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

Area of Science:

• Environmental Microbiology
• Molecular Biology
• Agricultural Science

Background:

• Monitoring beneficial fungal strain survival and abundance in new environments is crucial for their functional efficacy.
• Quantitative PCR (qPCR) methods for strain-specific fungal monitoring are underdeveloped, limiting ecological and agricultural applications.
• The Trichoderma guizhouense strain NJAU 4742, known for growth promotion via phytohormones and antagonism, serves as a model organism.

Purpose of the Study:

• To design and validate strain-specific primer pairs for detecting the abundance of Trichoderma guizhouense strain NJAU 4742 in soil.
• To establish a reliable qPCR methodology for monitoring the population dynamics of specific fungal strains.

Main Methods:

• Strain-specific primer pairs were designed through two rounds of genomic sequence comparison, including comparison with closely related species (T. harzianum CBS 226-95).
• Primers were designed from both the whole genome and an inserted marker gene (gfp).
• Primer efficacy was verified in three soil types and a pot experiment using pepper rhizosphere soil.

Main Results:

• Ten whole-genome-derived primer pairs and three gfp-derived primer pairs (P11, P12, P13) were designed.
• Primer pairs P6, P7, and P8 were selected after comparison with gfp-derived primers.
• Pot experiments confirmed stable colonization of gfp-NJAU 4742 in pepper rhizosphere soil using primers P6 and P7, with results comparable to P11 and P12.

Conclusions:

• The developed strategy for designing fungal-strain-specific primers is theoretically applicable to other fungi with available whole genome sequences.
• The qPCR methodology enables effective monitoring of fungal strain population dynamics.
• This approach supports the ecological and agricultural application of beneficial fungi by enabling precise tracking of their establishment and survival.