Detection of Polymyxa betae in sugar beet roots using microscopical, serological and molecular methods

Abstract

In order to design a precise diagnostic and quantitative system to detect of P. betae in sugar beet roots, three procedures including microscopical, serological and molecular methods were compared on a sugar beet cultivar susceptible to causal agent and vector of rhizomania disease. At first, susceptible cultivar Regina was cultivated in infested soil in greenhouse condition. After 5 weeks, roots of plants were stained with fuchsin- acid- lactophenol solution. Quantitative ELISA test was optimized with specific antiserum against recombinant protein glutathione s- transferase (GST) of P. betae. Running ELISA test, extracts of health and infected plants were prepared and used in DAS- ELISA test. Presence of P. betae in roots of plants was detected with amplification of rDNA of Plasmodiophoromycetes and P. betae species, simultaneously. Existence of cystosori of P. betae was observed in all of the plants planting in infested soil. Results of ELISA test were distinguished healthy plants from infected plants. Average of optical density at 405 nanometer for healthy plants, infected plants, protein PBS (negative control) and GST (positive control) was 0.126, 0.75, 0.11 and 2.45 respectively. Duplex PCR method was amplified two fragments of 454bp and 170bp in infected samples relating to rDNA region and specific region of the P. betae, respectively. Based on the results, it seems that, rapid detection and identification of P. betae in sugar beet and other hosts of the vector could be done on the basis of PCR method. Evaluating of resistance germplasm of sugar beet to P. betae could be recognized using quantitative ELISA tests.

Keywords

• rhizomania
• glutathione- s- transferase
• elisa
• pcr
• its