Rapid Visual LAMP-CRISPR/Cas12a Detection System for Cytospora Pathogens Associated with Pear Canker Integrating Isothermal Amplification with Programmable Secondary Recognition

Abstract

Pear canker, caused by Cytospora pathogens, is a devastating branch and trunk disease threatening the sustainable development of the fragrant pear industry in Korla, Xinjiang, China. Owing to the pathogen’s latent infection capability and the inconspicuous nature of early symptoms, symptom-based diagnosis is inadequate for early warning and precision management. Here, we developed a hybrid LAMP-CRISPR/Cas12a fluorescence detection system targeting Cytospora pyri and its closely related congener C. mali, the dominant Cytospora pathogens associated with pear canker. LAMP primers and Cas12a crRNA recognition sites were designed from conserved sequences of the β-tubulin (TUB2) gene, enabling rapid isothermal amplification and sequence-specific secondary confirmation. The optimized LAMP module amplifies the target within 30 min at 65 °C, and the CRISPR/Cas12a module produces stable fluorescence after approximately 15 min of trans-cleavage at 37 °C. The system yields positive signals for both C. pyri and the closely related C. mali, with no cross-reactivity against other Cytospora species or common fruit tree pathogenic fungi, supporting rapid screening of pome fruit canker pathogens. The limit of detection is 1 copy/μL of target template. Validation using Cytospora strains from diverse geographic and host origins demonstrated complete concordance between LAMP gel electrophoresis, CRISPR/Cas12a real-time fluorescence detection, and UV-assisted visual evaluation. Preliminary results from field-collected Korla fragrant pear branch and trunk samples were consistent with conventional PCR, supporting applicability to orchard samples. By integrating rapid isothermal amplification, secondary sequence verification, and simple visual evaluation, this system provides a robust platform for early screening, pathogen surveillance, and precision management of Cytospora canker in Korla fragrant pear orchards.