Rapid and sensitive diagnosis of plant quarantine fungi Alternaria triticina and Plenodomus libanotidis based on the RPA-CRISPR/Cas12a system

With the increase in cross-border transmission in the context of globalization, the necessity for developing rapid and accurate detection methods for plant pathogens has become critical. This study introduces a recombinase polymerase amplification (RPA) technique combined with CRISPR/Cas12a cleavage and fluorescence-based detection systems (FRB) or paper-based lateral flow strips (PLFS) for the rapid on-site detection of invasive alien fungi, specifically Alternaria triticina and Plenodomus libanotidis, which pose significant threats to agriculture and biodiversity. The results demonstrate that either RPA-CRISPR/Cas12a-FRB or RPA-CRISPR/Cas12a-PLFS can accurately detect the target species within 30 min, with a sensitivity of up to 10 pg/μL. These portable and easy-to-use assays are suitable for rapid on-site screening of plant pathogenic fungi in plant tissues, enabling applications in disease control and port quarantine.