Bactericidal activity of some plant essential oils against Ralstonia solanacearum infection

Potato plants and their tubers in Egypt are affected by one of the most renowned soil-borne pathogen, Ralstonia solanacearum, that caused brown rot in potato tubers and wilt in plants. There is no efficient therapeutic bactericide so; control of bacterial wilt is very rough.The study investigated three different concentrations of seven essential plant oils under in vitro and in vivo conditions as a result of their effects on Ralstonia solanacearum growth and their possibility use as potato seed pieces dressing for controlling bacterial wilt disease incidence. In vitro, anise oil at the three tested different concentrations (0.04, 0.07, and 0.14% vol/vol) was the most effective one inhibiting the growth of T4 and W9 isolates of Ralstonia solanacearum then pursued by thyme, lemongrass, and clove oils. On the other hand, rocket oil at the tested concentration was the least effective one followed by fennel oil. However, wheat germ oil was not completely effective. In vivo, experiment revealed that anise oil at the three concentrations significantly reduced disease incidence and severity in sponta and hermes potato cultivars and their effect was associated with increase of peroxidase, polyphenoloxidase, phenols and the foliar fresh weight of treated plants as well as the weight of tubers/plant followed by thyme and lemongrass oils compared to the infected untreated control.Morphological differences in bacterial cell structure have been observed using a transmission electron microscope (TEM). Anise oil at higher concentration caused of cell wall rupture and degraded cellular components.     

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.     

Eco-friendly application of nano-chitosan for controlling potato and tomato bacterial wilt

Bacterial wilt is one of the main diseases of Solanum spp., which caused by Ralstonia solanacearum (RS), formerly known as Pseudomonas solanacearum. Different concentrations of chitosan nanoparticles have been evaluated as one of the alternative methods of disease management in vitro and in vivo to reduce the risks of pesticide residues. Results in vitro experiment indicated that RS5 isolate was the most virulence one compared to RS1 and RS3. Increasing concentration of nano-chitosan, lead to increase inhibition zone, and this was observed at higher concentrations (100 and 200 µg/ml). In vivo results showed the highest concentration of spraying chitosan nanoparticles increase percentage reduction of disease incidence and severity, in effected potato and tomato plants. Recorded data of disease incidence and severity in treated potato plants were 78.93% and 71.85%, while on tomato plants were 81.64% and 77.63%, respectively compared to untreated infected potato plants were recorded 15.38%, 20.87%, and tomato plants were 20.98% and 28.64%. Results also revealed that 100 µg/ml of chitosan nanoparticles the lowest treatments used as soil amended curative treatments led to incease percentage reduction of disease incidence and severity, respectively on potato and tomato plants, but less than preventive treatment. The results registered that on potato plant were 54.93% and 52.65%, whilst recorded on tomato plants were 59.93% and 56.74%. Transmission electron microscopy (TEM) micrpgraphs illustrated that morphological of healthy R. solanacearum cells were undesirably stained with uranyl. The electron-dense uranyl acetate dye was limited to the cell surface slightly than the cytoplasm, which designated the integrity of the cell film of healthy cells. While bacterial cells treated with nano-chitosan, showed modification in the external shape, such as lysis of the cell wall and loss of cell flagella. Also, the result of using Random amplified polymorphic DNA (RAPD)-PCR observed that differences in treated Ralstonia solanancearum genotype by nano-chitosan compared to the genotype of the same untreated isolate.     

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.     

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.     

Genetic structure and phylogenetic relationships of Ralstonia solanacearum strains from diverse origins in Guangdong Province,China

Bacterial wilt, caused by Ralstonia solanacearum species complex is a key yield‐limiting factor on crops in Guangdong province, China. The genetic diversity of 110 R. solanacearum strains collected from 16 host plants in different areas of Guangdong province was analysed using biovar and phylotype classification schemes. Of 110 strains, fifty‐five strains belong to biovar 3, fifty‐two strains belong to biovar 4, two strains belong to biovar 2 and one strain belonged to biovar 1. Phylotype‐specific multiplex PCR showed that 108 strains belonged to phylotype I (biovars 1, 3, 4) and two strains belonged to phylotype II (biovar 2). The result of phylogenetic relationships analysis based on egl gene sequences demonstrated that 108 strains of phylotype I were grouped into nine previously described sequevars and a new sequevar 57, and two strains of phylotype II were grouped into sequevar 1. Sequevars 15, 34 and 44 widely distributed in Guangdong were predominant sequevars. Sequevar 45 was first reported on potato and pumpkin in China. These results revealed the genetic structure and phylogenetic relationships of R. solanacearum population in Guangdong and will be helpful in bacterial wilt‐resistance breeding.     

Developing a bacteriophage cocktail for biocontrol of potato bacterial wilt

Bacterial wilt is a devastating disease of potato and can cause an 80% production loss. To control wilt using bacteriophage therapy, we isolated and characterized twelve lytic bacteriophages from different water sources in Kenya and China. Based on the lytic curves of the phages with the pathogen Ralstonia solanacearum, one optimal bacteriophage cocktail, P1, containing six phage isolations was formulated and used for studying wilt prevention and treatment efficiency in potato plants growing in pots. The preliminary tests showed that the phage cocktail was very effective in preventing potato bacterial wilt by injection of the phages into the plants or decontamination of sterilized soil spiked with R. solanacearum. Eighty percent of potato plants could be protected from the bacterial wilt (caused by R. solanacearum reference strain GIM1.74 and field isolates), and the P1 cocktail could kill 98% of live bacteria spiked in the sterilized soil at one week after spraying. However, the treatment efficiencies of P1 depended on the timing of application of the phages, the susceptibility of the plants to the bacterial wilt, as well as the virulence of the bacteria infected, suggesting that it is important to apply the phage therapy as soon as possible once there are early signs of the bacterial wilt. These results provide the basis for the development of bacteriophagebased biocontrol of potato bacterial wilt as an alternative to the use of antibiotics.

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Genetic and pathogenic diversity of Ralstonia solanacearum species complex strains isolated in Burkina Faso

Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is a disease that negatively affects the cultivation of Solanaceae crops in Burkina Faso. Knowledge of the pathogen diversity is essential to deploy locally adapted control methods. In this study, diseased plants showing typical bacterial wilt symptoms were collected in the three main agroclimatic zones of Burkina Faso for the detection of RSSC isolates. Strain characterization was achieved through a phylogenetic and pathogenicity diversity assessment. A total of 102 isolates were sampled, and Phylotype I (Ralstonia pseudosolanacearum) was predominant (n = 101; sequevars 14, 31, 34, and 46). The remaining isolate was characterized as Phylotype IIA-35 (Ralstonia solanacearum). Phylotypes I-31 and I-46 were predominant and both characterized as the most the aggressive group of strains amongst a subset of 33 representative isolates. Our findings provide valuable information as regard RSSC diversity that breeders and resistance programme should target in order to fight this pathogen in Burkina Faso and around the world.     

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.     

Evaluation of rhizosphere bacterial antagonists for their potential to bioprotect potato (Solanum tuberosum) against bacterial wilt (Ralstonia solanacearum)

Bacterial wilt (Ralstonia solanacearum) is one of the production constraints of potato (Solanum tuberosum). The intent of the study was to evaluate potential of bacterial antagonists to suppress bacterial wilt disease development and evaluate the role of the strains as plant growth-promoting rhizobacteria (PGPR) in potato. One hundred-twenty rhizosphere bacterial isolates were screened against virulent strain of Ralstonia solanacearum PPRC-Rs. After in vitro screening, six antagonistic strains (PFMRI, BS-DFS, PF9, PF20, BC, and BS-wly) with inhibition diameter >11 mm were selected and studied further in the greenhouse, in vivo. During in vivo study, the strains were evaluated for their effect in suppressing disease development in terms of area under disease progress curve (AUDPC) and increasing biomass (plant height and dry weight) of potato. Accordingly, PFMRI, BS-DFS, and PF9, significantly reduced AUDPC by 78.6, 66, and 64.3%, and wilt incidence by 82.7, 66.2, and 65.7%, respectively, compared to the control. During the sole application, the strains significantly (P < 0.0001) increased plant height by 35.6, 45.9, and 45%, and dry matter by 111, 130.4, and 129%, respectively compared to non-bacterized control. In the presence of the pathogen strain PFMRI, BS-DFS, and PF9 increased plant height by 66, 50, and 48.2%, and dry matter by 153.8, 96.8, and 92.5%, respectively compared to the pathogen treated control. Hence, the study shows that PFMRI, BS-DFS, and PF9 strains have potential use in potato bioprotection, as PGPR or in an integrated bacterial wilt management; whose effectiveness under a variety of field conditions should be investigated.     

Detection of Ralstonia solanacearum Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5′ nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to ≥10² cells ml⁻¹ was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.     

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.     

Introgression of bacterial wilt resistance from eggplant to potato via protoplast fusion and genome components of the hybrids

Key message

Bacterial wilt resistant somatic hybrids were obtained via protoplast fusion between potato and eggplant and three types of nuclear genomes were identified in the hybrids through GISH and SSR analysis.

Abstract

Cultivated potato (Solanum tuberosum L.) lacks resistance to bacterial wilt caused by Ralstonia solanacearum. Interspecific symmetric protoplast fusion was conducted to transfer bacterial wilt resistance from eggplant (S. melongena, 2n = 2x = 24) into dihaploid potato (2n = 2x = 24). In total, 34 somatic hybrids were obtained, and of these, 11 rooted and were tested for genome components and resistance to race 1 of R. solanacearum. The hybrids exhibited multiple ploidy levels and contained the dominant nuclear genome from the potato parent. Three types of nuclear genomes were identified in the hybrids through genomic in situ hybridization (GISH) and simple sequence repeat (SSR) analysis, including (1) the potato type of the tetraploids in which eggplant chromosomes could not be detected by GISH but their nuclear DNA was confirmed by SSR, (2) the biased type of the hexaploids in which the chromosome dosage was 2 potato:1 eggplant, and (3) the chromosome translocation type of the mixoploids and aneuploids that was characterized by various rates of translocations of nonhomologous chromosomes. Cytoplasmic genome analysis revealed that mitochondrial DNA of both parents coexisted and/or recombined in most of the hybrids. However, only potato chloroplast DNA was retained in the hybrids speculating a compatibility between cpDNA and nuclear genome of the cell. The pathogen inoculation assay suggested a successful transfer of bacterial wilt resistance from eggplant to the hybrids that provides potential resistance for potato breeding against bacterial wilt. The genome components characterized in present research may explain partially the inheritance behavior of the hybrids which is informative for potato improvement.     

Role of Trehalose Synthesis in Ralstonia syzygii subsp. indonesiensis PW1001 in Inducing Hypersensitive Response on Eggplant (Solanum melongena cv. Senryo-nigou)

Ralstonia syzygii subsp. indonesiensis (Rsi, former name: Ralstonia solanacearum phylotype IV) PW1001, a causal agent of potato wilt disease, induces hypersensitive response (HR) on its non-host eggplant (Solanum melongena cv. Senryo-nigou). The disaccharide trehalose is involved in abiotic and biotic stress tolerance in many organisms. We found that trehalose is required for eliciting HR on eggplant by plant pathogen Rsi PW1001. In R. solanacearum, it is known that the OtsA/ OtsB pathway is the dominant trehalose synthesis pathway, and otsA and otsB encode trehalose-6-phosphate (T6P) synthase and T6P phosphatase, respectively. We generated otsA and otsB mutant strains and found that these mutant strains reduced the bacterial trehalose concentration and HR induction on eggplant leaves compared to wild-type. Trehalose functions intracellularly in Rsi PW1001 because addition of exogenous trehalose did not affect the HR level and ion leakage. Requirement of trehalose in HR induction is not common in R. solanacearum species complex because mutation of otsA in Ralstonia pseudosolanacearum (former name: Ralstonia solanacearum phylotype I) RS1002 did not affect HR on the leaves of its non-host tobacco and wild eggplant Solanum torvum. Further, we also found that each otsA and otsB mutant had reduced ability to grow in a medium containing NaCl and sucrose, indicating that trehalose also has an important role in osmotic stress tolerance.     

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.     

Role of hydrogen peroxide and ascorbate-glutathione pathway in host resistance to bacterial wilt of eggplant

Ralstonia solanacearum, a soil-borne bacterium causes bacterial wilt, is a lethal disease of eggplant (Solanum melongena L.). However, the first line of defense mechanism of R. solanacearum infection remains unclear. The present study focused on the role of induced H₂O₂, defense-related enzymes of ascorbate-glutathione pathway variations in resistant and susceptible cultivars of eggplant under biotic stress. Fifteen cultivars of eggplant were screened for bacterial wilt resistance, and the concentration of antioxidant enzymes were estimated upon infection with R. solanacearum. A quantitative real-time PCR was also carried out to study the expression of defense genes. The concentration of H₂O₂ in the pathogen inoculated seedlings was two folds higher at 12 h after pathogen inoculation compared to control. Antioxidant enzymes of ascorbate-glutathione pathway were rapidly increased in resistant cultivars followed by susceptible and highly susceptible cultivars upon pathogen inoculation. The enzyme activity of ascorbate-glutathione pathway correlates by amplification of their defense genes along with pathogenesis-related protein-1a (PR-1a). The expressions of defense genes increased 2.5?3.5 folds in resistant eggplant cultivars after pathogen inoculation. The biochemical and molecular markers provided an insight to understand the first line of defense responses in eggplant cultivars upon inoculation with the pathogen.     

The Relation of Different Crop Roots Exudates to the Survival and Suppressive Effect of Stenotrophomonas maltophilia (PD4560), Biocontrol Agent of Bacterial Wilt of Potato

Experiments were performed under greenhouse conditions to control bacterial wilt of potato (potato brown rot), caused by Ralstonia solanacearum race 3 biovar 2, Phylotype II, sequevar 1 using various biocontrol strategies. These strategies involved the use of the bacterial biocontrol agent Stenotrophomonas maltophilia (PD4560), in clay or sandy soils, planted with cowpea, maize or tomato which was grown separately in different pots in the inoculated soils. After harvest, the soil derived from each cultivated crop was inoculated with a mixture of three virulent R. solanacearum strains (K3, K10 and K16) to achieve a final concentration of 5 × 10⁸ cfu/g dry soil and used in pots under greenhouse conditions to cultivate potato seed tubers. The highest survival of S. maltophilia in soil (more than 160 days) coincided with a remarkable suppressing effect on disease incidence caused by R. solanacearum that expressed by wilt severity (up to 100% reduction), area under disease progress curve (AUDPC) (up to 99% reduction) and counts of the pathogen in soil (up to 75% reduction), rhizosphere (up to 80% reduction) and plant tissue (up to 97% reduction) of potato plants. The amino acid analysis of root exudates of crops under investigation revealed high percentages of asparagines (15.5–21%), glutamine (16–20%) and sulphur‐containing methionine (7–9%) in both of the cowpea and maize, respectively. In tomato root exudates, high percentages of arginine (around 26%) and lysine (around 23%) were detected. Methionine is known to favour the growth of S. maltophilia suggesting that especially cowpea and maize are suitable for crop rotation with potato and will enhance the sustainability of the biocontrol agent S. maltophilia.     

Detection,Identification and Phylogenetic Analysis of Indian Ralstonia solanacearum Isolates by Comparison of Partial hrpB Gene Sequences

A species‐specific Polymerase Chain Reaction (sPCR) method was developed to identify and detect isolates of Ralstonia solanacearum, the cause of bacterial wilt disease in chilli. PCR primers for R. solanacearum were identified by alignment of hrpB gene sequences and selection of sequences specific for R. solanacearum at their 3′ ends. The primers were shown to be specific for R. solanacearum, as no PCR product was obtained when genomic DNA from other bacterial species including closely related Ralstonia species, were used as test species. Lone pair of primers (RshrpBF and RshrpBR) was designed using hrpB gene sequence, unique to R. solanacearum which amplified a predicted PCR product of 810 bp from 20 different isolates. Phylogenetic analysis was also attempted to understand the evolutionary divergence of Indian R. solanacearum isolates. Based on phylogenetic analysis, Indian isolates showed homology with the standard reference isolates from other countries but, interestingly, one new isolate showed complete evolutionary divergence by forming an out‐group.     

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.     

Evaluation of rhizosphere bacteria and derived bio-organic fertilizers as potential biocontrol agents against bacterial wilt (Ralstonia solanacearum) of potato

Aims

Potato bacterial wilt (Ralstonia solanacearum) is a soil-borne disease that affects the potato plant (Solanum tuberosum) worldwide and causes serious economic losses in southern China. The objective of this study is to study the effect of bacterial antagonists and bio-organic fertilizers on potato bacterial wilt and rhizosphere soil microbial population.

Methods

In the present study, pot and field experiments were conducted to evaluate the LH23 (Bacillus amyloliquefaciens) and LH36 (Bacillus subtilis) strains and their derived bio-organic fertilizers (BIO23 and BIO36) as potential biocontrol agents against potato bacterial wilt.

Results

BIO23 and BIO36 decreased the incidence of bacterial wilt disease and increased potato yields. In pot experiments, the disease incidence of BIO23 and BIO36 was 8.9 % and 11.1 % respectively, much lower than the control (57.7 %). The biocontrol efficiency of BIO23 was 84.6 %, which was the most successful treatment and BIO36 was the second with a biocontrol efficiency of 80.8 %. The increased percentages of potato yields when compared with the control were 63.5 % (BIO23), 64.7 % (BIO36) 34.8 % (LH23), 33.6 % (LH36) and 20.7 % (OF). The counts of antagonists, bacteria and actinobacteria in the rhizosphere soil were significantly increased in BIO23 and BIO36 treatments, whereas the counts of R. solanacearum and fungi in the soil in the both treatments decreased. In field experiments, 70 days after treatment, the biocontrol efficacies of BIO23 and BIO36 treatments were 92.0 % and 84.0 %, and the yield increases of BIO23 and BIO36 treatments were 42.3 % and 28.8 %, respectively, when compared with the organic fertilizer treatment. In addition, the changes in the microbial populations were the same as those observed in the greenhouse experiment.

Conclusions

Potato bacterial wilt could be well controlled by the application bio-organic fertilizer containing a specific antagonist, mainly through the alternation of soil microbial community