Development of a Real-Time Quantitative PCR Assay for the Specific Detection of Bacillus velezensis and Its Application in the Study of Colonization Ability

Bacillus velezensis is a widely used biocontrol agent closely related to B. amyloliquefaciens, and the two species cannot be distinguished by universal primers that are currently available. The study aimed to establish a rapid, specific detection approach for B. velezensis. Many unique gene sequences of B. velezensis were selected through whole genome sequence alignment of B. velezensis strains and were used to design a series of forward and reverse primers, which were then screened by PCR and qPCR using different Bacillus samples as templates. The colonization ability of B. velezensis ZF2 in different soils and different soil environmental conditions was measured by qPCR and a 10-fold dilution plating assay. A specific primer pair targeting the sequence of the D3N19_RS13500 gene of B. velezensis ZF2 was screened and could successfully distinguish B. velezensis from B. amyloliquefaciens. A rapid specific real-time qPCR detection system for B. velezensis was established. B. velezensis ZF2 had a very strong colonization ability in desert soil, and the optimal soil pH was 7-8. Moreover, the colonization ability of strain ZF2 was significantly enhanced when organic matter from different nitrogen sources was added to the substrate. This study will provide assistance for rapid specificity detection and biocontrol application of B. velezensis strains.