Title : Rapid and sensitive detection of Diaporthe citri causing melanose in Australian finger lime (Citrus australasica) using an RPA-CRISPR/Cas12a molecular diagnostic assay
Abstract:
Melanose, caused by the fungus Diaporthe citri F.A. Wolf, is a fungal pathogen accounting for major preharvest and postharvest loss of all citrus species in the world. This fungal infection harms the tree and leaves and reduces fruit marketability. Rapid and accurate identification of D. citri makes early intervention possible and supports sustainable orchard management and effective control of its dispersion. However, conventional diagnostic approaches such as morphological identification or PCR-based methods are time-consuming and lab-dependent and may lack specificity when other closely related species are present. In this study, we develop and validate a Rapid Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a-based diagnostic assay for the early detection of this fungal pathogen in finger lime (Citrus australasica), which is a citrus species endemic to Australia. In this method, the combination of Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a is used to rapidly amplify the target region, and to recognise and cleave specific nucleic acid sequences to produce visible fluorescence signals within 30 minutes. Upon binding of the guide RNA (gRNA)–Cas12a complex to the target D. citri DNA, the activated Cas12a enzyme cleaves a fluorescent single-stranded DNA reporter, which generates a detectable fluorescence signal. This diagnostic method shows high specificity and sensitivity and can be completed within 30 minutes with no cross-reactions with other fungal pathogens. The RPA amplification and CRISPR-Cas12a diagnostic tool can be used as a novel and promising means for sustainable detection of this fungal pathogen. This study provides a reproducible approach for developing CRISPR-based point-of-care testing platforms for other fungal pathogens to improve disease management enhance biosecurity and support sustainable crop protection strategies.
