Development of a SCAR marker and a strain-specific genomic marker for the detection of the biocontrol agent strain CPA-8 Bacillus amyloliquefaciens (formerly B. subtilis)

In this work, reliable tools were developed to detect and identify the biocontrol strain CPA-8 using DNA amplification techniques. As a first approach, the RAPD (random amplified polymorphic DNA) technique was applied to a collection of 77 related Bacillus species. Among the primers tested, the primer pair OPG1/OPG6 amplified a 668 bp specific product to the strain CPA-8 that was sequenced and used to design SCAR (sequence-characterised amplified regions) primer pairs. The SCAR-4 marker amplified a semi-specific fragment of 665 bp not only for the strain CPA-8 but also for other 12 strains whose morphology was completely different from CPA-8. Another approach was developed to obtain a strain-specific genomic marker related to ecological adaptations of Bacillus amyloliquefaciens species. The primer pair F2/R2 obtained from RBAM 007760, a gene involved in surface adhesion, amplified a 265 bp fragment unique for strain CPA-8. Our results revealed that these two molecular markers, SCAR-4 and RBAM 007760 F2/R2 provide suitable monitoring tools to specifically identify the biocontrol CPA-8 when applied against brown rot caused by Monilinia spp. in stone fruit. Moreover, our findings demonstrate that the strain CPA-8 is affiliated with B. amyloliquefaciens species that was formerly designated as Bacillus subtilis.