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MOLECULAR CHARACTERIZATION AND IDENTIFICATION OF THE NEW ROOT-LESION NEMATODE SPECIES ON SOYBEAN IN NORTH DAKOTA
EXECUTIVE SUMMARY
NORTH DAKOTA SOYBEAN COUNCIL
JUNE 2020
Dr. Guiping Yan, Principal Investigator, Dept. Plant Pathology, NDSU
Research Conducted
New primers were designed for a molecular detection assay (real-time PCR) and tested with various nematode species. Melting curve analysis was also performed to assess amplification specificity. To determine amplification efficiency, DNA from four nematodes were subjected to 2-fold serial dilutions to generate a standard curve. Detection sensitivity was also determined. Twenty-seven fields were sampled, DNA was extracted from root-lesion nematodes, and nematode species was confirmed for each sample. To characterize the new species, three genomic regions of the nematode were amplified and sequenced, and DNA sequences of other root-lesion nematode species were retrieved from GenBank for phylogenetic analysis.
Why the research is important to ND soybean farmers
Root-lesion nematode is widely distributed in the U.S. and can cause significant yield suppression on soybean. Recently, soil samples were collected from a soybean field in North Dakota, and all samples contained root-lesion nematodes. These nematodes were identified as a new root-lesion nematode species that has not been reported in the literature. Our greenhouse bioassay demonstrated this new species infected and reproduced well on soybean. Thus, it is important to detect and characterize this new root-lesion nematode species discovered in North Dakota and determine its relationship with other species of root-lesion nematodes.
Final findings of the research
A new real-time PCR assay for detecting the new species was successfully developed. The primers were highly specific to the species by testing other nematode species and by melting curve analysis. The standard curve showed a strong negative correlation between real-time PCR and known nematode numbers, with high amplification efficiency. The assay was sensitive and could detect an equivalent to 1/32 of the DNA of a single nematode. Twenty out of the 27 soil samples collected were infested with root-lesion nematodes and their species identities were determined. As expected, the assay only amplified the sample with the new species but not the samples with other species. Three phylogeny trees were constructed and the new species was found to be present in a separate clade with the divergence from other root-lesion nematode species.
Benefits/Recommendations to North Dakota soybean farmers and industry
Results of this research will expand our knowledge of root-lesion nematode DNA sequence divergence, and improve detection efficiency of this new species in soybean fields. Sensitive and accurate detection of root-lesion nematodes is critical for nematode risk assessment and management to improve soil health and increase soybean yield.