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

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 BacLight, 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.

Surface sterilization for isolation of endophytes: Ensuring what (not) to grow

“…In the past few years, the plant-endophyte association has gained immense popularity owing to its widespread applications such as enhancing plant growth and development, alleviation of biotic and abiotic stresses, and production of economically important plant metabolites [4][5][6][7]. Different mechanisms of the endophytes could also be useful against pathogens with variable virulence behavior [8]. Endophytes produce an array of bioactive compounds having pharmaceutical importance like taxol [9], vinblastine, vincristine [10], ethyl acetate [11], camptothecin [12], podophyllotoxin [13], and so forth.…”

“…The NaOCl is used at a concentration of 1%-5% for surface-sterilization of plant tissues [20,55] and is commonly used in combination with other chemicals. However, some of the reports indicated the use of NaOCl alone for surface sterilization [3,8,69]. Zinniel et al [121] studied endophytes from soybean, wheat, and prairie plants in which leaves and stem tissue were surface sterilized using 2% NaOCl containing 0.1% Tween 20 for 10 s followed by SDW wash.…”

“…Zinniel et al [121] studied endophytes from soybean, wheat, and prairie plants in which leaves and stem tissue were surface sterilized using 2% NaOCl containing 0.1% Tween 20 for 10 s followed by SDW wash. Likewise, Sahu et al [3,8] reported the use of 2% NaOCl containing 0.1% Tween 20 for 10 s for isolation of bacterial endophytes from tomato and Ocimum tenuiflorum. Similar protocols were endorsed by some other workers [69,122].…”