Two-step genomic sequence comparison strategy to design Trichoderma strain-specific primers for quantitative PCR

Survival of inoculated fungal strains in a new environment plays a critical role in functional performance, but few studies have focused on strain-specific quantitative PCR (qPCR) methods for monitoring beneficial fungi. In this study, the Trichoderma guizhouense strain NJAU 4742 (transformed with the gfp gene and named gfp-NJAU 4742), which exhibits a growth-promoting effect by means of phytohormone production and pathogen antagonism, was selected as a model to design strain-specific primer pairs using two steps of genomic sequence comparison to detect its abundance in soil. After a second comparison with the closely related species T. harzianum CBS 226-95 to further differentiate the strain-specific fragments that had shown no homology to any sequence deposited in the databases used in the first comparison, ten primer pairs were designed from the whole genome. Meanwhile, 3 primer pairs, P11, P12 and P13, were also designed from the inserted fragment containing the gfp gene. After verification testing with three types of field soils, primer pairs P6, P7 and P8 were further selected by comparison with P11, P12 and P13. A practical test using a pot experiment showed that stable colonization of gfp-NJAU 4742 in pepper rhizosphere soil could be detected using primer pairs P6 and P7, showing no significant difference from the results of primers P11 and P12. Hence, the strategy described here for designing fungal-strain-specific primers may theoretically be used for any other fungi for which the whole genome sequence is available in a database, and the qPCR methodology developed can also be used to further monitor the population dynamics of different strains based on the designed primers.