phlF− mutant of Pseudomonas fluorescens J2 improved 2,4-DAPG biosynthesis and biocontrol efficacy against tomato bacterial wilt

Pseudomonas fluorescens J2 can produce 2,4-diacetylphloroglucinol (2,4-DAPG) as the main antibiotic compound and effectively inhibits the wilt pathogens Ralstonia solanacearum and Fusarium oxysporum. The phlF which negatively regulates the 2,4-DAPG synthesis in strain J2 was disrupted by homologous recombination to construct a mutant strain J2-phlF⁻. The mutant J2-phlF⁻ produced much more 2,4-DAPG and showed higher inhibitory effect on R. solanacearum than the wild type strain J2 in vitro. The mutant J2-phlF⁻ also showed more colonization of tomato roots and higher inhibition to R. solanacearum in soil than wild type strain J2. The biocontrol efficiency of mutant J2-phlF⁻ was higher against tomato bacterial wilt than wild type strain J2, but the differences were not significant. However, the application of both strains with organic fertilizer improved the colonization and biocontrol efficiency against tomato bacterial wilt and mutant strain J2-phlF⁻ showed higher biocontrol efficiency against tomato bacterial wilt than wild type strain J2. Both strains, J2 and J2-phlF⁻, could also promote the growth of tomato plants.     

Suppression of bacterial wilt in Eucalyptus urophylla by fluorescent Pseudomonas spp. in China

Bacterial wilt caused by Ralstonia solanacearum race 1, biovar III has become a severe problem in Eucalyptus plantations in south China. The disease mainly attacks young eucalypt trees, and no effective control measures are available yet. To explore possibilities to develop biological control of the disease, strains of fluorescent Pseudomonas spp. that are effective in suppressing plant diseases by known mechanisms, were tested for their potential to control bacterial wilt in Eucalyptus. Pseudomonas putida WCS358r, Pseudomonas fluorescens WCS374r, P. fluorescens WCS417r, and Pseudomonas aeruginosa 7NSK2 antagonize R. solanacearum in vitro by siderophore-mediated competition for iron, whereas inhibition of pathogen growth by P. fluorescens CHA0r is antibiosis-based. No correlations were found between antagonistic activities of these Pseudomonas spp. in vitro and biocontrol of bacterial wilt in Eucalyptus in vivo. None of the strains suppressed disease when mixed together with the pathogen through the soil or when seeds or seedlings were treated with the strains one to four weeks before transfer into soil infested with R. solanacearum. However, when the seedlings were dipped with their roots in a bacterial suspension before transplanting into infested soil, P. fluorescens WCS417r significantly suppressed bacterial wilt. P. putida WCS358r was marginally effective, whereas its siderophore-minus mutant had no effect at all, indicating that siderophore-mediated competition for iron can contribute but is not effective enough to suppress bacterial wilt in Eucalyptus. A derivative of P. putida WCS358r, constitutively producing 2,4-diacetylphloroglucinol (WCS358::phl) reduced disease. Combined treatment with P. fluorescens WCS417r and P. putida WCS358::phl did not improve suppression of bacterial wilt.     

Bacterial wilt of tomato in Karnataka and its management by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Field surveys undertaken in major tomato growing districts of the Karnataka state, located in southern part of India, revealed a high incidence of bacterial wilt caused by Ralstonia solanacearum and it is one of the most destructive bacterial diseases of economically important crops. Across all the tomato cultivars under evaluation, the disease incidence in plants ranged from 9% to 39% whereas the incidence in seeds ranged from 4% to 18%. The effects of tomato seed treatments with Pseudomonas fluorescens in the control of bacterial wilt under greenhouse conditions revealed that the treatments protected plants against soil-borne infections of the bacterial wilt organism. Seed treatment with antagonistic P. fluorescens strain significantly improved the quality of seed germination and seedling vigour. The disease incidence was significantly reduced in plants raised from P. fluorescens treated seeds followed by challenge inoculation with R. solanacearum. Periodic field surveys for the incidence of bacterial wilt of tomato could be recommended to monitor the populations of the bacterial wilt pathogen. Workable measures are presented that could lead to the reduction of the prevalence of this serious disease in affected fields of the small farm-holders.     

Ralstonia solanacearum colonization of tomato roots infected by Meloidogyne incognita

Bacterial wilt, caused by Ralstonia solanacearum, is one of the most serious diseases of tomato (Solanum lycopersicum). Concomitant infection of R. solanacearum and root‐knot nematode Meloidogyne incognita increases the severity of bacterial wilt in tomato, but the role of this nematode in disease complexes involving bacterial pathogens is not completely elucidated. Although root wounding by root‐knot nematode infection seems to play an important role, it might not entirely explain the increased susceptibility of plants to R. solanacearum. In the present study, green fluorescent protein (GFP)‐labelled R. solanacearum distribution was observed in the root systems of the tomato cultivar Momotaro preinoculated with root‐knot nematode or mock‐inoculated with tap water. Fluorescence microscopy revealed that GFP‐labelled R. solanacearum mainly colonized root‐knot nematode galls, and little or no green fluorescence was observed in nematode‐uninfected roots. These results suggest that the gall induced by the nematode is a suitable location for the growth of R. solanacearum. Thus, it is crucial to control both R. solanacearum and root‐knot nematode in tomato production fields to reduce bacterial wilt disease incidence and effects.     

Evaluation of bacterial antagonists of Ralstonia solanacearum,causal agent of bacterial wilt of potato

Bacterial wilt of potato caused by Ralstonia solanacearum is one of the most destructive diseases in Kurdistan province, Iran. The objective of the present study was to evaluate antagonistic effects of some rhizobacteria isolated from the rhizosphere of potato plants against R. solanacearum, the agent of potato bacterial wilt. A total of 52 rhizobacteria were isolated and screened for in vitro antagonistic activity against R. solanacearum. Seven isolates with inhibiting effects of the pathogen were identified by phenotypic properties and partial sequencing of 16s rRNA as Pseudomonas fluorescens Pf11, P. fluorescens Pf16, Pseudomonas putida Pp17, Paenibacillus sp. Pb28 and Enterobacter sp. En38, Pseudomonas fluorescens Pp23 and Serratia sp. Se40. Strains Pf11, Pf16, Pp17 and Pb28 significantly inhibited the growth of the pathogen. Strains En38, Pp23 and Se40 showed a moderate or weak inhibition. During greenhouse study, strains were evaluated for their effects in reducing of disease and increasing biomass of potato plants. In according to greenhouse experiment results, isolates Pb28, Pp17 and Pf11significantly reduced disease by 55.56%, 51.50% and 38.58%, respectively. In addition, plant biomass significantly increased in plants treated with Pb28, Pp17, Pf11 and Pf16, compared to the control. Therefore, this study shows that these four strains have potential to be used as biocontrol agents against R. solanacearum. To confirm their effectiveness as commercial biocontrol agent, it is necessary to evaluate their efficiency in the field conditions in the next studies.     

Characterization of PhlG, a Hydrolase That Specifically Degrades the Antifungal Compound 2,4-Diacetylphloroglucinol in the Biocontrol Agent Pseudomonas fluorescens CHA0

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative ΔphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a ΔphlA ΔphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a k_cat of 33 s⁻¹ and a K_m of 140 μM at 30°C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.     

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.     

Potential Role of Pathogen Signaling in Multitrophic Plant-Microbe Interactions Involved in Disease Protection

Multitrophic interactions mediate the ability of fungal pathogens to cause plant disease and the ability of bacterial antagonists to suppress disease. Antibiotic production by antagonists, which contributes to disease suppression, is known to be modulated by abiotic and host plant environmental conditions. Here, we demonstrate that a pathogen metabolite functions as a negative signal for bacterial antibiotic biosynthesis, which can determine the relative importance of biological control mechanisms available to antagonists and which may also influence fungus-bacterium ecological interactions. We found that production of the polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) was the primary biocontrol mechanism of Pseudomonas fluorescens strain Q2-87 against Fusarium oxysporum f. sp. radicis-lycopersici on the tomato as determined with mutational analysis. In contrast, DAPG was not important for the less-disease-suppressive strain CHA0. This was explained by differential sensitivity of the bacteria to fusaric acid, a pathogen phyto- and mycotoxin that specifically blocked DAPG biosynthesis in strain CHA0 but not in strain Q2-87. In CHA0, hydrogen cyanide, a biocide not repressed by fusaric acid, played a more important role in disease suppression.     

Biological control of bacterial wilt caused by Pseudomonas solanacearum in India with antagonistic bacteria

From among 125 strains of fluorescent and 52 strains of nonfluorescent bacteria initially screened in the laboratory for their antibiosis towards the bacterial wilt pathogen, Pseudomonas solanacearum, strain Pfcp of Pseudomonas fluorescens and strains B33 and B36 of Bacillus spp., were chosen and evaluated further in greenhouse and field tests. Pfcp treated banana (Musa balbisiana), eggplant and tomato plants were protected from wilt upto 50, 61 and 95% in greenhouse and upto 50, 49 and 36% respectively in field. Protection afforded by the Bacillus strains was lower. In bacteria-treated plants which were subsequently inoculated with P. solanacearum plant height and biomass values increased and were close to those of nontreated and noninoculated control plants.     

Evaluation of the strains of Acinetobacter and Enterobacter as potential biocontrol agents against Ralstonia wilt of tomato

Bacterial wilt (Ralstonia solanacearum) of tomato, Lycopersicon esculentum, causes a considerable amount of damage to tomato in Southern China. Biological control is one of the more promising approaches to reduce the disease incidence and yield losses caused by this disease. Based on antagonistic activity against R. solanacearum and three soil-borne fungal pathogens as well as biocontrol efficacy in the greenhouse, two bacterial strains Xa6 (Acinetobacter sp.) and Xy3 (Enterobacter sp.) were selected out of fourteen candidates as potential biocontrol agents. In order to find a suitable antagonist inoculation method, we compared the methods of root-dipping with soil-drenching in the aspects including rhizocompetence, biocontrol efficacy, and effect of promoting plant growth under greenhouse conditions. The drenching treatment resulted in a higher biocontrol efficacy and plant-yield increase, and this method was also easier to operate in the field on a large scale. Field trials were conducted for further evaluation of these two antagonistic strains. In both greenhouse and field experiments, the strain Xy3 had a better control effect against bacterial wilt than Xa6 did, while Xa6 caused higher biomass or yield increases. As recorded on the 75th day after treatment in two field experiments, biocontrol efficacy of Xy3 was about 65% in both field trials, and the yield increases caused by Xa6 were 32.4 and 40.7%, respectively, in the two trials. This is the first report of an Acinetobacter sp. strain used as a BCA against Ralstonia wilt of tomato.     

Bioorganic Fertilizer Enhances Soil Suppressive Capacity against Bacterial Wilt of Tomato

Tomato bacterial wilt caused by Ralstonia solanacearum is one of the most destructive soil-borne diseases. Many strategies have been taken to improve soil suppressiveness against this destructive disease, but limited success has been achieved. In this study, a novel bioorganic fertilizer revealed a higher suppressive ability against bacterial wilt compared with several soil management methods in the field over four growing seasons from March 2011 to July 2013. The application of the bioorganic fertilizer significantly (P<0.05) reduced disease incidence of tomato and increased fruit yields in four independent trials. The association among the level of disease incidence, soil physicochemical and biological properties was investigated. The soil treated with the bioorganic fertilizer increased soil pH value, electric conductivity, organic carbon, NH₄ ⁺-N, NO₃ ^--N and available K content, microbial activities and microbial biomass carbon content, which were positively related with soil suppressiveness. Bacterial and actinomycete populations assessed using classical plate counts were highest, whereas R. solanacearum and fungal populations were lowest in soil applied with the bioorganic fertilizer. Microbial community diversity and richness were assessed using denaturing gel gradient electrophoresis profile analysis. The soil treated with the bioorganic fertilizer exhibited higher bacterial community diversity but lower fungal community diversity. Redundancy analysis showed that bacterial community diversity and richness negatively related with bacterial wilt suppressiveness, while fungal community richness positively correlated with R. solanacearum population. We concluded that the alteration of soil physicochemical and biological properties in soil treated with the bioorganic fertilizer induced the soil suppressiveness against tomato bacterial wilt.     

Characterization of antifungal metabolite phenazine from rice rhizosphere fluorescent pseudomonads (FPs) and their effect on sheath blight of rice

We have shown, the outcome of antifungal activity of phenazine derivatives which is produced by fluorescent pseudomonads (FPs) for the control of sheath blight of rice. A total of 50 fluorescent pseudomonads (FPs) were isolated from rice rhizosphere. Off which, 36 FPs exhibited antagonistic activity against Rhizoctonia solani, Macrophomina phaseolina, Fusarium oxysporum, Alternaria alternata and Sclerotium rolfsii up to 70–80% compared to control by dual culture method. BOX-PCR analyses of antagonistic isolates indicated that two phylogenetic group, where group I consisted of 28 isolates and eight isolates belongs to group II. Among 36 FPs, a total of 10 FPs revealed that the presence of phenazine derivatives on thin layer chromatography (TLC), which is coincided with that of authentic phenazine with R_f value 0.57. Similar to TLC analysis, antibiotic encoding gene phenazine-1-carboxamide (PCN) was detected in 10 FPs by PCR analysis with respective primer. Among, PCN detected isolates of FPs, a significant biocontrol potential possessing isolate designated as VSMKU1 and it was showed prominent antifungal activity against R. solani and other tested fungal pathogens. Hence, the isolate VSMKU1 was selected for further studies. The selected isolate VSMKU1 was identified as Pseudomonas aeruginosa by 16S rDNA sequence analysis. The antifungal metabolite phenazine like compound produced by VSMKU1 was confirmed by UV, FT-IR and HPLC analysis. The phenazine compound from VSMKU1 significantly arrest the growth of R. solani compared to carbendazim by well diffusion method. The detached leaf assay showed remarkable inhibition of lesion height 80 to 85% by the treatments of culture (VSMKU1), cell free culure filtrate and phenazine like compound compared to control and other treatments was observed in detached leaves of rice. These results emphasized that VSMKU1 isolate can be used as an alternative potential biocontrol agent against sheath blight of rice, instead of using commercial fungicide such as validamycin and carbendazim which cause environmental pollution and health hazards.     

Biological control of eucalyptus bacterial wilt with rhizobacteria

The antagonistic potential of 298 rhizobacteria obtained from the rhizosphere and rhizoplane of tomato and eucalyptus plants was assessed for the control of bacterial wilt of eucalyptus caused by Ralstonia solanacearum. Several tests were performed using tomato plants as a screening system to select efficient rhizobacteria. Different methods for antagonist delivery and pathogen inoculation were evaluated: (1) seeds were microbiolized (soaked for 12 h in a suspension of the antagonist propagules) and germinated seedlings had their roots immersed in the pathogen inoculum suspension; (2) seedlings originated from microbiolized seeds were transplanted to soil infested with R. solanacearum and (3) roots of seedlings were immersed in a suspension of propagules of the antagonist and subsequently in a suspension of R. solanacearum. Nine isolates (UFV-11, 32, 40, 56, 62, 101, 170, 229, and 270) were selected as potential antagonists to R. solanacearum as they suppressed bacterial wilt in at least one of the methods assessed. The selected antagonists were evaluated against two isolates of R. solanacearum using in vitro and in vivo (inoculated eucalyptus) tests. Isolates UFV-56 (Bacillus thuringiensis), UFV-62 (Bacillus cereus) and a commercial formulation of several rhizobacteria (Rizolyptus®) suppressed bacterial wilt in eucalyptus protecting the plants during the early stages of development.     

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.     

Secondary Metabolite- and Endochitinase-Dependent Antagonism toward Plant-Pathogenic Microfungi of Pseudomonas fluorescens Isolates from Sugar Beet Rhizosphere

Forty-seven isolates representing all biovars of Pseudomonas fluorescens (biovars I to VI) were collected from the rhizosphere of field-grown sugar beet plants to select candidate strains for biological control of preemergence damping-off disease. The isolates were tested for in vitro antagonism toward the plant-pathogenic microfungi Pythium ultimum and Rhizoctonia solani in three different plate test media. Mechanisms of fungal inhibition were elucidated by tracing secondary-metabolite production and cell wall-degrading enzyme activity in the same media. Most biovars expressed a specific mechanism of antagonism, as represented by a unique antibiotic or enzyme production in the media. A lipopeptide antibiotic, viscosinamide, was produced independently of medium composition by P. fluorescens bv. I, whereas the antibiotic 2,4-diacetylphloroglucinol was observed only in glucose-rich medium and only in P. fluorescens bv. II/IV. Both pathogens were inhibited by the two antibiotics. Finally, in low-glucose medium, a cell wall-degrading endochitinase activity in P. fluorescens bv. I, III, and VI was the apparent mechanism of antagonism toward R. solani. The viscosinamide-producing DR54 isolate (bv. I) was shown to be an effective candidate for biological control, as tested in a pot experiment with sugar beet seedlings infested with Pythium ultimum. The assignment of different patterns of fungal antagonism to the biovars of P. fluorescens is discussed in relation to an improved selection protocol for candidate strains to be used in biological control.     

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.     

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.