuarina equisetifolia is crucial in protecting coastal regions of China against typhoon attacks, but has faced a substantial challenge due to wilt disease caused by pathogens of the Ralstonia solanacearum species complex (RSSC). Although the initial outbreak of Casuarina wilt in 1970s was effectively controlled by disease-resistant C. equisetifolia varieties, the disease has recently re-emerged in coastal regions of Guangdong. In this study, we report the isolation, characterization, and comparative genomic analysis of 11 RSSC strains from diseased C. equisetifolia at various locations along the coast of Guangdong. Phylogenomic analysis showed that the strains were closely related and clustered with phylotype I strains previously isolated from peanuts. Single-gene based analysis further suggested these strains could be derived from strains present in Guangdong since the 1980s, indicating a historical context to their current pathogenicity. Casuarina-isolated strains exhibited notably higher virulence against C. equisetifolia and peanuts than representative RSSC strains GMI1000 and EP1, suggesting host-specific adaptations which possibly contributed to the recent outbreak. Comparative genomic analysis among RSSC strains revealed a largely conserved genome structure and high levels of conservation in gene clusters encoding extracellular polysaccharides biosynthesis, secretion systems, and quorum sensing regulatory systems. However, we also found a number of unique genes in the Casuarina-isolated strains that were absent in GMI1000 and EP1, and vice versa, pointing to potential genetic factors underpinning their differential virulence. These unique genes offer promising targets for future functional studies. Overall, our findings provide crucial insights into the RSSC pathogens causing Casuarina wilt in Guangdong, guiding future efforts in disease control and prevention.

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.

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.

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 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.

Genome-wide identification of type III effectors and other virulence factors in Ralstonia pseudosolanacearum causing bacterial wilt in ginger (Zingiber officinale).

Ralstonia pseudosolanacearum causes bacterial wilt in ginger, reducing ginger production worldwide. We sequenced the whole genome of a highly virulent phylotype I, race 4, biovar 3 Ralstonia pseudosolanacearum strain GRsMep isolated from a severely infected ginger field in India. R. pseudosolanacearum GRsMep genome is organised into two replicons: chromosome and megaplasmid with a total genome size of 5,810,605 bp. This strain encodes approximately 72 effectors which include a combination of core effectors as well as highly variable, diverse repertoire of type III effectors. Comparative genome analysis with GMI1000 identified conservation in the genes involved in the general virulence mechanism. Our analysis identified type III effectors, RipBJ and RipBO as present in GRsMep but absent in the reported genomes of other strains infecting Zingiberaceae family. GRsMep contains 126 unique genes when compared to the pangenome of the Ralstonia strains that infect the Zingiberaceae family. The whole-genome data of R. pseudosolanacearum strain will serve as a resource for exploring the evolutionary processes that structure and regulate the virulence determinants of the strain. Pathogenicity testing of the transposon insertional mutant library of GRsMep through virulence assay on ginger plants identified a few candidate virulence determinants specific to bacterial wilt in ginger.

Biocontrol of bacterial wilt in tomato with a cocktail of lytic bacteriophages.

Bacteriophages (phages) have been proposed as promising alternative pesticides against various bacterial diseases of crops. However, the efficacy of phages in managing plant bacterial diseases is variable and poorly understood in natural settings. In this study, two lytic phages, RpT1 and RpY2, were investigated for their biocontrol potential against bacterial wilt by Ralstonia pseudosolanacearum invasion in tomato plants. The two phages possess similar morphology and genome organization to those of the Autographiviridae family with a broad host range. Treatment with the two phages (alone or in combination) resulted in a significant reduction in bacterial wilt incidence. Three days post-treatment with phages, which was performed after R. pseudosolanacearum inoculation with a specified density of 108 PFU (plaque forming units)/g of soil, led to the most effective biocontrol activity compared to other treatments and a lower density of phage. A phage cocktail containing both RpT1 and RpY2 suppressed disease symptoms in agricultural soils, mimicking their ability to control diseases in natural settings. Furthermore, supplementation with specific adjuvants enhanced the biocontrol potential of both phages. The persistence of the two phages under various environmental conditions indicates their stable activity in soils. Consequently, the consistent biocontrol activity of these phages provides insights into the proper application, timing, and density of phages for effective phage therapy in bacterial wilt control in tomato. KEY POINTS: • Biocontrol potential of phages in natural settings individually and as a cocktail. • Apparent long-term persistence of phages in natural soils, various temperatures, and pH. • An effective approach for developing phages for biocontrol.

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.

Elucidation of antibacterial effect of calcium chloride against Ralstonia pseudosolanacearum race 4 biovar 3 infecting ginger (Zingiber officinale Rosc.).

Bacterial wilt incited by Ralstonia pseudosolanacearum (Rps) race 4 biovar 3 is a serious threat to ginger (Zingiber officinale Rosc.) cultivation throughout the ginger growing tracts and warrants effective remedial measures since most of the strategies failed at field level implementation. After a series of experiments, calcium chloride was found to be effective against Rps both in vitro and in planta and its prophylactic effect has been successfully demonstrated under field conditions. CaCl2 at a concentration of > 2% significantly inhibited Rps under in vitro conditions. Calcium is an important nutritional element imparts a major role in plant disease resistance, and numerous studies have demonstrated the mitigating effect of calcium for disease management. CaCl2 being inhibitory to Rps, the mechanism of inhibition by CaCl2 against Rps was elucidated by a series of in vitro assays including swarming motility and biofilm formation. Direct inhibition was also studied using Scanning Electron Microscopy (SEM). The minimum bactericidal concentration and minimum inhibitory concentration were found to be around 3% while the EC 90 value was found to be 2.25%. The SEM analysis revealed the destruction of cell structure by making perforations on the cell surface. CaCl2 at the targeted concentrations inhibited biofilm formation as well as swarming motility of Rps. These findings suggest that CaCl2 exhibits strong antibacterial activity against Rps and has the potential to be used as an effective bactericide for Rps in managing bacterial wilt in ginger.

Chemotactic disruption as a method to control bacterial wilt caused by Ralstonia pseudosolanacearum.

We have demonstrated that chemotaxis to l-malate facilitated motility of Ralstonia pseudosolanacearum MAFF 106611, a causative agent of bacterial wilt, to plant roots. Here, we evaluated the assumption that the disruption of chemotaxis to l-malate leads to inhibition of plant infection by R. pseudosolanacearum MAFF 106611. Chemotactic assays revealed that chemotaxis to l-malate was completely or partially inhibited in the presence of l-, d-, and dl-malate, respectively. Moreover, l-malate served as a carbon and energy source for R. pseudosolanacearum MAFF 106611, while d-malate inhibited the growth of this bacterium. In the sand-soak inoculation virulence assay for tomato plants, the addition of l-, d-, and dl-malate to sand suppressed the plant infection. We concluded that supplementation of l- and dl-malate suppresses tomato plant infection with R. pseudosolanacearum MAFF 106611 by disrupting its chemotaxis to l-malate, while d-malate suppresses it by both the disruption of l-malate chemotaxis and inhibition of growth.

Enhanced resistance to fungal and bacterial diseases in tomato and Arabidopsis expressing BSR2 from rice.

KEY MESSAGE: The overexpression of rice BSR2 would offer a simple and effective strategy to protect plants from multiple devastating diseases in tomato and Arabidopsis. Many devastating plant diseases are caused by pathogens possessing a wide host range. Fungal Botrytis cinerea and Rhizoctonia solani, as well as bacterial Pseudomonas syringae and Ralstonia pseudosolanacearum are four such pathogens that infect hundreds of plant species, including agronomically important crops, and cause serious diseases, leading to severe economic losses. However, reports of genes that can confer resistance to broad host-range pathogens via traditional breeding methods are currently limited. We previously reported that Arabidopsis plants overexpressing rice BROAD-SPECTRUM RESISTANCE2 (BSR2/CYP78A15) showed tolerance not only to bacterial P. syringae pv. tomato DC3000 but also to fungal Colletotrichum higginsianum and R. solani. Rice plants overexpressing BSR2 displayed tolerance to two R. solani anastomosis groups. In the present study, first, BSR2-overexpressing (OX) Arabidopsis plants were shown to be additionally tolerant to B. cinerea, R. solani, and R. pseudosolanacearum. Next, tomato 'Micro-Tom' was used as a model to determine whether such tolerance by BSR2 can be introduced into dicot crops to prevent infection from pathogens possessing wide host range. BSR2-OX tomato displayed broad-spectrum disease tolerance to fungal B. cinerea and R. solani, as well as to bacterial P. syringae and R. pseudosolanacearum. Additionally, undesirable traits such as morphological changes were not detected. Thus, BSR2 overexpression can offer a simple and effective strategy to protect crops from multiple destructive diseases.

Different Sequevars of Ralstonia pseudosolanacearum Causing Bacterial Wilt of Bidens pilosa in China.

Bidens pilosa is an invasive weed that threatens the growth of crops and biodiversity in China. In 2017, suspected bacterial wilt of B. pilosa was discovered in Qinzhou and Beihai, Guangxi, China. A variety of weeds are considered as reservoirs harboring bacterial wilt pathogens, but most do not show obvious symptoms in the field. Identifying the classification status of the B. pilosa bacterial wilt pathogen and exploring its geographical origin might be helpful for clarifying the role of weeds in the circulation of the disease. Phylotyping, sequevar analysis, and cross inoculation of pathogens isolated from B. pilosa and nearby peanut (Arachis hypogaea), balsam gourd (Momordica charantia), and eucalyptus (Eucalyptus robusta) plants were carried out. Three isolates of B. pilosa (Bp01, Bp02, and Bp03) were identified as Ralstonia pseudosolanacearum, race 1, biovar 3, and phylotype I, and belonged to sequevars 17 and 44, and an unknown sequevar. The sequevars isolated from B. pilosa were not completely consistent with those of the nearby hosts, and the virulence of these isolates differed when cross inoculated. The Bp03 sequevar was different from peanut isolate sequevars in the same field and was not identical to any previously designated sequevars. The isolates from B. pilosa and other nearby hosts displayed low or no virulence toward their cross hosts (with wilt incidences less than 33.33%). An exception to this was the isolates from B. pilosa, which displayed high virulence toward eucalyptus (with a wilt incidence of 70.00 to 100.00%). This is the first report of different sequevars of R. pseudosolanacearum causing typical bacterial wilt symptoms in B. pilosa in the field.

Involvement of many chemotaxis sensors in negative chemotaxis to ethanol in Ralstonia pseudosolanacearum Ps29.

Ralstonia pseudosolanacearum Ps29 showed repellent responses to alcohols including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1,3-propanediol and prenol. R. pseudosolanacearum Ps29 possesses 22 putative chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). To identify a MCP involved in negative chemotaxis to ethanol, we measured ethanol chemotaxis of a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29. However, all the mutants showed repellent responses to ethanol comparable to that of the wild-type strain. We constructed a stepwise- and multiple-mcp gene deletion mutant collection of R. pseudosolanacearum Ps29. Analysis of the collection found that an 18-mcp-knockout mutant (strain POC18) failed to respond to ethanol. Complementation analysis using POC18 as the host strain found that introduction of mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 restored the ability of POC18 to respond to ethanol. However, unexpectedly, strain POC10II, harbouring unmarked deletions in 10 mcp genes including mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 showed repellent responses to ethanol comparable to that of wild-type Ps29. We hypothesised that multiple mcp mutations in POC18 led to a shortage of MCPs required for formation of functional chemoreceptor arrays. When pPS16 (encoding McpP involved in phosphate chemotaxis) was introduced into POC18, POC18(pPS16) did not respond to phosphate. This result supports the hypothesis. But, genetic analysis revealed that MCPs (Mcp07, Mcp13, Mcp20 and Mcp21) are not essential for ethanol chemotaxis. Thus, we conclude that many and unspecified MCPs are involved in negative chemotaxis to ethanol in R. pseudosolanacearum Ps29.

Development of a tomato xylem-mimicking microfluidic system to study Ralstonia pseudosolanacearum biofilm formation.

The bacterial wilt pathogen Ralstonia pseudosolanacearum (Rps) colonizes plant xylem vessels and blocks the flow of xylem sap by its biofilm (comprising of bacterial cells and extracellular material), resulting in devastating wilt disease across many economically important host plants including tomatoes. The technical challenges of imaging the xylem environment, along with the use of artificial cell culture plates and media in existing in vitro systems, limit the understanding of Rps biofilm formation and its infection dynamics. In this study, we designed and built a microfluidic system that mimicked the physical and chemical conditions of the tomato xylem vessels, and allowed us to dissect Rps responses to different xylem-like conditions. The system, incorporating functional surface coatings of carboxymethyl cellulose-dopamine, provided a bioactive environment that significantly enhanced Rps attachment and biofilm formation in the presence of tomato xylem sap. Using computational approaches, we confirmed that Rps experienced linear increasing drag forces in xylem-mimicking channels at higher flow rates. Consistently, attachment and biofilm assays conducted in our microfluidic system revealed that both seeding time and flow rates were critical for bacterial adhesion to surface and biofilm formation inside the channels. These findings provided insights into the Rps attachment and biofilm formation processes, contributing to a better understanding of plant-pathogen interactions during wilt disease development.

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.

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 through discerning 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.

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

The gram-negative plant-pathogenic ß-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.