Molecular Detection of bae16 and bace16 of Bacillus subtilis and their Bio-control Efficiency Against Nematodes that Infect Cucumber Plants

Abstract

This study aims to detect the qualitative detection of Npb and Apb genes responsible for producing neutral proteases (Bea16) and alkaline serine proteases (Bace16), which are among the main proteases produced by Bacillus subtilis that affect nematodes, and determine the extent of their effect on nematode cuticle degradation with increasing exposure time.  B. subtilis is a wild type of bacteria present in the soil and can produce various proteases, some of which may be effective against nematodes. In the current study, B. subtilis was isolated from the soil and identified molecularly based on a conserved region in the 16S rRNA. Real-time PCR amplified the Npb and Apb genes extracted from B. subtilis for the qualitative detection of their genes, and the results of the qualitative detection showed the presence of genes at C_t 27.91, 27.33 for Npb, and C_t 33.51, 35.09 for Apb, respectively. After nematodes (second-stage juvenile) isolates were treated with 10, 20, 30, 40, and 100% crude extracts at 24, 48, and 72 h, the in vitro experiments after 72 h of exposure showed that 30, 40, and 100% crude dilutions had the highest event mortality rates, with averages of 90.7%, 97.3%, and 96.7%, respectively. This is due to the crude extract containing some proteases produced by B. subtilis that can affect nematodes, including neutral and alkaline serine. This study found that among the various proteases produced by B. subtilis that can impact nematodes, B. subtilis can make two intriguing ones: Bae16 and Bace16. These proteases, along with others, may degrade the nematode's cuticle.