Quantitative detection of Heterodera avenae and H. latipons using qPCR

Twelve Heterodera species are considered of major economic significance in cereals; Heterodera avenae, H. latipons and H. filipjevi are the most important ones. Precise identification and quantification of these nematodes are necessary to develop effective integrated pest control. This study reports the use of the mitochondrial cytochrome oxidase subunit 1 (COI) gene to develop qPCR assays that could be used for the identification and quantification of H. avenae and H. latipons. Two qPCR primer sets comprising two primers and a probe, were designed and optimized. The qPCR assays that we developed for the quick detection and quantification of H. avenae and H. latipons were able to detect a single second-stage juvenile (J2). Their specificity was confirmed by the lack of amplification of 13 other Heterodera species. A qPCR using DNA extracted from 120 J2 + eggs of H. avenae and H. latipons were resulted in steady Ct-values (Ct = 22.33 ± 0.1 and Ct= 18.6 ± 0.12, respectively). Dilution series of DNA extracted from 120 of J2 + eggs of the two species were made. The assays resulted in a standard curve showing a highly significant linearity between the Ct-values and the dilution rates (R² = 0.99; slope = -3.05 and R² = 0.99; slope = -3.5 for H. avenae and H. latipons respectively). The two qPCR assays provide a sensitive and valid tool for the rapid detection and quantification of the two species whether they occur alone or in mixtures with other species.