Rapid and Efficient Molecular Detection of Phytophthora nicotianae Based on RPA-CRISPR/Cas12a

Abstract

Phytophthora nicotianae is a global and polyphagous pathogen with a wide host range. P. nicotianae can infect Areca catechu, Durio zibethinus L., Psidium guajava L., Hevea brasiliensis, and other tree species. The pathogen is capable of inducing butt rot and affecting aerial parts, including stems, leaves, and fruits. Compared to other Phytophthora species, P. nicotianae is more adaptable to abiotic stress. In this study, recombinase polymerase amplification (RPA) in combination with the CRISPR/Cas12a system was used for the detection of P. nicotianae, and achieved rapid and efficient detection of P. nicotianae. The assay was highly specific to P. nicotianae. All 4 tested isolates of P. nicotianae yielded positive results, whereas 30 isolates belonging to 17 other Phytophthora species, 8 fungal species, and 4 Bursaphelenchus xylophilus vermicules lacked detection. Under the conditions of 37 °C, after 20 min of RPA reaction and 25 min of Cas12a cleavage, a DNA concentration as low as 10 pg·μL−1 could be detected. In addition, it detected P. nicotianae from artificially inoculated leaves of Fatsia japonica. In this study, a novel method was established for the efficient and accurate detection of P. nicotianae based on the combination of RPA and the CRISPR/Cas12a system.