Evaluation of inhibitory potential of rhizosphere and endophytic bacteria againstErwinia carotovora

Erwinia carotovora is a pathogen of many important plant species and infects a large number of ornamental and agricultural plants such as corn, rice, tomatoes, onions, sugar beets and vegetables. This bacterium is one of the most important factors that greatly reduces crop yields by causing soft rot in the stems and tubers of potatoes before and after harvest. Although the disease has been reported in most potato-growing areas of the country, no environmentally friendly control measure has been taken to prevent this disease. Therefore, identifying the biological factors effective in controlling soft rot disease, especially in agricultural hotspots such as Golestan province, seems to be essential. This study aimed to isolate and identify rhizosphere and endophytic bacteria with antagonistic activity against E. carotovora in potato fields of Marzankola and Fazelabad villages of Gorgan city located in Golestan province.

Soil, root, leaf and stem samples of potatoes were cultured on nutrient agar medium and after incubating at 26°C for 3 -5 days the grown colonies were morphologically examined. After purification of the colonies, the initial identification of the isolates was performed by Gram staining and the ability to produce spores. The antagonistic activity of the isolates was determined by agar well diffusion method. In this method, the plates were incubated at room temperature for 3-5 days and the growth inhibition zone was measured around the wells. Chloroform test was used to evaluate the production of antimicrobial agent by antagonist isolates. Catalase and protease sensitivity tests were used to determine the nature of the antimicrobial agent. Then, the thermal stability of the antimicrobial agent and the effect of pH on its inhibitory activity were measured. Isolates with more antagonistic activity were identified based on 16S rRNA sequencing.

A total of 42 bacterial strains were isolated from roots (13), leaves (7), stems (5) and rhizosphere soil (17). Among the isolates, 33 isolates were Gram-positive and 9 isolates were Gram-negative. Based on the results of antagonistic activity of isolates, the 8 isolates showed antagonistic activity against E. carotovora, all of which were Gram-positive spore-forming bacilli and belonged to the genus Bacillus. The inhibitory activity of the isolates was consistent with the results obtained from the chloroform test. That is, compared to the control, the isolate with a greater amount of antimicrobial agent had the larger growth inhibition zone. In the catalase test, it was found that the antimicrobial substance produced by the isolates is of a nature other than hydrogen peroxide and it was inactivated after treatment with proteases, indicating their protein nature. The inhibitory activity of the supernatant of these isolates was lost at 100 ° C and had the best inhibitory effect at pH 7 to 8. The isolates that had the highest inhibitory activity against E. carotovora were identified based on the sequence of 16 S rRNA gene. After sequencing, these isolates were found to be more than 90% similar to Bacillus subtilis strain ZWQ-1 and Bacillus Mycoides strain 29B-B9, respectively.

Considering that in the present study, the isolates with the best inhibitory effect against E. carotovora belonged to the genus Bacillus, it seems that the antagonistic potential of these bacteria can be used for biological control of plant diseases, of course, to better judge about their performance, it is necessary to conduct further experiments in greenhouse and field conditions.