Strain-specific monitoring of Bacillus amyloliquefaciens AH2 in strawberry fields using a specific molecular marker

The biocontrol agent Bacillus amyloliquefaciens AH2 is effective against Botrytis cinerea in strawberry crops. However, evaluating its actual field establishment is challenging owing to variable environmental stressors and the presence of diverse native Bacillus populations. Because conventional markers often fail to distinguish introduced commercial strains from native microbiota, this study aimed to develop a highly specific TaqMan quantitative polymerase chain rection (qPCR) assay to monitor B. amyloliquefaciens AH2 under real field conditions.

The assay targets a unique polymorphic region of the class I S-adenosyl-l-methionine DNA methyltransferase (hsdM) gene. Analytical validation demonstrated high sensitivity, with a limit of detection of 14 fg of genomic DNA. Specificity was confirmed against 30 bacterial strains, differentiating AH2 from closely related non-target Bacillus ecotypes. Field application (15 L/ha; 10¹¹ colony-forming units per L of formulation) yielded 100% diagnostic sensitivity. Normalizing bacterial abundance against the host plant internal transcribed spacer region allowed accurate relative quantification. The analysis revealed significantly higher relative abundance in leaves versus fruits. Crucially, absolute zero amplification in untreated controls confirmed the assay's robust in planta specificity against complex environmental backgrounds.

This strain-specific qPCR assay provides a foundational molecular tool for tracking B. amyloliquefaciens AH2 population dynamics and ensuring product traceability. Although future studies incorporating disease incidence data are required, this monitoring capability represents a crucial first step toward optimizing application strategies within the framework of precision agriculture. Furthermore, this hsdM-targeted approach serves as a blueprint for developing analogous tracking tools for other commercial microbial inoculants. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.