Characterisation of Ralstonia solanacearum (Smith) Yabuuchi et al. infecting solanaceous vegetables in relation to physico-chemical and biological properties of soil

Abstract

Ralstonia solanacearum, the causal agent of vascular wilt disease of crop plants has been ranked as the second most important bacterial pathogen in the world next to Pseudomonas syringae. The high diversity exhibited by the pathogen hampers the breeding for resistance, consequently the resistant varieties developed may not express uniform level of resistance in different geographical locations. Being a soil inhabitant, the survival of R. solanacearum is influenced by physico-chemical and biological properties of soil. Considering these facts, present investigation was carried out with the objective of characterisation of R. solanacearum from different agro ecological units (AEUs) of Kerala and to determine the effect of soil physical, chemical and biological properties on the pathogen. Purposive sampling survey was conducted in four AEUs of Kerala viz., North Central laterite (NCL), Marayur hills (MH), Southern laterite (SL) and Palakkad central plains (PCP) from March to September 2018. Two locations from each AEUs were selected for the survey. The per cent incidence in different locations ranged from 20 to 88 per cent. The pathogen was isolated from infected plant samples collected during the survey on triphenyl tetrazolium chloride (TZC) agar and pathogenicity was proved by inoculation on respective hosts. The rhizosphere soil samples of healthy and diseased plants were collected from each location. The population of the pathogen present in the soil was quantified and it ranged from 20.66 x 104 cfu g-1of soil to 137.66 x 104 cfu g-1 of soil. A strong positive correlation was observed between pathogen density in soil and per cent disease incidence (PDI). A total of eight isolates were collected, purified and maintained for all the experiments. The colony characters of different isolates on TZC agar showed considerable variation and the morphology of the bacterial cells was studied using scanning electron microscopy. Typical rod shaped cells with size 0.3-0.5 µm x 1.2-1.7 µm were observed. The molecular characterization of the isolates was done by PCR amplification and sequencing of 16S rRNA. The sequences were subjected to in-silico analysis which confirmed the identity of all isolates as R. solanacearum (Smith) Yabuuchi et al. The phylogenetic analysis revealed that the eight isolates collected from different AEUs clustered on different branches of the tree while those from the same AEUs clustered together. This indicates considerable variation among the isolates in accordance with location which can be attributed to the difference soil parameters in these locations. The isolates of the pathogen were further categorized into races and biovars based on pathogenicity on differential hosts and utilization of disaccharides and hexose-alcohols respectively. The results revealed that two isolates from Marayur hills (MH 1 and MH 2) belong to race 3, biovar II whereas two from Palakkad central plains (PCP 1 and PCP 2) belong to race 1 biovar III A. The other four isolates collected from Northern central laterite (NCL 1 and NCL 2) and Southern laterite (SL 1 and SL 2) were identified as race 1, biovar III. The physico-chemical and biological properties of the soil samples collected during the survey were analysed using standard protocols. The statistical analysis using paired sample t-test revealed significantly higher soil pH, organic carbon, available K, Ca and Fe content and soil microflora in rhizosphere soil of healthy plant compared to diseased. A significant positive correlation was observed between PDI and soil parameters viz., water holding capacity and bulk density whereas soil pH and available Ca content exhibited a negative correlation with PDI. A similar trend was observed in the case of pathogen population also. Further, multiple regression analysis was performed to assess the extent of variation contributed by different soil parameters on PDI and pathogen population. The results indicate that 96.8 per cent variation in the bacterial wilt incidence is explained by soil pH and available Ca content in the rhizosphere soil with negative correlation whereas bulk density and Ca content contributed 92.2 per cent in the build-up of population of Ralstonia solanacearum in soil. The study revealed the influence of soil factors on bacterial wilt disease incidence, population of R. solanacearum and pathogen variability. Hence, it is concluded that, manipulation of soil factors can play a major role in integrated management of the disease. Furthermore, the variability of pathogen according to geographical region, may be considered while planning resistance breeding programmes against bacterial wilt disease.