A rapid and visual detection method for Alternaria alternata, the causal agent of leaf spot disease on yam, based on RPA-CRISPR/Cas12a

Abstract

Yam (Dioscorea opposita Thunb.) is an important crop that is highly valuable as both food and medicine in China. However, fungal diseases pose a significant threat to the safety and reliability of its production. In this study, a strain designated 23HNTG4-2 was isolated and shown to play predominant role in the occurrence of leaf spot disease on D. opposita. Its pathogenicity was determined based on Koch's postulates. Further analysis based on a multigene phylogenetic tree identified the 23HNTG4-2 as Alternaria alternata. Considering that the traditional methods for detecting pathogens are often time-consuming, we developed a platform based on RPA-CRISPR/Cas12a to detect A. alternata. Our research revealed that the CRISPR RNA (crRNA) that contained two bases (Al-crRNA3) of the protospacer adjacent motif (PAM) site exhibited a maximum fluorescence value (1.37 ± 0.052) during the CRISPR/Cas12a reaction. Compared with PCR and qPCR assays, this platform was much more sensitive (3 × 10¹ copies/μL) and enhanced the reliability of the detection results. The platform can be utilized in field tests without the need for intricate instruments. In summary, we verified that A. alternata produces necrotic spots on the yam leaves and provided an RPA-CRISPR/Cas12a detection platform, which is suitable for field tests owing to significant advances in sensitivity and specificity, visible results, and its lack of reliance on complicated instruments.