D.K. Yadav

Potential of Bacillus amyloliquefaciens for Biocontrol of Bacterial Wilt of Tomato Incited by Ralstonia solanacearum

Fifty seven rhizobacteria were isolated from rhizospheric soil of wilted tomato plants and among them two strains of rhizobacteria, having better antagonistic and plant growth promoting ability were characterized them as Bacillus amyloliquefaciens DSBA-11 and DSBA-12 based on morphological, biochemical, partial gene sequence analysis of 16S rRNA and fatty acid methyl ester analysis. Antagonistic activity of these strains DSBA-11, DSBA-12 was compared with other Bacillus species such as B. subtilis DTBS-5, B. cereus JHTBS-7, B. pumilus MTCC-7092 strains, against Ralstonia solanacearum race 1, bv 3, phylotype I, inciting bacterial wilt of tomato underin vitro conditions. B. amyloliquefaciens DSBA-11 showed maximum growth inhibition of R. solanacearum (4.91cm2) followed by strains DSBA-12 (3.31cm2) and B. subtilis (3.07 cm2). Moreover, strains DSBA-11 was also have better phosphorus solubilizing ability (42.6 μg/ml) and indole acetic acid (95.4 μg/ml) production than other strains of Bacillus spp. in vitro conditions. Biocontrol efficacy and plant growth ability of these bacterial antagonists was tested against bacterial wilt of tomato cv. Pusa Ruby under glasshouse conditions. Minimum bacterial wilt disease incidenceincultivar Pusa Ruby (17.95%) was recorded in B. amyloliquefaciens DSBA-11followed by B. amyloliquefaciens DSBA-12 after 30 days of inoculation.The bio-control efficacy was higher in B. amyloliquefaciens DSBA -12 treated plants, followed by B. pumilus MTCC- 7092.

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.

Genetic diversity of iturin producing strains of Bacillus species antagonistic to Ralstonia solanacerarum causing bacterial wilt disease in tomato

Bacillus spp. is a potential bacterial antagonist to manage bacterial wilt disease of tomato incited by Ralstonia solanacearum, which is one of the most threatening diseases of tomato in India. Genetic diversity of Bacillus strains and their potentiality to control bacterial wilt of tomato isolated from rhizospheric soil and endophytic tomato plants from different agro-climatic regions of India were studied. Rhizospheric soil and plants of tomato were pasteurized at 80°C for 15 min before dilution and then inoculated onto the Petri plates containing tryptic soy agar medium and incubated at 28± 2°C. Out of 250 isolates of Bacillus species, 47 strains showed antagonistic ability against R. solanacearum.

Biological Characterization and Genetic Diversity of Indian Strains of Ralstonia solanacearum Biovars 3 and 4 Causing Bacterial Wilt of Tomato

Ralstonia solanacearum biovars 3 and 4 causing bacterial wilt of tomato (Solanum lycopersicum L.) is a devastating soil borne plant pathogen across the world. Eighty seven isolates of R. solanacearum were isolated from wilted tomato plants from Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Jharkhand, Orissa states of India and characterized them by traditional and molecular methods. Biovar of R. solanacearum was determined using set of carbon sources and it showed that biovar 3 of R. solanacearum were found most prominent (90.2 per cent) in all the states of India, whereas biovar 4 was found in states of Jharkhand and Himachal Pradesh. The phylotype specific multiplex PCR assigned all 87 the isolates of R. solanacearum infecting tomato under phylotype I. To study the genetic diversity, BOX-PCR and multilocus sequence typing approaches were used. Amplification products yielded in BOX-PCR fingerprint pattern ranging from 500 bp -4 kb and found 23 DNA typing groups of 87 isolates of R. solanacearum at 50% similarity coefficient. Under multilocus sequence typing, three virulence genes viz., hrp (regulatory transcription regulation) and egl (endoglucanase precursor) and fli C genes of 18 strains of R. solanacearum belonging to different agro-climatic zones was done. Based on sequence analysis of egl gene, majority of the Indian strains of R. solanacearum were very close to each other except ORT-8, UTT-23 and JHT2 and there were very close to strain GMI1000. A lot of genetic variability was found in Indian isolates of R. solanacearum irrespective of place of isolation and climatic conditions.

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

Abstract 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.

Evaluation of crucifer germplasm for black rot (Xanthomonas campestris pv. campestris) resistance

Black rot is a devastating bacterial disease caused by Xanthomonas campestris pv. campestris (Pam.) Dowson, inflicting 10–50% losses in cauliflower production. Therefore, 41 accessions/varieties of five crucifer species including Brassica oleracea var. botrytis L. (C genome), B. rapa (A genome), B. napus (AC genome), B. carinata (BC genome) and Eruca sativa were screened in field conditions against artificial inoculation of Xcc race 1. Mean disease severity and disease incidence (%) were observed in different accessions/varieties of cauliflower (7.51 to 8.46, 88 to 94), B. rapa (4.81 to 8.8, 66.33 to 98.00), B. carinata (0.07 to 6.48, 7.33 to 87), B. napus (1.46 to 7.33, 31.00 to 96.66) and E. sativa (7.10 to 7.24, 86 to 88.33), respectively. All the varieties/accessions of cauliflower and E. sativa were observed highly susceptible. Three accessions of B. rapa were found to be susceptible and remaining were classified into very susceptible types. Out of 16 accessions of B. carinata screened, 12 were susceptible, two were very susceptible, one was partially resistant and one was fully resistant. While in B. napus, three accessions as partial resistant, seven as susceptible and four were very susceptible against Xcc race 1. Newly identified Xcc resistant and partially resistant/tolerant sources can be used for breeding black rot resistant cole crops by introgressing gene(s) of interest into B. oleracea.