Detection and quantification of the nematophagous fungus Hirsutella minnesotensis in soil with real-time PCR

A real-time PCR assay was developed to quantify in soil the fungus Hirsutella minnesotensis, an important parasite of secondary-stage juvenile (J2) of the soybean cyst nematode. A primer pair 5′-GGGAGGCCCGGTGGA-3′ and 5′-TGATCCGAGGTCAACTTCTGAA-3′ and a TaqMan probe 5′-CGTCCGCCGTAAAACGCCCAAC-3′ were designed based on the sequence of the ITS region of the rRNA gene. The primers were highly species-specific. The PCR reaction system was very sensitive and able to detect as few as 4 conidia g^?1 soil. Regression analysis showed similar slopes and efficiency on DNA from pure culture (y = ?3.587x + 41.017, R² = 0.9971, E = 0.9055) and from Log conidia g^?1 soil (y = ?3.855x + 37.669, R² = 0.9139, E = 0.8172), indicating that the real-time PCR protocol can reliably quantify H. minnesotensis in the soil. The real-time PCR assay was applied to 20 soil samples from soybean fields, and compared with a parasitism assay. The real-time PCR assay detected H. minnesotensis in six of the soils, whereas the parasitism assay detected H. minnesotensis in the same six soils and three additional soils. The real-time PCR assay was weakly correlated (R² = 0.49) with the percentage of parasitized J2 in the six soils, indicating that different types of soil may interfere the efficiency of the real-time PCR assay, possibly due to the effect of soil types on efficacy of DNA extraction. The parasitism assay appeared to be more sensitive than real-time PCR in detecting presence of H. minnesotensis, but real-time PCR was much faster and less costly and provided a direct assessment of fungal biomass. Using the two assays in combination can obtain more complete information about the fungus in soil than either assay alone. Hirsutella parasitism was widespread and detected in 13 of the 20 field soils, indicating that these fungi may contribute to suppressiveness of soybean cyst nematode in nature and likely have high biological control potential for the nematode.