Rapid Detection of Frogeye Leaf Spot Pathogen in Seeds by LAMP Assays to Protect Soybean Production.

Abstract

Frogeye leaf spot (FLS), caused by Cercospora sojina, is a significant disease affecting soybean crops economically. Seed inspection plays a vital role in reducing the incidence of this disease. However, limited research has been conducted on the fast and accurate identification of C. sojina in soybean seeds. In this study, we developed a rapid diagnostic method to detect C. sojina in soybean seeds using loop-mediated isothermal amplification (LAMP) assays. A software application called K-mer Elimination by Cross-reference was utilized to identify specific sequences belonging to C. sojina. The LAMP primer set designed based on the selected specific sequence proved to be highly specific, distinguishing C. sojina from other common soybean pathogens and two maize pathogens of the Cercospora genus. The primer set exhibited remarkable sensitivity, detecting 16 copies per reaction, which is approximately 1,000 times more sensitive than conventional PCR. The LAMP assays were applied for direct inspection of soybean seeds, revealing that 20 of the 30 tested soybean varieties were contaminated with C. sojina, with contamination rates ranging from 10 to 60%. The seeds of TongNong 943 and JiNong 38 had the highest contamination rate at 60%. Seeds coated with fungicide showed a reduced disease incidence, as demonstrated on PDA medium and in soil pots. In summary, our results indicate that rapid seed diagnosis using LAMP assays can effectively screen out seeds carrying C. sojina, providing a preventive strategy in managing FLS disease.