Protease enzymes from Pseudomonas fluorescens and Pseudomonas aeruginosa as potential bioagents against Pratylenchus loosi

Root lesion nematode of tea (Pratylenchus loosi) is one of the most dangerous and destructive pests in all areas of the world where tea grows. Rhizobacteria with nematicidal properties were screened using the soils of root lesion nematode-infected tea fields located in Guilan province, Iran. Effective bacteria were identified based on molecular and biochemical methods. Protease enzyme production was investigated on skim milk agar (1%) medium. The effects of temperature and pH on the amount of protease production by bacteria were investigated. Three protease inhibitors, namely PMSF, EDTA and 1,10-phenanthroline, were investigated. In total, 150 bacterial strains were isolated from tea rhizosphere using the spread-plating technique and nutrient agar medium, of which four bacterial isolates of Pseudomonas spp. had nematicidal properties in the range of 84.98% to 95.24%. Two of the bacterial isolates, RH25 (Pseudomonas aeruginosa) and RH37 (Pseudomonas fluorescens), had the largest clear zone of proteolysis on skim milk agar and were subjected to various biochemical tests. The influences of time (1 to 120 h), temperature (22 to 37degree celsius) and pH (7.0 to 8.5) were investigated in the production of protease. The highest enzyme production was obtained from growing RH37 and RH25 on mineral medium when incubated at temperatures of 26degree celsius and 30degree celsius for 72 and 96 h and at initial pH values of 7.2 and 7.6, respectively. Proteases obtained from RH37 (P. fluorescens) belonged to the metalloprotease and serine protease families and the one produced by RH25 (P. aeruginosa) was a metalloprotease. According to the results of this research, the most effective control mechanism of these bacteria against nematodes is the production of bacterial protease. This mechanism can be exploited to control nematodes.