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 50-GGGAGGCCCGGTGGA-30 and 50-TGATCCGAGGTCAACTTCTGAA-30 and a TaqMan probe 50-CGTCCGCCGTAAAACGCCCAAC-30 were designed based on the sequence of the ITS region of the rRNA gene. The primers were highly species-specific. The PCR reaction systemwas 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, R = 0.9971, E = 0.9055) and from Log conidia g 1 soil (y = 3.855x + 37.669, R = 0.9139, E = 0.8172), indicating that the real-time PCR protocol can reliably quantify H. minnesotensis in thesoil. The real-timePCRassaywasapplied to20 soil samples fromsoybeanfields, and comparedwitha parasitism assay. The real-time PCR assay detected H. minnesotensis in six of the soils, whereas the parasitismassaydetectedH.minnesotensis in thesamesixsoils and threeadditional soils. Thereal-timePCR assaywasweakly correlated (R = 0.49)with thepercentage of parasitized J2 in the six soils, indicating that different typesof soilmay interfere theefficiencyof thereal-timePCRassay, possiblydue to theeffect of soil types on efficacy of DNAextraction. Theparasitismassay appeared to bemore sensitive than real-time PCR in detecting presence ofH.minnesotensis, but real-time PCRwasmuch 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 in13of the20field soils, indicating that these fungimaycontribute to suppressivenessof soybean cyst nematode in nature and likely have high biological control potential for the nematode. 2009 Elsevier B.V. All rights reserved.