The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection

The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.     

Induction of the Viable but Nonculturable State of Ralstonia solanacearum by Low Temperature in the Soil Microcosm and Its Resuscitation by Catalase

Ralstonia solanacearum is the causal agent of bacterial wilt on a wide variety of plants, and enters a viable but nonculturable (VBNC) state under stress conditions in soil and water. Here, we adopted an artificial soil microcosm (ASM) to investigate the VBNC state of R. solanacearum induced by low temperature. The culturability of R. solanacearum strains SL341 and GMI1000 rapidly decreased at 4°C in modified ASM (mASM), while it was stably maintained at 25°C in mASM. We hypothesized that bacterial cells at 4°C in mASM are viable but nonculturable. Total protein profiles of SL341 cells at 4°C in mASM did not differ from those of SL341 culturable cells at 25°C in mASM. Moreover, the VBNC cells maintained in the mASM retained respiration activity. Catalase treatment effectively restored the culturability of nonculturable cells in mASM, while temperature increase or other treatments used for resuscitation of other bacteria were not effective. The resuscitated R. solanacearum from VBNC state displayed normal level of bacterial virulence on tomato plants compared with its original culturable bacteria. Expression of omp, oxyR, rpoS, dps, and the 16S rRNA gene quantified by RT-qPCR did not differ significantly between the culturable and VBNC states of R. solanacearum. Our results suggested that the VBNC bacterial cells in mASM induced by low temperature exist in a physiologically unique state.     

Silicon-Mediated Tomato Resistance Against Ralstonia solanacearum is Associated with Modification of Soil Microbial Community Structure and Activity

Bacterial wilt caused by Ralstonia solanacearum is a serious soil-borne disease of Solanaceae crops. In this study, the soil microbial effects of silicon-induced tomato resistance against R. solanacearum were investigated through pot experiment. The results showed that exogenous 2.0 mM Si treatment reduced the disease index of bacterial wilt by 19.18 % to 52.7 % compared with non-Si-treated plants. The uptake of Si was significantly increased in the Si-treated tomato plants, where the Si content was higher in the roots than that in the shoots. R. solanacearum inoculation resulted in a significant increase of soil urease activity and reduction of soil sucrase activity, but had no effects on soil acid phosphatase activity. Si supply significantly increased soil urease and soil acid phosphatase activity under pathogen-inoculated conditions. Compared with the non-inoculated treatment, R. solanacearum infection significantly reduced the amount of soil bacteria and actinomycetes by 52.5 % and 16.5 %, respectively, but increased the ratio of soil fungi/soil bacteria by 93.6 %. After R. solanacearum inoculation, Si amendments significantly increased the amount of soil bacteria and actinomycetes and reduced soil fungi/soil bacteria ratio by 53.6 %. The results suggested that Si amendment is an effective approach to control R. solanacearum. Moreover, Si-mediated resistance in tomato against R. solanacearum is associated with the changes of soil microorganism amount and soil enzyme activity.     

Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment,Stem Colonization,and Virulence

Ralstonia solanacearum, an economically important plant pathogen, must attach, grow, and produce virulence factors to colonize plant xylem vessels and cause disease. Little is known about the bacterial metabolism that drives these processes. Nitrate is present in both tomato xylem fluid and agricultural soils, and the bacterium''s gene expression profile suggests that it assimilates nitrate during pathogenesis. A nasA mutant, which lacks the gene encoding the catalytic subunit of R. solanacearum''s sole assimilatory nitrate reductase, did not grow on nitrate as a sole nitrogen source. This nasA mutant exhibited reduced virulence and delayed stem colonization after soil soak inoculation of tomato plants. The nasA virulence defect was more severe following a period of soil survival between hosts. Unexpectedly, once bacteria reached xylem tissue, nitrate assimilation was dispensable for growth, virulence, and competitive fitness. However, nasA-dependent nitrate assimilation was required for normal production of extracellular polysaccharide (EPS), a major virulence factor. Quantitative analyses revealed that EPS production was significantly influenced by nitrate assimilation when nitrate was not required for growth. The plant colonization delay of the nasA mutant was externally complemented by coinoculation with wild-type bacteria but not by coinoculation with an EPS-deficient epsB mutant. The nasA mutant and epsB mutant did not attach to tomato roots as well as wild-type strain UW551. However, adding either wild-type cells or cell-free EPS improved the root attachment of these mutants. These data collectively suggest that nitrate assimilation promotes R. solanacearum virulence by enhancing root attachment, the initial stage of infection, possibly by modulating EPS production.     

Detection of Ralstonia solanacearum in Soil and Weeds from Commercial Tomato Fields Using Immunocapture and the Polymerase Chain Reaction

A detection assay for Ralstonia solanacearum in soil and weeds was developed by combining immunocapture and the polymerase chain reaction (IC‐PCR). Anti‐R. solanacearum polyclonal antibodies were produced in a white female rabbit and Dynal^® super‐paramagnetic beads were coated with purified immunoglobulinG (IgG). Using IC‐PCR, the 718 bp target DNA was amplified at a detection threshold of approximately 10⁴ colony‐forming units (CFU) bacteria per millilitre of suspension. DNA was not amplified in soil suspensions derived from autoclaved and non‐autoclaved soils, which contained R. solanacearum at 1–10⁵ CFU/g soil. However, a positive PCR result was obtained when bacteria in the soil suspensions were first enriched in nutrient broth. IC‐PCR detected R. solanacearum in tomato stems 24 h after inoculation by stem puncture with a suspension containing approximately 10⁵ CFU/ml. IC‐PCR detected the bacterium in 28 of 55 (51%) weeds and 10 of 32 (31%) soil samples. Of the weeds, Physalis minima, Amaranthus spinosus and Euphorbia hirta had the highest incidence of infection. R. solanacearum was not detected in soil taken from fallow fields, but it was discovered in some weed species. Symptomless tomato and pepper plants collected from the fields in which tomato bacterial wilt had previously occurred were found to contain R. solanacearum. These discoveries suggest that weeds and latent hosts may play a role in the survival of R. solanacearum between cropping cycles.     

The Ralstonia solanacearum csp22 peptide,but not flagellin‐derived peptides,is perceived by plants from the Solanaceae family

Ralstonia solanacearum, the causal agent of bacterial wilt disease, is considered one of the most destructive bacterial pathogens due to its lethality, unusually wide host range, persistence and broad geographical distribution. In spite of the extensive research on plant immunity over the last years, the perception of molecular patterns from R. solanacearum that activate immunity in plants is still poorly understood, which hinders the development of strategies to generate resistance against bacterial wilt disease. The perception of a conserved peptide of bacterial flagellin, flg22, is regarded as paradigm of plant perception of invading bacteria; however, no elicitor activity has been detected for R. solanacearum flg22. Recent reports have shown that other epitopes from flagellin are able to elicit immune responses in specific species from the Solanaceae family, yet our results show that these plants do not perceive any epitope from R. solanacearum flagellin. Searching for elicitor peptides from R. solanacearum, we found several protein sequences similar to the consensus of the elicitor peptide csp22, reported to elicit immunity in specific Solanaceae plants. A R. solanacearum csp22 peptide (csp22^Rsol) was indeed able to trigger immune responses in Nicotiana benthamiana and tomato, but not in Arabidopsis thaliana. Additionally, csp22^Rsol treatment conferred increased resistance to R. solanacearum in tomato. Transgenic A. thaliana plants expressing the tomato csp22 receptor (SlCORE) gained the ability to respond to csp22^Rsol and became more resistant to R. solanacearum infection. Our results shed light on the mechanisms for perception of R. solanacearum by plants, paving the way for improving current approaches to generate resistance against R. solanacearum.     

Disruption of Firmicutes and Actinobacteria abundance in tomato rhizosphere causes the incidence of bacterial wilt disease

Enrichment of protective microbiota in the rhizosphere facilitates disease suppression. However, how the disruption of protective rhizobacteria affects disease suppression is largely unknown. Here, we analyzed the rhizosphere microbial community of a healthy and diseased tomato plant grown <30-cm apart in a greenhouse at three different locations in South Korea. The abundance of Gram-positive Actinobacteria and Firmicutes phyla was lower in diseased rhizosphere soil (DRS) than in healthy rhizosphere soil (HRS) without changes in the causative Ralstonia solanacearum population. Artificial disruption of Gram-positive bacteria in HRS using 500-μg/mL vancomycin increased bacterial wilt occurrence in tomato. To identify HRS-specific and plant-protective Gram-positive bacteria species, Brevibacterium frigoritolerans HRS1, Bacillus niacini HRS2, Solibacillus silvestris HRS3, and Bacillus luciferensis HRS4 were selected from among 326 heat-stable culturable bacteria isolates. These four strains did not directly antagonize R. solanacearum but activated plant immunity. A synthetic community comprising these four strains displayed greater immune activation against R. solanacearum and extended plant protection by 4 more days in comparison with each individual strain. Overall, our results demonstrate for the first time that dysbiosis of the protective Gram-positive bacterial community in DRS promotes the incidence of disease.Subject terms: Plant sciences, Microbiome     

Detection of Ralstonia solanacearum from Asymptomatic Tomato Plants,Irrigation Water,and Soil Through Non-selective Enrichment Medium with hrp Gene-Based Bio-PCR

Bacterial wilt of tomato caused by Ralstonia solanacearum (Smith) Yabuuchi et al. (Microbiol Immunol 39:897–904, 1995) is a serious disease, which causes losses up to 60 % depending on environmental conditions, soil property, and cultivars. In present investigation, nucleotide sequences of virulence, hypersensitive response and pathogenicity (hrp) gene were used to design a pair of primer (Hrp_rs 2F: 5′-AGAGGTCGACGCGATACAGT-3′ and Hrp_rs 2R: 5′-CATGAGCAAGGACGAAGTCA-3′) for amplification of bacterial genome. The genomic DNA of 27 isolates of R. solanacearum race 1 biovar 3 & 4 was amplified at 323 bp. The specificity of primer was tested on 13 strains of R. solanacearum with other group of bacteria such as Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, and X. citri subsp. citri. Primer amplified DNA fragment of R. solanacearum at 323 bp. The sensitivity of the primer was 200 cfu/ml and improved further detection level by using non-specific enrichment medium casamino acids-pepton-glucose broth followed by PCR (BIO-PCR). Out of 130 samples of asymptomatic tomato plants, irrigation water, and soil collected from bacterial wilt infested field in different agro-climatic regions of India, R. solanacearum was detected from 86.9, 88.5, and 90.9 per cents samples using BIO-PCR, respectively. The primer was found specific for detecting viable and virulent strains of R. solanacearum and useful for the diagnosis of R. solanacearum in tomato seedlings and monitoring of pathogen in irrigation water and soil.     

Survival Strategy of Erwinia amylovora against Copper: Induction of the Viable-but-Nonculturable State

Copper compounds, widely used to control plant-pathogenic bacteria, have traditionally been employed against fire blight, caused by Erwinia amylovora. However, recent studies have shown that some phytopathogenic bacteria enter into the viable-but-nonculturable (VBNC) state in the presence of copper. To determine whether copper kills E. amylovora or induces the VBNC state, a mineral medium without copper or supplemented with 0.005, 0.01, or 0.05 mM Cu²⁺ was inoculated with 10⁷ CFU/ml of this bacterium and monitored over 9 months. Total and viable cell counts were determined by epifluorescence microscopy using the LIVE/DEAD kit and by flow cytometry with 5-cyano-2,3-ditolyl tetrazolium chloride and SYTO 13. Culturable cells were counted on King's B nonselective solid medium. Changes in the bacterial morphology in the presence of copper were observed by scanning electron microscopy. E. amylovora entered into the VBNC state at all three copper concentrations assayed, much faster when the copper concentration increased. The addition of different agents which complex copper allowed the resuscitation (restoration of culturability) of copper-induced VBNC cells. Finally, copper-induced VBNC cells were virulent only for the first 5 days, while resuscitated cells always regained their pathogenicity on immature fruits over 9 months. These results have shown, for the first time, the induction of the VBNC state in E. amylovora as a survival strategy against copper.     

Composition and activity of rhizosphere microbial communities associated with healthy and diseased greenhouse tomatoes

Aims

The goal of this study was to investigate the structure and functional potential of microbial communities associated with healthy and diseased tomato rhizospheres.

Methods

Composition changes in the bacterial communities inhabiting the rhizospheric soil and roots of tomato plants were detected using 454 pyrosequencing. Microbial functional diversity was investigated with BIOLOG technology.

Results

There were significant shifts in the microbial composition of diseased samples compared with healthy samples, which had the highest bacterial diversity. The predominant phylum in both diseased and healthy samples was Proteobacteria, which accounted for 35.7–97.4 % of species. The class Gammaproteobacteria was more abundant in healthy than in diseased samples, while the Alphaproteobacteria and Betaproteobacteria were more abundant in diseased samples. The proportions of pathogenic Ralstonia solanacearum and Actinobacteria species were also elevated in diseased samples. The proportions of the various bacterial populations showed a similar trend both in rhizosphere soil and plant roots in diseased versus disease-free samples, indicating that pathogen infection altered the composition of bacterial communities in both plant and soil samples. In terms of microbial activity, functional diversity was suppressed in diseased soil samples. Soil enzyme activity, including urease, alkaline phosphatase and catalase activity, also declined.

Conclusions

This is the first report that provides evidence that R. solanacearum infection elicits shifts in the composition and functional potential of microbial communities in a continuous-cropping tomato operation.     

Rhizocompetence and antagonistic activity towards genetically diverse Ralstonia solanacearum strains – an improved strategy for selecting biocontrol agents

Bacterial wilt caused by Ralstonia solanacearum is a serious threat for agricultural production in China. Eight soil bacterial isolates with activity against R. solanacearum TM15 (biovar 3) were tested in this study for their in vitro activity towards ten genetically diverse R. solanacearum isolates from China. The results indicated that each antagonist showed remarkable differences in its ability to in vitro antagonize the ten different R. solanacearum strains. Strain XY21 (based on 16S rRNA gene sequencing affiliated to Serratia) was selected for further studies based on its in vitro antagonistic activity and its excellent rhizocompetence on tomato plants. Under greenhouse conditions XY21 mediated biocontrol of tomato wilt caused by seven different R. solanacearum strains ranged from 19 to 70 %. The establishment of XY21 and its effects on the bacterial community in the tomato rhizosphere were monitored by denaturing gradient gel electrophoresis of 16S rRNA gene fragments PCR-amplified from total community DNA. A positive correlation of the in vitro antagonistic activities of XY21 and the actual biocontrol efficacies towards seven genetically different R. solanacearum strains was found and further confirmed by the efficacy of XY21 in controlling bacterial wilt under field conditions.     

Using the Ralstonia solanacearum Tat Secretome To Identify Bacterial Wilt Virulence Factors

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.     

Effect of temperature,cultivars, injury of root and inoculums load of Ralstonia solanacearum to cause bacterial wilt of tomato

Bacterial wilt, caused by Ralstonia solanacearum , is responsible for severe losses in tomato crops in the world. In the present study, the effect of temperature, cultivars of tomato, injury of root system and inoculums load of R. solanacearum to cause bacterial wilt disease under control conditions was undertaken. Three strains UTT-25, HPT-3 and JHT-5 of R. solanacearum were grown at 5–40?°C in vitro to study, the effect of temperature on the growth of bacteria and maximum growth was found at 30?°C after 72?h in all the strains. Twenty-one days old seedlings of two cultivars of tomato i.e. N-5 (moderately resistant) and Pusa Ruby (highly susceptible) were transplanted into the pots and inoculated with R. solanacearum strain UTT-25 (5 × 10⁸?cfu/ml), mechanically injured and uninjured roots of the plant. The plants were allowed to grow at 20, 25, 30 and 35?°C at National Phytotron Facility, IARI, New Delhi to study the effect of temperature on intensity of bacterial wilt disease. Maximum wilt disease intensity was found 98.73 and 95.9 % in injured roots of Pusa Ruby and N-5 cultivars of tomato at 35?°C on 11th days of inoculation, respectively. However, no wilt disease was observed in both the cultivars at 20?°C up to 60?days. For detection of R. solanacearum from asymptomatic tomato plants, hrpB-based sequence primers (Hrp_rs2F and Hrp_rs2R) amplified at 323?bp was used in bio-PCR to detect R. solanacearum from crown, mid part of stem and upper parts of the plant. Another experiment was conducted to find out the inoculum potential of R. solanacearum strain UTT-25 to cause bacterial wilt in susceptible cultivar Pusa Ruby. The bacteria were inoculated at concentration of bacterial suspension 10 to 10¹⁰?cfu/ml in injured and uninjured roots of the plants separately and injured root accelerated wilt incidence and able to cause wilt disease 63.3% by 100?cfu/ml of R. solanacearum, while no disease appeared at 10?cfu/ml on the 11th day of inoculation in injured and uninjured roots of the plant.     

细菌有活力但不可培养状态及其机制研究进展

有活力但不可培养(viable but non-culturable,VBNC)状态是细菌遭遇逆境时进入的一种特殊状态,该状态下的菌体在条件适宜时可复苏并恢复其致病性,被认为是细菌躲避不良环境的一种生存策略。VBNC状态菌体对人类医学和工农业生产具有巨大的潜在威胁,开展关于VBNC状态的检测及诱导、复苏及其机制研究可为减少或避免该状态细菌的危害提供理论基础。本文简要综述了细菌VBNC状态在诱导、复苏及致病性等方面的研究进展,并结合本实验室及国内外相关团队近年来在植物病原细菌VBNC状态研究中的结果,详细总结了VBNC状态细菌的形成和复苏机制,对植物病原细菌在环境胁迫下的存活机制、病害田间初侵染来源分析及VBNC状态菌体在病害循环中的作用等相关研究具有重要参考意义。     

Effect of Seed Treatment by Cold Plasma on the Resistance of Tomato to Ralstonia solanacearum (Bacterial Wilt)

This study investigated the effect of cold plasma seed treatment on tomato bacterial wilt, caused by Ralstonia solanacearum (R. solanacearum), and the regulation of resistance mechanisms. The effect of cold plasma of 80W on seed germination, plant growth, nutrient uptake, disease severity, hydrogen peroxide (H₂O₂) concentration and activities of peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.2) and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) were examined in tomato plants. Plasma treatment increased tomato resistance to R. solanacearum with an efficacy of 25.0%. Plasma treatment significantly increased both germination and plant growth in comparison with the control treatment, and plasma-treated plants absorbed more calcium and boron than the controls. In addition, H₂O₂ levels in treated plants rose faster and reached a higher peak, at 2.579 µM gFW⁻¹, 140% greater than that of the control. Activities of POD (421.3 U gFW⁻¹), PPO (508.8 U gFW⁻¹) and PAL (707.3 U gFW⁻¹) were also greater in the treated plants than in the controls (103.0 U gFW⁻¹, 166.0 U gFW⁻¹ and 309.4 U gFW⁻¹, respectively). These results suggest that plasma treatment affects the regulation of plant growth, H₂O₂ concentration, and POD, PPO and PAL activity in tomato, resulting in an improved resistance to R. solanacearum. Consequently, cold plasma seed treatment has the potential to control tomato bacterial wilt caused by R. solanacearum.     

Evaluation of Rpf protein of Micrococcus luteus for cultivation of soil actinobacteria

Rpf protein, a kind of resuscitation promoting factor, was first found in the culture supernatant of Micrococcus luteus. It can resuscitate the growth of M. luteus in “viable but non-culture, VBNC” state and promote the growth of Gram-positive bacteria with high G + C content. This paper investigates the resuscitating activity of M. luteus ACCC 41016^T Rpf protein, which was heterologously expressed in E. coli, to cells of M. luteus ACCC 41016^T and Rhodococcus marinonascens HBUM200062 in VBNC state, and examines the effect on the cultivation of actinobacteria in soil. The results showed that the recombinant Rpf protein had resuscitation effect on M. luteus ACCC 41016^T and R. marinonascens HBUM200062 in VBNC state. 83 strains of actinobacteria, which were distributed in 9 families and 12 genera, were isolated from the experimental group with recombinant Rpf protein in the culture medium. A total of 41 strains of bacteria, which were distributed in 8 families and 9 genera, were isolated from the control group without Rpf protein. The experimental group showed richer species diversity than the control group. Two rare actinobacteria, namely HBUM206391^T and HBUM206404^T, were obtained in the experimental group supplemented with Rpf protein. Both may be potential new species of Actinomadura and Actinokineospora, indicating that the recombinant expression of M. luteus ACCC 41016^T Rpf protein can effectively promote the isolation and culture of actinobacteria in soil.     

Pathogenesis and stress related,as well as metabolic proteins are regulated in tomato stems infected with Ralstonia solanacearum

A comparative proteome analysis was initiated to systematically investigate the physiological response of tomato (Solanum lycopersicum) to infection with Ralstonia solanacearum, causal agent of bacterial wilt. Plants of the susceptible tomato recombinant inbred line NHG3 and the resistant NHG13 were either infected or not infected with R. solanacearum and subsequently used for proteome analysis. Two-dimensional isoelectric focussing/sodium dodecyl-sulphate polyacrylamide gel electrophoresis (2-D IEF/SDS-PAGE) allowed the separation of about 650–690 protein spots per analysis. Twelve proteins were of differential abundance in susceptible plants in response to bacterial infection, while no differences were observed in the resistant genotype. LC-MS/MS analysis of these spots revealed 12 proteins, six of which were annotated as plant and six as bacterial proteins. Among the plant proteins, two represent pathogenesis related (PR) proteins, one stress response protein, one enzyme of carbohydrate and energy metabolism, and one hypothetical protein. A constitutive difference between resistant and susceptible lines was not found.