A Rapid Pathogenicity assay of Ralstonia Solanacearum in Early Seedling Stage of Brinjal Under Soil Free Medium Niraj Singh Department of Microbiology, Royal Global University , Guwahati, Assam, 781035 Department of Molecular Biology and Biotechnology, Tezpur University, Assam, 784028
Abstract Ralstonia solanacearum causes a lethal bacterial wilt disease in more than 400 plant species that includes many important crops such as tomato, potato, eggplant, peanut, pepper etc. This bacterium lives in soil, when favorable condition comes it enters through root and infects the host plant. This work reporting, standardization of an easy and rapid pathogenicity assay of Ralstonia solanacearum in 10 to 12 days old seedlings of Brinjal( Solanum melongena) by root inoculation approach. Wilting symptom started within 70 hrs post inoculation in pathogen inoculated seedling and it takes only less than three weeks to complete the virulence assay. By using this method, R. solanacearum mutants for different virulence functions such as hrpB, phcA, pilT could be clearly distinguished from the wild type counterpart. Colonization study of bacterium in the infected seedlings performed by gus staining as well as by fluorescent microscopy. This newly standardized method is very much economical in terms of space, labor, cost as well as the quantity of bacterial inoculum required. It can also be very helpful in large scale screening by maintaining natural mode of entry and it may turn out to be very useful and promising system to understand virulence functions and pathogen adaptation.
Introduction
Material and Method
Although several studies have been investigated the virulence of R. solanacearum on adult host plants by using different method such as by soil drenching, stem pricking etc., while infection studies of this pathogen on the seedling stages of hosts are less common, In this work, it have reported an efficient and rapid infection method to assay pathogenicity of this bacterium, which mimics the natural mode of infection. In this pathogenicity assay we inoculated R. solanacearum in 10-12 days old brinjal seedling through root and observed the wilt disease progression in infected seedlings under growth chamber ,through this method disease progression rate was observed very high, wilting symptom started within 70 hrs post inoculation and whole pathogenicity assay require only 3 week for complition. which was subsequently validated on different brinjal cultivars , whereas the mutants like hrpB and phcA , pilT were shown reduced in virulence. In addition to infection assay it can help significantly in understanding intricate molecular mechanism of Ralstonia solanacearum and its host adaptability in near future. It may turn out to be an easy and useful approach in understanding virulence functions of the pathogen in seedling stages of hosts that may mimic pathogenicity mechanisms in the adult plant.
Ralstonia solanacearum(F1C1)
Virulence Assay Of F1C1& Mutant
hrpB mutant of F1C1(hrpB mutant is deficient in type III protein secretion system and in virulence) Genin et al. (1992) phcA mutant of F1C1(phcA mutant is deficient in exopolysaccharide and in virulence) Genin et al. (2005) pilT Mutant (twitching motility and virulence deficient) Liu et al.( 2001) rpoN2 mutant of F1C1( virulence proficient) Ray et al. (2016)
Mutants phenotype of hrpB, phcA,pilT
.
Result F1C1 F1C1
phcA
pilT mutant
F1C1
hrpB
Hyper motility
Hypersensitive response
Twitching motility
Pathogenicity assay in different brinjal cultivar Bacterial wilts disease progression of R.
solanacearum(F1C1) in 10-12 days old Wilts disease progression of R. solanacearum(F1C1) P. putida, B. subtilis and E.coli strain in 10-12 days old brinjal seedlings by root inoculation.
Control
Infected
seedling of four different brinjal cultivar by root inoculation approach, here this graph is clearly indicating that seedling stage assay is highly reproducible in all
four employed brinjal cultivar.
Conclusion & Discussion
Wilts disease progression of R. Gus staining of wilted Colonization study by solanacearum(F1C1) hrpB, phcA and pilT seedling & control fluorescent tag (mcherry) mutants strain in brinjal seedlings.
References
N. Singh, T.Phukan, P.Sarma, K. Kabyashree, A. Barman, R. Kumar, R. V. Sonti , S. Genin and S. Kumar Ray , An innovative root inoculation method to study R. solanacearum pathogenicity in tomato seedling, 2018, 108, Phytopathology(APS ) Niraj Singh (2020) A Rapid Pathogenicity Assay of R. solanacearum in 10-12 Days Old Eggplant (Solanum melongena) Seedlings by Root Inoculation Approach, bioRxiv .
In this study we are reporting a rapid and soil free root inoculation method to study Ralstonia solanacearum pathogenicity in the early seedlings stages of brinjal under gnotobiotic conditions. This approach is also helpful in resistant cultivar screening and biocontrol development at early seedling stage. The method described here is economic in terms of space, labor, cost as well as the quantity of bacterial inoculum required. It may turn out to be an easy and useful approach in understanding virulence functions of the pathogen in seedling stages of hosts that may mimic pathogenicity mechanisms in the adult plant. Acknowledgement I am very much thankful to Prof. Suvendra k. Ray and I am also thankfull to DBT and other organizers for providing me opportunity to present my work.