Authors
Horacio D. Lopez-Nicora, Department of Plant Pathology, Ohio State University, Columbus 43210;
James P. Craig, Department of Plant and Microbial Biology, University of California Berkeley, Berkeley 94720;
Xuebiao Gao, Pasteuria Bioscience, Inc. 12085 Research Drive, Suite 185 Alachua, FL 32615;
Kris N. Lambert, Department of Crop Science, University of Illinois, Urbana 61801 and
Terry L. Niblack, Department of Plant Pathology, Ohio State University, Columbus 43210
Abstract
Heterodera glycines, the soybean cyst nematode, is a major pathogen of soybean. Effective management of this pathogen is contingent on the use of resistant cultivars; thus, screening for resistant cultivars is essential. The purpose of this research was to develop a method to assess infection of soybean roots by H. glycines with real-time quantitative polymerase chain reaction (qPCR). This method will serve as a prelude to differentiation of resistance levels in soybean cultivars. A reproducible inoculation method was developed by means of a sand column to provide active second-stage juveniles (J2). Two-day-old soybean roots were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours after infestation, the roots were surface-sterilized and genomic DNA (gDNA) was extracted. For the qPCR assay, a primer pair for the single copy gene HgSNO, which codes for a protein involved in the production of vitamin B6, was selected for H. glycines gDNA amplification within soybean roots. Compatible ‘Lee 74’, incompatible ‘Peking’, and cultivars with different levels of resistance to H. glycines were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours postinfestation, infected seedlings were transplanted into pasteurized soil. Subsequently, they were harvested at 1, 7, 10, 14, and 21 days postinfestation for gDNA extraction. With the qPCR assay, the time needed to differentiate highly resistant cultivars from the rest was reduced. Quantification of H. glycines infection by traditional means (numbers of females produced in 30 days) is a time-consuming practice. This qPCR assay has the potential to replace the traditional Female Index-based screening and improve precision in determining infection levels.
