High-Resolution Melting (HRM) Assay for Molecular Detection of Calonectria ilicicola in Soybean

Calonectria ilicicola, a newly-emerging soilborne pathogen posing significant economic risk in the U.S. Midwest, leads to red crown rot (RCR) in soybean crops. Accurate detection of this disease is crucial, as symptoms are often indistinguishable from those of other soybean diseases. To address this need, researchers have developed and validated a high-resolution melting (HRM) assay targeting the Translation Elongation Factor 1α (TEF-1α) gene of C. ilicicola.

The assay was subjected to rigorous evaluation using a specificity panel of 78 DNA samples. This panel comprised 67 DNA samples from various fungi and oomycetes, along with 11 DNA samples from soybean host tissue. Additionally, 56 DNA samples from both symptomatic and asymptomatic soybean plants were used for testing. The HRM assay produced unique melting profiles, with a clear distinction between the C. ilicicola target and non-target fungi and oomycetes.

In certain cases, the absence or delayed amplification further aided in differentiating between the target pathogen and other microorganisms. Furthermore, the assay successfully identified three distinct haplotypes among C. ilicicola isolates, which corresponded to specific nucleotide polymorphisms in the TEF-1α target region.

The assay's quantitative performance was exemplary, displaying a high linearity (R^2= 0.9992) and maintaining an acceptable amplification efficiency (94.05%) across a wide range of DNA concentrations. In field samples, the HRM-based detection method confirmed the presence of C. ilicicola in symptomatic plants and yielded no detection in asymptomatic tissues, accurately reflecting the disease status of the plants.

In conclusion, this HRM assay offers a rapid, sensitive, and reliable diagnostic tool for the routine molecular detection of Calonectria ilicicola. Its ability to accurately differentiate C. ilicicola from other fungi and oomycetes, as well as its strong quantitative performance, make it an invaluable resource for managing RCR in soybean fields.