A Simplified Protocol for Reversing Phenotypic Conversion of Ralstonia solanacearum during Experimentation

Pramod Kumar Sahu, Shailendra Singh, Amrita Gupta, Udai B. Singh, Surinder Paul, Diby Paul, Pandiyan Kuppusamy, Harsh V. Singh and Anil Kumar Saxena
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Pramod Kumar Sahu: ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan UP-275103, India
Shailendra Singh: ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan UP-275103, India
Amrita Gupta: ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan UP-275103, India
Udai B. Singh: ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan UP-275103, India
Surinder Paul: ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan UP-275103, India
Diby Paul: Pilgram Marpeck School of Science, Technology, Engineering and Mathematics, Truett McConnel University, 100 Alumni Dr. Cleveland, GA 30528, USA
Pandiyan Kuppusamy: ICAR-Central Institute for Research on Cotton Technology, Ginning Training Centre, Nagpur, Maharashtra 440023, India
Harsh V. Singh: ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan UP-275103, India
Anil Kumar Saxena: ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan UP-275103, India

IJERPH, 2020, vol. 17, issue 12, 1-15

Abstract: Background: Ralstonia solanacearum has the problem of losing the virulence in laboratory conditions, during prolonged experimentation. Since pure colonies of R. solanacearum contain cell fractions differing in virulence, it was considered worthwhile to find a way of selecting the cells with lower attenuation. Therefore, a methodology for inducing virulent-type colonies occurrence in Ralstonia solanacearum was developed. Methods: Nutrient gradient was created by swabbing R. solanacearum culture in a slanted KMTTC medium, and Phyllanthus emblica extract was given by well diffusion. Live–dead cell imaging using Bac Light, effects of ascorbic acid on cell viability, and production of virulence factors (exopolysaccharides, cellulase, and pectinase) supported this hypothesis. The tagging of R. solanacearum with green fluorescent protein and further confocal scanning laser microscopic visualization confirmed the colonization in vascular bundles of tomato. Results: P. emblica extract suppressed R. solanacearum initially in well diffusion, but further developed virulent-type colonies around the wells. Nutrient deprivation was found to have synergistic effects with P. emblica extract. The converted fluidal (virulent type) colonies could be able to colonize vascular bundles and cause wilting symptoms. Conclusion: This method will be useful in the laboratories working on biocontrol of R. solanacearum for maintaining virulent-type colonies. Moreover, it could form the basis for studies on the stability of phenotypic conversion and cell fractions in R. solanacearum .

Keywords: Ralstonia solanacearum; virulence; virulence induction protocol; Phyllanthus emblica; nutrient deprivation (search for similar items in EconPapers)
JEL-codes: I I1 I3 Q Q5 (search for similar items in EconPapers)
Date: 2020
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