Bacterial Wilt Pathogen Ralstonia Solanacearum Research Articles

The tomato P69 subtilase family is involved in resistance to bacterial wilt.

The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases-subtilases-may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post-translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D-deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum.

Complete genome sequence resource of Pantoea anthophila CL1 causing soft rot disease in Clausena lansium (Wampee) in China.

Pantoea anthophila CL1 is a causal agent of soft rot disease in Clausena lansium (Wampee) in China, and has inhibitory activity against the bacterial wilt pathogen Ralstonia solanacearum. Here we report the genome sequencing and analysis of P. anthophila CL1, representing the first complete genome resource of the species. The CL1 genome consists of four circular replicons (one chromosome and three plasmids) with a total size of 4,594,065 bp, and contains 4,109 protein-coding genes and 106 RNA genes. Our bioinformatic analysis of CL1 predicted 228 virulence factors, two Type VI Secretion Systems, and six secondary metabolite biosynthesis gene clusters producing saccharides, siderophores, and terpene. The complete genome sequence of P. anthophila CL1 provides a solid foundation for further investigation of its pathogenesis and antimicrobial activity, and also represents a valuable resource for the comparative genomics of Pantoea.

Cell Density-Regulated Adhesins Contribute to Early Disease Development and Adhesion in Ralstonia solanacearum.

Adhesins (adhesive proteins) help bacteria stick to and colonize diverse surfaces and often contribute to virulence. The genome of the bacterial wilt pathogen Ralstonia solanacearum (Rs) encodes dozens of putative adhesins, some of which are upregulated during plant pathogenesis. Little is known about the role of these proteins in bacterial wilt disease. During tomato colonization, three putative Rs adhesin genes were upregulated in a ΔphcA quorum-sensing mutant that cannot respond to high cell densities: radA (Ralstonia adhesin A), rcpA (Ralstonia collagen-like protein A), and rcpB. Based on this differential gene expression, we hypothesized that adhesins repressed by PhcA contribute to early disease stages when Rs experiences a low cell density. During root colonization, Rs upregulated rcpA and rcpB, but not radA, relative to bacteria in the stem at mid-disease. Root attachment assays and confocal microscopy with ΔrcpA/B and ΔradA revealed that all three adhesins help Rs attach to tomato seedling roots. Biofilm assays on abiotic surfaces found that Rs does not require RadA, RcpA, or RcpB for interbacterial attachment (cohesion), but these proteins are essential for anchoring aggregates to a surface (adhesion). However, Rs did not require the adhesins for later disease stages in planta, including colonization of the root endosphere and stems. Interestingly, all three adhesins were essential for full competitive fitness in planta. Together, these infection stage-specific assays identified three proteins that contribute to adhesion and the critical first host-pathogen interaction in bacterial wilt disease. IMPORTANCE Every microbe must balance its need to attach to surfaces with the biological imperative to move and spread. The high-impact plant-pathogenic bacterium Ralstonia solanacearum can stick to biotic and abiotic substrates, presumably using some of the dozens of putative adhesins encoded in its genome. We confirmed the functions and identified the biological roles of multiple afimbrial adhesins. By assaying the competitive fitness and the success of adhesin mutants in three different plant compartments, we identified the specific disease stages and host tissues where three previously cryptic adhesins contribute to success in plants. Combined with tissue-specific regulatory data, this work indicates that R. solanacearum deploys distinct adhesins that help it succeed at different stages of plant pathogenesis.

Evaluation of Culture Filtrates of Endophytic Microorganisms from Tomato against Ralstonia solanacearum

A novel approach to biological control using endophytes has gained immense potential in disease management. The endophytes constitute a valuable source of secondary metabolites such as alkaloids, terpenoids, sterols and phenolic compounds which play a major role in plant defense mechanisms. Therefore an experiment was carried out to study the effect of secondary metabolites of endophytes from tomatoes against the bacterial wilt pathogen Ralstonia solanacearum both in vitro and in vivo conditions using their culture filtrates. In vitro evaluation of the culture filtrates of the endophytes was carried out by inoculating the pathogen on a five per cent culture filtrate amended Potato Dextrose Agar medium. Complete inhibition of the pathogen was observed in all endophytes indicating the inhibitory effect of their culture filtrates. In order to study the effect of secondary metabolites of endophytes on R. solanacearum under in planta conditions, culture filtrates along with culture suspensions for comparison were used. The tomato seedlings dipped first in culture filtrate/suspension then in bacterial ooze showed lowest wilt incidence and among the two, seedling dip in filtrate was the effective one. The minimum wilt incidence was noticed with seedlings dipped first in culture filtrate of endophyte Trichoderma viride-2 recording 10.92, 15.68 and 19.84 per cent followed by T. harzianum-1 and Bacillus subtilis, which showed 12.5, 16.67 and 22.22 per cent at 7, 10 and 14 days after inoculation respectively. Thus the above findings clearly indicated the inhibitory effect of secondary metabolites of the endophytes on bacterial wilt pathogen.

Scot Marker as a Tool to Determine the Genetic Diversity of Bacterial Wilt Pathogen Ralstonia solanacearum

Scot Marker as a Tool to Determine the Genetic Diversity of Bacterial Wilt Pathogen Ralstonia solanacearum

Antibacterial activity and mechanism of ginger extract against Ralstonia solanacearum.

The current study aimed to determine the chemical compositions of ginger extract (GE) and to assess the antibacterial activities of GE against the ginger bacterial wilt pathogen Ralstonia solanacearum and to screen their mechanisms of action. A total of 393 compounds were identified by using ultra-performance liquid chromatography and tandem-mass spectrometry. The antibacterial test indicated that GE had strong antibacterial activity against R. solanacearum and that the bactericidal effect exhibited a dose-dependent manner. The minimum inhibitory concentration and minimum bactericidal concentration of R. solanacearum were 3.91 and 125 mg/ml, respectively. The cell membrane permeability and integrity of R. solanacearum were destroyed by GE, resulting in cell content leakage, such as electrolytes, nucleic acids, proteins, extracellular adenosine triphosphate and exopoly saccharides. In addition, the activity of cellular succinate dehydrogenase and alkaline phosphatase of R. solanacearum decreased gradually with an increase in the GE concentration. Scanning electron microscopy analysis revealed that GE treatment changed the morphology of the R. solanacearum cells. Further experiments demonstrated that GE delayed or slowed the occurrence of bacterial wilt on ginger. GE has a significant antibacterial effect on R. solanacearum, and the antibacterial effect is concentration dependent. The GE treatments changed the morphology, destroyed membrane permeability and integrity, reduced key enzyme activity and inhibit the synthesis of the virulence factor EPS of R. solanacearum. GE significantly controlled the bacterial wilt of ginger during infection. This research provides insight into the antimicrobial mechanism of GE against R. solanacearum, which will open a new application field for GE.

Induced ligno‐suberin vascular coating and tyramine‐derived hydroxycinnamic acid amides restrict Ralstonia solanacearum colonization in resistant tomato

Tomato varieties resistant to the bacterial wilt pathogen Ralstonia solanacearum have the ability to restrict bacterial movement in the plant. Inducible vascular cell wall reinforcements seem to play a key role in confining R. solanacearum into the xylem vasculature of resistant tomato. However, the type of compounds involved in such vascular physico-chemical barriers remain understudied, while being a key component of resistance. Here we use a combination of histological and live-imaging techniques, together with spectroscopy and gene expression analysis to understand the nature of R. solanacearum-induced formation of vascular coatings in resistant tomato. We describe that resistant tomato specifically responds to infection by assembling a vascular structural barrier formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties, effectively contributing to resistance.

The role of the MAP kinase\u2212kinase protein StMKK1 in potato immunity to different pathogens

Mitogen-activated protein kinase (MAPK) cascades play important roles in plant immunity. Previously, we reported that the potato StMKK1 protein negatively regulates Nicotiana benthamiana resistance to Phytophthora infestans. However, the functions of StMKK1 in potato immunity are unknown. To investigate the roles of StMKK1 in potato resistance to different pathogens, such as the potato late-blight pathogen P. infestans, the bacterial wilt pathogen Ralstonia solanacearum, and the gray-mold fungal pathogen Botrytis cinerea, we generated StMKK1 transgenic lines and investigated the response of potato transformants to destructive oomycete, bacterial, and fungal pathogens. The results showed that overexpression and silencing of StMKK1 do not alter plant growth and development. Interestingly, we found that StMKK1 negatively regulated potato resistance to the hemibiotrophic/biotrophic pathogens P. infestans and R. solanacearum, while it positively regulated potato resistance to the necrotrophic pathogen B. cinerea. Further investigation showed that overexpression of StMKK1 suppressed potato pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and salicylic acid (SA)-related responses, while silencing of StMKK1 enhanced PTI and SA-related immune responses. Taken together, our results showed that StMKK1 plays dual roles in potato defense against different plant pathogens via negative regulation of PTI and SA-related signaling pathways.

Extracellular protease from Bacillus subtilis B315 with antagonistic activity against bacterial wilt pathogen (Ralstonia solanacearum) of chili

Abstract. Prihatiningsih N, Asnani A, Djatmiko HA. 2021. Extracellular protease from Bacillus subtilis B315 with antagonistic activity against bacterial wilt pathogen (Ralstonia solanacearum) of chili. Biodiversitas 22: 1291-1295. Antagonistic bacteria isolated from a healthy potato are potential biocontrol agents against a number of bacterial pathogens. The capability of antagonistic bacteria to produce extracellular protease could be considered as an indicator of their antagonistic capacity. The objective of this research was to study the optimum activity of protease produced by Bacillus subtilis B315 to control Ralstonia solanacearum causing wilt of chili. In this research were optimum incubation time, pH and crude extract protease concentration of B. subtilis B315 examined for maximum production of extracellular protease. The results showed that B. subtilis B315 was capable of producing protease at optimum incubation time of 30 min, pH 9, and crude extract protease concentration 0.50% with an activity of 1.3185 U/mL. Protease-producing capability of B. subtilis B315 was shown by the formation of a clear zone around the colony of B. subtilis B315 grown on skim-milk agar medium. B. subtilis B315 was capable of inhibiting the growth of R. solanacearum in vitro as indicated by 32 mm clear zone. Furthermore, in chili application of B subtilis showed that this bacteria was capable of controlling chili bacterial wilt caused by R. solanacearum with effectiveness of 60.89%. The prospective of B. subtilis B315 could be formulated as biopesticide.

Endophytes from Gnetum gnemon L. can protect seedlings against the infection of phytopathogenic bacterium Ralstonia solanacearum as well as promote plant growth in tomato

Endophytes are beneficial plant microbes which help the plants by producing various plant growth promoting substances and also by acting as biocontrol agents against various plant pathogens. In the present study, evaluation of endophytic bacteria isolated from Gnetum gnemon, an ethnomedicinal plant was carried out for their plant growth promoting (PGP) activity and antagonistic potential against bacterial wilt pathogen Ralstonia solanacearum. Initially a total of 40 endophytic bacteria were isolated which were clustered into 13 groups based on RFLP and BOX-PCR fingerprinting. These 13 representative isolates belonged to different genera of Bacillus (9), Solibacillus (1), Staphylococcus (2) and Caballeronia (1). Among them, six isolates were positive for production of IAA, the value of which ranged from 11.16 to 27.63 μg mL−1. Phosphate solubilisation in the range of 106.4 to –212.7 μg mL−1 was shown by three isolates. Eight isolates producedammonia, the value of which ranged from 1.3 to 6.1 μmol mL−1. All tested isolates were positive for siderophore production. For extracellular enzyme production, 7 isolates were positive for protease, 8 for cellulase and 10 for amylase production. The isolates were also tested for their antagonistic activity against R. solanacearum in vitro and in planta assay using tomato seedlings. Staphylococcus warneri GL1 showed the highest biocontrol efficacy of 77.67 % followed by Bacillus velezensis GL3 i.e. 70.1 %. R. solanacearum antagonistic isolates were analysed for the presence of antimicrobial peptide biosynthesis genes bmyB, srfAA, fenD and ituC. All the antagonistic isolates showed the presence of all four genes, except the isolate Bacillus velezensis GMC2, where the gene for fengycin synthetase (fenD) was absent. Based on in vitro PGP traits, three isolates Bacillus velezensis GL3, Bacillus atrophaeus GMC1 and Bacillus megaterium GS2 were selected, these three endophytic bacteria individually and their consortia were tested for in planta PGP activities in tomato plants. Application of Bacillus velezensis GL3 alone and consortia of three isolates showed significant improvement in growth parameters such as shoot length, fresh weight and dry weight in a pot experiment. Colonization of endosphere of treated tomato seedlings by the endophytic isolate Bacillus velezensis GL3 was confirmed by visualization of colony morphology and BOX-PCR fingerprinting. Our study highlights the potential of endophytes associated with unexplored plants like G. gnemon for development of bioformulation aimed at enhancing plant growth and bacterial wilt disease control.

A Susceptible Scion Reduces Rootstock Tolerance to Ralstonia solanacearum in Grafted Eggplant

The bacterial wilt pathogen (Ralstonia solanacearum) is a highly pathogenic soil-borne bacterium that invades the vascular system of a host plant leading to plant wilting and death. In agricultural systems, tolerant rootstocks are usually used to enhance disease resistance and tolerance in crop plants to soil-borne pathogens. Here, two distinct eggplant cultivars with different tolerances to R. solanacearum infection, the disease-tolerant cultivar ‘S21′ and the disease-susceptible cultivar ‘Rf’, were used to investigate if scion tolerance level can affect tolerance of rootstock upon an infection of the same pathogen. Three scion/rootstock grafted combinations were considered: Rf/S21, S21/S21, and Rf/Rf. Plants that resulted from the combination Rf/S21, composed of the susceptible scion grafts, showed weak tolerance to R. solanacearum infection, and exhibited the poorest growth compared to the tolerant scion grafts (S21/S21). As expected, the combination Rf/Rf showed the lowest level of disease tolerance. Furthermore, a high level of exopolysaccharides (EPSs) and cell wall degrading enzymes (CWDEs) were detected in susceptible scion grafts. These factors are involved in plant growth inhibition due to blocking transport between scion and rootstock and damage of vascular tissues in the plant. A high level of reactive oxygen species (ROS) and active oxygen scavenging enzymes were also detected in susceptible scion grafts. Excess accumulation of these substances harms the dynamic balance in plant vascular systems. These results indicated that the use of a susceptible scion in scion/rootstock eggplant grafts contributed to a reduction in rootstock tolerance to Ralstonia solanacearum.

Biogenic synthesis of iron oxide nanoparticles via Skimmia laureola and their antibacterial efficacy against bacterial wilt pathogen Ralstonia solanacearum

Plant diseases are threat to global food security. The excessive use of agrochemicals is the leading cause of pesticides resistance and toxicity to beneficial life forms. The quest for innocuous and alternate antimicrobial agent is crucial in order to overcome the pathogen resistance and the birth of nanotech offers pledge to combat pathogenic organisms. In this study, a facile benign biogenic approach was adopted for the synthesis of biocompatible iron oxide nanoparticles (Fe2O3−NPs) via Skimmia laureola leaf extract and the synthesized nanoparticles were evaluated for their antibacterial efficacy against bacterial wilt pathogen Ralstonia solanacearum in vitro and in planta. Physico-chemical characterization of the synthesized nanoparticles was performed through UV–Visible spectroscopy, Fourier Transform Infrared Spectroscopy, X-Ray Diffraction, Energy Dispersive X-ray Spectroscopy and Scanning Electron Microscopy. The results revealed polydisperse nanoparticles in the size range of 56 nm to 350 nm. The culture media containing 6 mg/mL of Fe2O3−NPs dramatically inhibited the bacterial growth in vitro. Scanning electron microscopy revealed degenerative characteristics including degraded, shriveled and concentrated cell walls. Diseases severity was effectively reduced with 6% w/v of Fe2O3−NPs treated root zone in planta. Plant shoots, root length and fresh biomass were enhanced with Fe2O3−NPs treatments. The results indicated that the biosynthesized Fe2O3−NPs have the potential to control agriculturally important phytopathogen Ralstonia solanacearum in vitro and in planta.

NtPR1a regulates resistance to Ralstonia solanacearum in Nicotiana tabacum via activating the defense-related genes

Pathogenesis-related proteins (PRs) are associated with the development of systemic acquired resistance (SAR) against further infection enforced by fungi, bacteria and viruses. PR1a is the first PR-1 member that could be purified and characterized. Previous studies have reported its role in plants’ resistance system against oomycete pathogens. However, the role of PR1a in Solanaceae plants against the bacterial wilt pathogen Ralstonia solanacearum remains unclear. To assess roles of NtPR1a in tobacco responding to R. solanacearum, we performed overexpression experiments in Yunyan 87 plants (a susceptible tobacco cultivar). The results illuminated that overexpression of NtPR1a contributed to improving resistance to R. solanacearum in tobacco Yunyan 87. Specifically speaking, NtPR1a gene could be induced by exogenous hormones like salicylic acid (SA) and pathogenic bacteria R. Solanacearum. Moreover, NtPR1a-overexpressing tobacco significantly reduced multiple of R. solanacearum and inhibited the development of disease symptoms compared with wild-type plants. Importantly, overexpression of NtPR1a activated a series of defense-related genes expression, including the hypersensitive response (HR)-associated genes NtHSR201 and NtHIN1, SA-, JA- and ET-associated genes NtPR2, NtCHN50, NtPR1b, NtEFE26, and Ntacc oxidase, and detoxification-associated gene NtGST1. In summary, our results suggested that NtPR1a-enhanced tobacco resistance to R. solanacearum may be mainly dependent on activation of the defense-related genes.

Cooperative Reinforcement of Ionic Liquid and Reactive Solvent on Enzymatic Synthesis of Caffeic Acid Phenethyl Ester as an In Vitro Inhibitor of Plant Pathogenic Bacteria.

It is widely believed that lipases in ionic liquids (ILs) possess higher enzyme activity, stability and selectivity; however, reaction equilibrium is always limited by product inhibition, and the product is difficult to separate from non-volatile ILs using distillation. To solve this problem, using trialkylphosphine oxide (TOPO) as a complexing agent, a novel biphase of reactive solvent and IL was firstly reported for caffeic acid phenethyl ester (CAPE) production from methyl caffeate (MC) and 2-phenylethanol (PE) catalyzed by lipase via transesterification. The effects of the reaction parameters and their action mechanism were investigated, and the inhibition of CAPE against bacterial wilt pathogen Ralstonia solanacearum was firstly measured. The MC conversion of 98.83% ± 0.76% and CAPE yield of 96.29% ± 0.07% were obtained by response surface methodology in the 25 g/L TOPO-cyclohexane/[Bmim][Tf2N] (1:1, v/v); the complex stoichiometry calculation and FTIR spectrum confirmed that the reversible hydrogen-bond complexation between TOPO and caffeates significantly enhances the cooperative effect of two phases on the lipase-catalyzed reaction. The temperature was reduced by 14 °C; the MC concentration increased by 3.33-fold; the ratio of catalyst to donor decreased by 4.5-fold; and Km decreased 1.08-fold. The EC50 of CAPE against R. solanacearum was 0.17–0.75 mg/mL, suggesting that CAPE is a potential in vitro inhibitor of plant pathogenic bacteria.

Root-mediated signal transmission of systemic acquired resistance against above-ground and below-ground pathogens.

Background and Aims Plants modulate defence signalling networks in response to various biotic stresses via inter-organ communications. The root-mediated transmission of systemic acquired resistance (SAR) against soil-borne and air-borne plant pathogens from SAR-induced plants to neighbouring plants subjected to local chemical and pathogen treatments was evaluated. Methods The first two plants out of ten Nicotiana benthamiana seedlings were pre-treated with the SAR-triggering chemical benzothiadiazole (BTH). All ten seedlings were then challenged with two pathogenic bacteria, i.e. the root (bacterial wilt) pathogen Ralstonia solanacearum and the leaf (wildfire) pathogen Pseudomonas syringae pv. tabaci, at 7 d after SAR induction. Key Results Disease severity was noticeably lower in BTH-pre-treated plants than in the control. Surprisingly, two plants located next to BTH-treated plants exhibited reduced disease symptoms indicating that SAR signal transmission occurred through the root system. Determinant(s) secreted from the root system were search for and it was found that salicylic acid (SA) is a major molecule involved in SAR transmission through the root. Analysis of the expression of the defence-related genes N. benthamiana pathogenesis-related gene 1a (NbPR1a) and NbPR2 confirmed that BTH treatment elicited SAR via root-root transmission between plants. Plants with knock-down of the multiple resistance component SGT1 and SA biosynthesis-related gene ICS1 by Tobacco rattle virus-mediated virus-induced gene silencing exhibited a lack of root-mediated SAR transmission. The biological relevance of this finding was validated by challenge with the SAR-inducing avirulent pathogen P. syringae pv. syringae instead of BTH, which produced similar results. Conclusions Our findings demonstrated that SAR is transmissible through the root system from SAR-triggered plants to neighbouring plants.

Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum.

Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease.

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

ABSTRACTPlants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum. SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule.

Effects of volatile organic compounds produced by Bacillus amyloliquefaciens on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum.

The production of volatile organic compounds (VOCs) by microbes is an important characteristic for their selection as biocontrol agents against plant pathogens. In this study, we identified the VOCs produced by the biocontrol strain Bacillus amyloliquefaciens T-5 and evaluated their impact on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum. The results showed that the VOCs of strain T-5 significantly inhibited the growth of R. solanacearum in agar medium and in soil. In addition, VOCs significantly inhibited the motility traits, root colonization, biofilm formation, and production of antioxidant enzymes and exopolysaccharides by R. solanacearum. However, no effect of VOCs on the production of hydrolytic enzymes by R. solanacearum was observed. The strain T-5 produced VOCs, including benzenes, ketones, aldehydes, alkanes, acids, and one furan and naphthalene compound; among those, 13 VOCs showed 1-10% antibacterial activity against R. solanacearum in their produced amounts by T-5; however, the consortium of all VOCs produced on agar medium, in sterilized soil, and in natural soil showed 75, 62, and 85% growth inhibition of R. solanacearum, respectively. The real-time PCR analysis further confirmed the results when the expression of different virulence- and metabolism-related genes in R. solanacearum cells was decreased after exposure to the VOCs of strain T-5. The results of this study clearly revealed the significance of VOCs in the control of plant pathogens. This information would help to better comprehend the microbial interactions mediated by VOCs in nature and to develop safer strategies to control plant disease.

Biochemical and Molecular Variations of Guaiacol Peroxidase and Total Phenols in Bacterial Wilt Pathogenesis of Solanum melongena

Plants respond to bacterial pathogen attack by activating various defense responses, which are associated with the accumulation of several factors like defense-related enzymes and inhibitors which serve to prevent pathogen infection. The present study focused on the role of the defense-related enzyme and gene expression of Guaiacol preroxidase and Total phenol content in imparting activities was analyzed by selecting three different eggplant cultivars against bacterial wilt pathogen Ralstonia solanacearum. The temporal pattern of induction of these enzymes showed (42.72 U) maximum activity at 21 h after the pathogen inoculation (hpi) in resistant cultivars. The expressions of defense genes increased 5.5 folds in resistant eggplant cultivars after pathogen inoculation. The total phenol content increased significantly (P<0.05) in resistant cultivars upon pathogen inoculation compared to susceptible and highly susceptible cultivars. The biochemical and molecular markers provided an insight to understand the first line of defense responses in eggplant cultivars upon inoculation with the pathogen.

Towards the Identification of Type III Effectors Associated with Ralstonia solanacearum Virulence on Tomato and Eggplant.

For the development of pathogen-informed breeding strategies, identifying the microbial genes involved in interactions with the plant is a critical step. To identify type III effector (T3E) repertoires associated with virulence of the bacterial wilt pathogen Ralstonia solanacearum on Solanaceous crops, we used an original association genetics approach combining DNA microarray data and pathogenicity data on resistant eggplant, pepper, and tomato accessions. From this first screen, 25 T3Es were further full-length polymerase chain reaction-amplified within a 35-strain field collection, to assess their distribution and allelic diversity. Six T3E repertoire groups were identified, within which 11 representative strains were chosen to challenge the bacterial wilt-resistant egg plants 'Dingras multiple Purple' and 'AG91-25', and tomato Hawaii 7996. The virulence or avirulence phenotypes could not be explained by specific T3E repertoires, but rather by individual T3E genes. We identified seven highly avirulence-associated genes, among which ripP2, primarily referenced as conferring avirulence to Arabidopsis thaliana. Interestingly, no T3E was associated with avirulence to both egg-plants. Highly virulence-associated genes were also identified: ripA5_2, ripU, and ripV2. This study should be regarded as a first step toward investigating both avirulence and virulence function of the highlighted genes, but also their evolutionary dynamics in natural R. solanacearum populations.