Browsing by Author "Ajayasree, T S"

Filter results by typing the first few letters
Now showing 1 - 1 of 1
  • No Thumbnail Available
    Item
    Characterization and management of bacterial wilt of solanaceous vegetable in relation to physico-chemical and biological properties of soil
    (Department of Plant Pathology, College of Agriculture, Vellanikkara, 2025-11-15) Ajayasree, T S; Sible, George Varghese
    Solanaceous vegetables viz., tomato, chilli, and brinjal are widely cultivated in different agro-ecological units (AEUs) in Kerala. However, their cultivation is hampered by bacterial wilt disease. Being a soil-borne pathogen, the growth and survival of this bacterium may be greatly influenced by physico-chemical and biological properties of soil. Hence, the present investigation was carried out with the objectives of isolation and characterization of the bacteria responsible for bacterial wilt of solanaceous vegetables from four selected AEUs of Kerala; which were not studied earlier, to study the soil physico-chemical and biological properties influencing bacterial pathogen and to develop an integrated management strategy for bacterial wilt disease. A purposive sampling survey was conducted in four AEUs of Kerala viz., Southern Central Laterites (SCL), Southern and Central Foothills (SCFH), Northern Laterites (NL), and Northern Foothills (NFH). Three locations from each of these four AEUs were surveyed and samples of diseased plants and rhizosphere soil were collected during August to November 2019. Rhizosphere soil of healthy plants were also collected from each location. The per cent disease incidence in different locations ranged from 20.40 to 56.60 per cent. The bacterial wilt pathogen was isolated from infected plants using tetrazolium chloride (TZC) agar and Koch’s postulates were proved by raising seedling in polybags and further confirmed by inoculating seedlings in protrays. The pathogen population in the rhizosphere ranged from 1.33 to 102.67 x 106 cfu g-1 soil. The 12 isolates of bacterial pathogen collected from these locations were purified and maintained in sterile water for further experiments. Different isolates exhibited considerable variation in colony size and pigmentation. All the isolates were gram negative and scanning electron microscopy (SEM) revealed that, the bacterial cells are rod shaped with different sizes such as 0.47 to 0.69 µm x 1.46 to 1.94 µm in the case of NL 2; 0.50 to 0.52 µm x 1.48 to 1.69 µm in case of NL 1 and 0.52 to 0.59 µm x 1.50 to 1.94 µm in case of SCL 2. The molecular characterization of the isolates by 16S rRNA sequencing revealed that there are five different bacterial wilt pathogens present in four different AEUs of Kerala. The isolate SCL 1 was identified as Klebsiella variicola; SCL 2, SCFH 1, SCFH 2, and NL 3 were identified as Klebsiella pneumoniae; NFH 3, NL 2, SCFH 3, and SCL 3 were identified as Enterobacter mori. Further, NFH 1 and NL 1 were identified as Klebsiella oxytoca and NFH 2 was identified as Enterobacter cloacae. Phylogenetic analysis revealed that, the different isolates clustered together on the basis of the genus and species to which it belongs. However, due to absence of barcode gaps between Klebsiella and Enterobacter spp., at genus level using 16S rRNA sequencing, specific primer based molecular characterization by sequencing housekeeping genes rpoB, gyrB and hsp60 was carried out. In-silico analysis of rpoB gene sequences confirmed the identity of 11 isolates. Interestingly, SCFH 1, SCFH 2, SCFH 3, SCL 1, SCL 3 and NFH 2 were identified as Enterobacter sp. Whereas, NL 3, NFH 1 and NFH 3 were identified as Klebsiella sp. Moreover, SCL 2 and NL 1 were identified as Pantoea sp. In-silico analysis of gyrB gene sequences confirmed the identity of eight isolates. SCFH 1, SCFH 2, SCFH 3, SCL 1, SCL 3 and NFH 2 were identified as Enterobacter sp. Moreover, SCL 2 and NL 1 were identified as Pantoea sp. Similarly, In-silico analysis of hsp60 gene sequences also confirmed the identity of eight isolates. NL 3, NFH 1 and NFH 3 were identified as Klebsiella sp. Further, SCL 1, SCFH 1, SCFH 2 and SCFH 3 isolates were identified as Enterobacter sp. and SCL 2 isolate was identified as Pantoea sp. Concatenated phylogenetic analysis of rpoB, gyrB and hsp60 gene analysis depicted the grouping of different genera viz., Klebsiella sp., Enterobacter sp. and Pantoea sp. separately in different clusters. However, there were shift between different genera viz., Klebsiella to Pantoea sp., Klebsiella to Enterobacter sp. and Enterobacter to Klebsiella sp. within Enterobacteriaceae family while identifying using 16SrRNA and rpoB, gyrB, hsp60 gene sequencing. This indicates close genetic relatedness of these genera. This also indicates considerable genetic variation among the isolates present in different locations of AEUs. The isolates of these bacterial wilt pathogens were tested for pathogenicity on the reported host plants viz., tomato, brinjal, chilli, banana, potato, ginger and mulberry. However, the isolates infected only tomato, brinjal and chilli but could not infect the other host plants tested. These isolates were tested for their ability to utilize disaccharides and hexose-alcohols too. The isolates belonging to the same genus and species showed almost similar carbohydrate utilization patterns. The physico-chemical and biological properties of the soil samples collected were studied using standard protocols. Statistical analysis using paired sample t-test revealed that, there was no significant difference in chemical properties viz., pH, organic carbon, available P, K, Ca, Fe and Mg content between rhizosphere soil of healthy and diseased plants. Similarly, there was no significant difference in the physical properties of rhizosphere soil of healthy and diseased plants within the same location except for core bulk density. However, there is statistically significant difference in population of actinomycetes and fluorescent pseudomonads in rhizosphere of healthy and diseased soil. Moreover, dehydrogenase and catalase activity also showed a statistically significant difference between healthy and diseased plant rhizosphere soil. Metagenomic studies revealed that, rhizosphere soil of diseased plants has very high pathogen population compared to that of the healthy plants. However, bacterial diversity and richness was less in rhizosphere soil of wilted plants while that of healthy plants. Moreover, the microbial community structure in healthy and sick soil also showed difference. Even though three bacterial genera (Klebsiella, Enterobacter, Ralstonia) causing bacterial wilt disease were found in the soil of bacterial wilt infected and healthy plant’s rhizosphere, the bacterial population of Klebsiella genus was only significantly higher in diseased rhizosphere soil (SCFH1D) as compared with that of (SCFH1H) rhizosphere soil of the healthy plant. This suggests that an increase in the relative abundance of phytopathogenic bacteria K. pneumoniae is the cause of bacterial wilt disease in chilli. These findings are also evidently supported by the molecular characterization results of the bacterial wilt pathogen (SCFH1 isolate) obtained from this location which is also identified as K. pneumoniae based on 16S rRNA gene sequencing. These studies also revealed that some beneficial microbes were present in the rhizosphere of healthy chilli plant, however, shifts may occur between the rhizosphere bacterial flora of bacterial wilt infected and healthy plant’s rhizosphere. To develop an integrated management strategy for bacterial wilt disease, field experiments were conducted in a bacterial wilt sick plot at the Department of Vegetable Science, CoA, Vellanikkara. The 12 different treatments in the field experiment included application of bleaching powder, soil test-based lime application, seedling root dip in streptocycline, drenching of copper hydroxide, drenching of copper oxychloride, seed treatment and seedling dip with Pseudomonas fluorescens, drenching of P. fluorescens, planting grafted plants of tomato and other integration treatments. The results of the study revealed that to manage the bacterial wilt of tomato, an integration of different treatments viz., application of bleaching powder (15 kg/ ha) + soil test- based lime application + streptocycline (@ 2g/10L) root dip + drenching of copper oxychloride @ 0.3 per cent is required. Efficacy of silver nanoparticles against bacterial wilt pathogen was tested under in-vitro conditions and it was found that there is no antibacterial property against bacterial wilt pathogens Klebsiella sp., Enterobacter sp. and Pantoea sp. The study revealed that in all 12 different locations surveyed in four selected agro-ecological units, Klebsiella sp., Enterobacter sp. and Pantoea sp. cause bacterial wilt of solanaceous vegetables. There is statistically significant difference in core bulk density and various biological properties of rhizosphere soil of healthy and diseased plants. Metagenomic analysis revealed that, the density and diversity of bacterial population in rhizosphere soil of healthy plants are much higher compared to that of diseased plants. To manage bacterial wilt of tomato, integration of application of bleaching powder (15 kg/ ha) + soil test-based lime application + streptocycline (@ 2g/10L) root dip + drenching of copper oxychloride @ 0.3 per cent was found suitable.