Homology between the HrpO protein of Pseudomonas solanacearum and bacterial proteins implicated in a signal peptide-independent secretion mechanism

A region of approximately 22 kb of DNA defines the large hrp gene cluster of strain GMI1000 of Pseudomonas solanacearum. The majority of mutants that map to this region have lost the ability to induce disease symptoms on tomato plants and are no longer able to elicit a hypersensitive reaction (HR) on tobacco, a nonhost plant. In this study we present the complementation analysis and nucleotide sequence of a 4772 by region of this hrp gene cluster. Three complete open reading frames (ORFs) are predicted within this region. The corresponding putative proteins, HrpN, HrpO and HpaP, have predicted sizes of 357, 690 and 197 amino acids, respectively, and predicted molecular weights of 38607, 73 990 and 21959 dalton, respectively. HrpN and HrpO are both predicted to be hydrophobic proteins with potential membrane-spanning domains and HpaP is rich in proline residues. A mutation in hpaP (for hrp associated) does not affect the HR on tobacco or the disease on tomato plants. None of the proteins is predicted to have an N-terminal signal sequence, which would have indicated that the proteins are exported. Considerable sequence similarities were found between HrpO and eight known or predicted prokaryotic proteins: LcrD of Yersinia pestis and Y. enterocolitica, FlbF of Caulobacter crescentus, F1hA of Bacillus subtilis, MxiA and VirH of Shigella flexneri, InvA of Salmonella typhimurium and HrpC2 of Xanthomonas campestris pv. vesicatoria. These homologies suggest that certain hrp genes of phytopathogenic bacteria code for components of a secretory system, which is related to the systems for secretion of flagellar proteins, Ipa proteins of Shigella flexneri and the Yersinia Yop proteins. Furthermore, these homologous proteins have the common feature of being implicated in a distinct secretory mechanism, which does not require the cleavage of a signal peptide. The sequence similarity between HrpO and HrpC2 is particularly high (66% identity and 81 % similarity) and the amino acid sequence comparison between these two proteins presented here reveals the first such sequence similarity to be shown between Hrp proteins of P. solanacearum and X. campestris. An efflux of plant electrolytes was found to be associated with the interactions between P. solanacearum and both tomato and tobacco leaves. This phenomenon may be part of the mechanism by which hrp gene products control and determine plant-bacterial interactions, since hrpO mutants induced levels of leakage which were significantly lower than those induced by the wild type on each plant.     

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.     

The Pseudomonas syringae pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate

The host-specific plant pathogen Pseudomonas syringae elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in A₂₃₀ assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringae pathovars, Pseudomonas viridiflava, Ralstonia (Pseudomonas) solanacearum, and Xanthomonas campestris. ΔhrpZ::nptII or hrpW::ΩSp^r P. syringae pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a hrpZ hrpW double mutant. These features of hrpW and its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall.     

Characterization of the dTDP-rhamnose biosynthetic genes encoded in the rfb iocus of Shigella flexneri

The nucleotide sequence of the proximal half of the rfb region of Shigella flexneri has been determined, and the genes encoding enzymes involved in the biosynthesis of dTDP-rhamnose have been identified. These genes show strong homology to the rfb genes encoding dTDP-rhamnose biosynthesis in Salmonella enterica serovar typhimurium (strain LT2) and S. enterica serovar anatum (strain M32) (Jiang et al., 1991; Wang et al., 1992). An open reading frame upstream of rfbB was also identified which encoded a protein having strong similarity with GalU, and has been designated galF. GalF has 92% amino acid sequence identity with an S. enterica LT2 gene, orf2X8, which is similarly situated upstream of rfbB (Jiang et al., 1991). The T7 expression system was utilized to identify proteins corresponding to those predicted from DNA sequence analysis. The similarity of the predicted proteins with proteins that are functionally identical or related, and with others of unknown function from the Yersinia enterocolitica O3 rfb region, and in the Escherichia coli K-12 rff region are also described. We have re-addressed the assignment of each gene of the dTDP-rhamnose pathway with the known enzymes of the pathway, in particular rfbC and rfbD. A reporter plasmid to detect genes encoding enzymes of the dTDP-rhamnose pathway is described. An analysis of the intergenic region between galF and rfbB has been made, and comparison with the same region from S. enterica LT2 discussed.     

Elucidation of the hrp Clusters of Xanthomonas oryzae pv. oryzicola That Control the Hypersensitive Response in Nonhost Tobacco and Pathogenicity in Susceptible Host Rice

Xanthomonas oryzae pv. oryzicola, the cause of bacterial leaf streak in rice, possesses clusters of hrp genes that determine its ability to elicit a hypersensitive response (HR) in nonhost tobacco and pathogenicity in host rice. A 27-kb region of the genome of X. oryzae pv. oryzicola (RS105) was identified and sequenced, revealing 10 hrp, 9 hrc (hrp conserved), and 8 hpa (hrp-associated) genes and 7 regulatory plant-inducible promoter boxes. While the region from hpa2 to hpaB and the hrpF operon resembled the corresponding genes of other xanthomonads, the hpaB-hrpF region incorporated an hrpE3 gene that was not present in X. oryzae pv. oryzae. We found that an hrpF mutant had lost the ability to elicit the HR in tobacco and pathogenicity in adult rice plants but still caused water-soaking symptoms in rice seedlings and that Hpa1 is an HR elicitor in nonhost tobacco whose expression is controlled by an hrp regulator, HrpX. Using an Hrp phenotype complementation test, we identified a small hrp cluster containing the hrpG and hrpX regulatory genes, which is separated from the core hrp cluster. In addition, we identified a gene, prhA (plant-regulated hrp), that played a key role in the Hrp phenotype of X. oryzae pv. oryzicola but was neither in the core hrp cluster nor in the hrp regulatory cluster. A prhA mutant failed to reduce the HR in tobacco and pathogenicity in rice but caused water-soaking symptoms in rice. This is the first report that X. oryzae pv. oryzicola possesses three separate DNA regions for HR induction in nonhost tobacco and pathogenicity in host rice, which will provide a fundamental base to understand pathogenicity determinants of X. oryzae pv. oryzicola compared with those of X. oryzae pv. oryzae.     

Nutritional Similarity between Leaf-Associated Nonpathogenic Bacteria and the Pathogen Is Not Predictive of Efficacy in Biological Control of Bacterial Spot of Tomato

It has been demonstrated that for a nonpathogenic, leaf-associated bacterium, effectiveness in the control of bacterial speck of tomato is correlated with the similarity in the nutritional needs of the nonpathogenic bacterium and the pathogen Pseudomonas syringae pv. tomato. This relationship was investigated further in this study by using the pathogen Xanthomonas campestris pv. vesicatoria, the causal agent of bacterial spot of tomato, and a collection of nonpathogenic bacteria isolated from tomato foliage. The effects of inoculation of tomato plants with one of 34 nonpathogenic bacteria prior to inoculation with the pathogen X. campestris pv. vesicatoria were quantified by determining (i) the reduction in disease severity (number of lesions per square centimeter) in greenhouse assays and (ii) the reduction in leaf surface pathogen population size (log₁₀ of the number of CFU per leaflet) in growth chamber assays. Nutritional similarity between the nonpathogenic bacteria and X. campestris pv. vesicatoria was quantified by using either niche overlap indices (NOI) or relatedness in cluster analyses based upon in vitro utilization of carbon or nitrogen sources reported to be present in tomato tissues or in Biolog GN plates. In contrast to studies with P. syringae pv. tomato, nutritional similarity between the nonpathogenic bacteria and the pathogen X. campestris pv. vesicatoria was not correlated with reductions in disease severity. Nutritional similarity was also not correlated with reductions in pathogen population size. Further, the percentage of reduction in leaf surface pathogen population size was not correlated with the percentage of reduction in disease severity, suggesting that the epiphytic population size of X. campestris pv. vesicatoria is not related to disease severity and that X. campestris pv. vesicatoria exhibits behavior in the phyllosphere prior to lesion formation that is different from that of P. syringae pv. tomato.     

An AFLP-based database of Shigella flexneri and Shigella sonnei isolates and its use for the identification of untypable Shigella strains

Amplified fragment length polymorphism (AFLP) can be used to assess the genetic diversity of closely related microbial genomes. In this study, the first of its kind for identification of Shigella, the high discriminatory power of AFLP has been used to determine the genetic relatedness of 230 isolates of Shigella flexneri and Shigella sonnei strains. An AFLP database was generated to demonstrate its utility in the discrimination of closely related strains. Based on AFLP, S. flexneri strains could be grouped into separate clusters according to their serotypes. Within each serotype, strains demonstrated 80–100% similarity indicating that identical strains and closely related strains could be distinguished by this technique. S. flexneri 6 formed a distinct cluster with 55% similarity to the rest of the S. flexneri strains showing significant divergence from the rest of the S. flexneri strains. Significantly, S. sonnei isolates formed a distinct group and showed approximately the same level of genetic linkage to S. flexneri as Escherichia coli strains. Untypable isolates that showed conflicting agglutination reactions with conventional typing sera were identifiable by AFLP. Thus AFLP can be used for genetic fingerprinting of Shigella strains and aid in the identification of variant untypable isolates.     

Characterisation and diversity of Indian isolates of Ralstonia solanacearum causing bacterial wilt of Capsicum annuum L.

Genetic diversity of 13 isolates of Ralstonia solanacearum causing bacterial wilt in hot pepper and bell pepper (Capsicum annuum L.) from 6 states of India was assessed. All isolates of R. solanacearum belonged to biovar 3, race 1 and phylotype I. These isolates consisted of 4 distinct DNA types at 75% similarity coefficient using ERIC, BOX and REP-PCRs techniques. Multilocus sequence analysis of hrpB, fliC and egl genes of 6 isolates of R. solanacearum along with 2 out group bacteria was done and they showed high level of variability within these three regions of the genome involving in pathogenicity.     

The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice

The nucleotide sequence was determined for the genome of Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, a bacterium that causes bacterial blight in rice (Oryza sativa L.). The genome is comprised of a single, 4 941 439 bp, circular chromosome that is G + C rich (63.7%). The genome includes 4637 open reading frames (ORFs) of which 3340 (72.0%) could be assigned putative function. Orthologs for 80% of the predicted Xoo genes were found in the previously reported X.axonopodis pv. citri (Xac) and X.campestris pv. campestris (Xcc) genomes, but 245 genes apparently specific to Xoo were identified. Xoo genes likely to be associated with pathogenesis include eight with similarity to Xanthomonas avirulence (avr) genes, a set of hypersensitive reaction and pathogenicity (hrp) genes, genes for exopolysaccharide production, and genes encoding extracellular plant cell wall-degrading enzymes. The presence of these genes provides insights into the interactions of this pathogen with its gramineous host.     

HrpI of Erwinia amylovora functions in secretion of harpin and is a member of a new protein family.

HrpI, a 78-kDa protein, functions in the secretion of harpin, a proteinaceous elicitor of the hypersensitive response from Erwinia amylovora. The predicted amino acid sequence of HrpI is remarkably similar to that of LcrD of Yersinia species, the first member of a recently described protein family. Other proteins of the family are MixA from Shigella flexneri, InvA from Salmonella typhimurium, FlhA from Caulobacter crescentus, HrpI from Pseudomonas syringae pv. syringae, HrpO from Pseudomonas solanacearum, and HrpC2 from Xanthomonas campestris pv. vesicatoria. Cells of E. amylovora containing mutated hrpI genes or cells of Escherichia coli containing the cloned hrp gene cluster with mutated hrpI produce but do not export harpin. When similar cells with functional hrpI genes were grown at 25 degrees C, but not at 37 degrees C, harpin was exported to the culture supernatant. Direct evidence that HrpI is involved in the secretion of a virulence protein has been offered. Two other loci of the hrp gene cluster are involved in the regulation of harpin, and four other loci also are involved in the secretion of harpin. Since harpin and other proteins likely to be secreted by the LcrD family of proteins lack typical signal peptides, their secretion mechanism is distinct from the general protein export pathway.     

Shigella flexneri type-specific antigen V: cloning,sequencing and characterization of the glucosyl transferase gene of temperate bacteriophage SfV

With lysogeny by bacteriophage SfV, Shigella flexneri serotype Y is converted to serotype 5a. The glucosyl transferase gene (gtr) from bacteriophage SfV of S. flexneri, involved in serotype-specific conversion, was cloned and characterized. The DNA sequence of a 3.7 kb EcoRI–BamHI fragment of bacteriophage SfV which includes the gtr gene was determined. This gene, encoding a polypeptide of 417 aa with 47.67 kDa molecular mass, caused partial serotype conversion of S. flexneri from serotype Y to type V antigen as demonstrated by Western blotting and the sensitivity of the hybrid strain to phage Sf6. The deduced protein of the partially sequenced open reading frame upstream of the gtr showed similarity to various glycosyl transferases of other bacteria. Orf3, separated from the gtr by a non-coding region and transcribed convergently, codes for a 167 aa (18.8 kDa) protein found to have homology with tail fibre genes of phage lambda and P2.     

Simple Deletion: a vector- and marker-free method to generate and isolate site-directed deletion mutants

We have designed a new vector- and marker-free site-directed deletion system for gram-negative bacteria. In this system, a specific DNA fragment is amplified from a parental strain by using polymerase chain reaction (PCR), then circularized and introduced back into the parental strain for homologous recombination. The recombinant mutant is then detected and isolated by PCR-based sib selection. Unlike conventional methods, our Simple Deletion method requires no cloning procedures, and no foreign genes such as antibiotic-resistance genes are introduced as selection markers. The resulting mutant is, therefore, the same as the parental strain except for the lack of the target region. This method is categorized as a type of “self-cloning,” and the resulting mutant can be used for laboratory research without restrictions. Using this method, we generated a mutant of a plant pathogenic bacterium, Xanthomonas campestris pv. campestris, in which the 20.4-kb hrp gene cluster involved in the type III secretion system and in pathogenicity was deleted. In addition, we proved that this method can also be used to delete smaller DNA regions of X. campestris pv. campestris and to generate deletion mutants of the bacterium Ralstonia solanacearum.     

Molecular characterization of the genes involved in O-antigen modification,attachment, integration and excision in Shigella flexneri bacteriophage SfV

Bacteriophage SfV is a temperate phage of Shigella flexneri responsible for converting serotype Y (3,4) to serotype 5a (V; 3,4) through its glucosyl transferase gene. The glucosyl transferase (gtr) gene of SfV has been cloned and shown to partially convert S. flexneri serotype Y to serotype 5a. In this study, we found that the serotype-converting region of SfV was approximately 2.5 kb in length containing three continuous ORFs. The recombinant strain carrying the three complete ORFs expressed the type V and group antigen 3,4, both indistinguishable from that of S. flexneri 5a wild-type strain. The interruption of orf5 or orf6 gave partial conversion in the S. flexneri recombinant strain indicated by the incomplete replacement of group antigen 3,4. The region adjacent to the serotype-conversion genes was found to be identical to the attP-int-xis region of phage P22. Altogether, an approximately 2.2-kb sequence covering a portion of the serotype-conversion (approximately 500 nt)-attP-int-xis regions of SfV was remarkably similar to that of P22.     

Complete DNA sequence and gene analysis of the virulence plasmid pCP301 of Shigella flexneri 2a

The complete nucleotide sequence and organization of the large virulence plasmid pCP301 (termed by us) of Shigella flexneri 2a strain 301 were determined and analyzed. The result showed that the entire DNA sequence of pCP301 is composed of 221618 bp which form a circular plasmid. Sequence analysis identified 272 open reading frames (ORFs), among which, 194 correspond to the proteins described previously, 61 have low identity (<60%) to known proteins and the rest 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include the genes associated with bacterial virulence, the genes associated with regulation and the genes relating to plasmid maintenance, stability and DNA metabolism. Insertion sequence (IS) elements are 68 kb in length and account for 30 percent of complete sequence of the plasmid which indicates that gene multiple rearrangements of the pCP301 have taken place in Shigella flexneri evolution history. The research result is helpful for interpreting the pathogenesis of Shigella, as well as the genetics and evolution of the plasmid.     

Genetic Diversity of African and Worldwide Strains of Ralstonia solanacearum as Determined by PCR-Restriction Fragment Length Polymorphism Analysis of the hrp Gene Region

The genetic diversity among a worldwide collection of 120 strains of Ralstonia solanacearum was assessed by restriction fragment length polymorphism (RFLP) analysis of amplified fragments from the hrp gene region. Five amplified fragments appeared to be specific to R. solanacearum. Fifteen different profiles were identified among the 120 bacterial strains, and a hierarchical cluster analysis distributed them into eight clusters. Each cluster included strains belonging to a single biovar, except for strains of biovars 3 and 4, which could not be separated. However, the biovar 1 strains showed rather extensive diversity since they were distributed into five clusters whereas the biovar 2 and the biovar 3 and 4 strains were gathered into one and two clusters, respectively. PCR-RFLP analysis of the hrp gene region confirmed the results of previous studies which split the species into an “Americanum” division including biovar 1 and 2 strains and an “Asiaticum” division including biovar 3 and 4 strains. However, the present study showed that most of the biovar 1 strains, originating from African countries (Reunion Island, Madagascar, Zimbabwe, and Angola) and being included in a separate cluster, belong to the “Asiaticum” rather than to the “Americanum” division. These African strains could thus have evolved separately from other biovar 1 strains originating from the Americas.     

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.     

Ralstonia solanacearum Strains from Martinique (French West Indies) Exhibiting a New Pathogenic Potential

We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America.     

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.