Advancing strain-specific TaqMan assays for Trichoderma asperellum detection in commercial agricultural settings

• The agricultural sector is increasingly using biological control agents (BCA) to combat soil-borne plant diseases. • BCAs can/shall preserve soil quality and fertility while reducing reliance on synthetic pesticides. • Monitoring the fate and persistence of BCAs in the soil is crucial to optimise their application. • An improved TaqMan assay with high specificity and sensitivity in detecting Trichoderma asperellum in soil was developed. • The assay was successfully applied and validated on soils where tomatoes and strawberries were grown. The global agricultural sector is facing significant challenges in achieving higher sustainability, which has increased interest in using biological control agents (BCAs) to manage plant diseases. However, it is essential to ensure that microbial-based products, such as BCAs, are utilised in a manner that does not harm soil quality and fertility while decreasing reliance on synthetic pesticides. To accomplish this, it is crucial to monitor the fate and persistence of bioinoculants in the soil, which is essential for optimising their application over time, as well as for regulatory and commercial purposes and environmental risk assessment. A qPCR detection method utilising TaqMan chemistry is proposed, which has demonstrated high specificity and sensitivity in detecting Trichoderma asperellum, a common BCA species, in soil. The primers and probe were designed based on the βtubulin2 gene. The TaqMan-based assay was applied and validated on soils where tomatoes and strawberries were grown after a previous application of T. asperellum FC80 strain over three years. The TaqMan-based assay was able to detect the target strain accurately, meeting the stringent requirements for commercial and regulatory applications. The TaqMan assay developed here has the potential to impact the agricultural sector significantly. It can be used for regulatory, commercial, and scientific purposes to track, monitor, and determine the presence and fate of T. asperellum under field crop conditions, thereby contributing to adopting more sustainable and efficient agricultural practices.