Rapid and Sensitive On-Site Nucleic Acid Detection of Three Main Fusarium Pathogens of Maize Stalk Rot Based on RPA-CRISPR/Cas12a.

Maize stalk rot is a soilborne disease that poses a serious threat to maize production worldwide, with the most significant cause being fungal stalk rot. The development of a visual and rapid detection method for the maize stalk rot pathogen is significant for its prompt and accurate identification, enhancing agricultural production efficiency, and implementing timely preventive measures. These measures will help safeguard the maize yield and quality, ultimately reducing agricultural losses. In this study, we aimed to develop an efficient method to detect maize stalk rot pathogens. We focused on three pathogenic fungi commonly found in maize-producing regions worldwide: Fusarium verticillioides, F. proliferatum, and F. graminearum. Based on translation elongation factor 1-α, we developed a rapid detection technique using recombinase polymerase amplification-CRISPR/Cas12a, combined with test strips to develop an on-site rapid visual detection test for these pathogens. The method showed detection sensitivity for F. verticillioides, F. proliferatum, and F. graminearum within 20 min at concentrations of 7.8 pg/μl, 0.11 ng/μl, and 0.13 ng/μl, respectively. The sensitivity increased with increasing reaction time. Testing of field disease samples indicated that the method is effective in detecting nucleic acids obtained through crude extraction methods. In conclusion, we developed a visually rapid detection technology that does not rely on complex instruments and equipment for the on-site early detection of F. verticillioides, F. proliferatum, and F. graminearum in the field to implement effective control measures, ensuring stable and high maize yields.