Survey and development of a molecular test for Soybean Cyst Nematode (Heterodera glycines Ichinohe) in Manitoba

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Ghavami Shirehjin, Nazanin
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Soybean Cyst Nematode (SCN), Heterodera glycines (Ichinohe 1952), recognized as a major global soybean pest, is already established in the Canadian provinces of Ontario and Quebec. This study anticipated its imminent presence in Manitoba, driven by its rapid spread from U.S. counties bordering the province. The research objectives were to a) survey SCN presence in Manitoba soybean fields, b) develop a molecular method for direct soil quantification of H. glycines, and c) identify native cyst nematodes in Manitoba Prairie Preserves. The survey of 30 commercial soybean fields in Manitoba revealed four positive fields for SCN using morphological characters of cysts, PCR with species-specific primers for H. glycines (COXIII and SCAR), and DNA sequencing of four regions of the genome, marking the first report of H. glycines in Manitoba. In 2021, an additional soybean field in Manitoba showed SCN symptoms and H. glycines females on the soybean roots. A molecular test employing a SYBR Green I-based real-time qPCR assay using species-specific primers for H. glycines (COXIII and SCAR) was developed, offering a reliable, sensitive and specific means to detect and quantify H. glycines eggs directly in soil DNA extracts of Manitoba and Ontario samples. This developed method eliminates the need for labour-intensive microscopic identification and egg counting of H. glycines in soils. Soil sampling in three Prairie Preserves of Manitoba revealed that, out of the prairies sampled, only one had nematode cysts. The cysts were extracted from the rhizosphere soil of Prairie Cordgrass located in the Manitoba Tall Grass Prairie Preserve. Using morphological and molecular analysis, which involved PCR amplification and sequencing of four regions of the genome (ITS rDNA, D2-D3 expansion segments of 28S rDNA; 18S rDNA, and a fragment of Mt-DNA (COI)), the recovered cyst nematodes were confirmed to be Punctodera stonei. This finding marks the first report of P. stonei in Manitoba and only the second in Canada. The findings of this study provide valuable insights into SCN presence in Manitoba, present a reliable molecular method for H. glycines quantification in soils, and unveil the presence of a cyst nematode species in a native prairie of Manitoba.
Soybean, Soybean Cyst Nematode, SCN, Heterodera glycines, Species-specific primer, Real-time qPCR