Citrus Alternaria brown spot caused by the necrotrophic fungal pathogen of the tangerine pathotype of Alternaria alternata causes yield losses in global tangerine production. In this study, we focus on a cytochrome P450 monooxygenase encoding gene, Aacp1, for its role in the sporulation, toxin production, and virulence of the tangerine pathotype of Alternaria alternata. Aacp1-deficient mutants (∆Aacp1) produced significantly fewer conidia than the wild-type strain. Chemical assays demonstrated that Aacp1 plays a negative role in resistance to oxidant stress and biosynthesis of ACT toxin. Virulence assays revealed that ΔAacp1 fails to induce necrotic lesions on detached Hongjv leaves. Transcriptomic analyses of WT and ΔAacp1 revealed that many metabolic process genes were regulated. Furthermore, our results revealed a previously unrecognized Aacp1 affected the expression of the gene encoding a naphthalene dioxygenase (AaNdo1) for sporulation and full virulence. Overall, this study revealed the diverse functions of cytochrome P450 monooxygenase in the phytopathogenic fungus.

Citrus alternaria brown spot, caused by the tangerine pathotype of Alternaria alternata, is one of the most severe fungal diseases affecting citrus crops. Currently, there is a critical need for rapid and visual detection techniques to identify the tangerine pathotype of A. alternata. In this study, a novel detection system was developed by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology, targeting the ACTT3 gene specific to the tangerine pathotype of A. alternata. Through optimization of reaction time and component concentrations, the assay demonstrated a detection sensitivity of 1 pg μL⁻¹ within 40 min at a constant temperature of 37 °C. The results can be visually interpreted using nucleic acid test strips, offering advantages in specificity, sensitivity, and speed. This system has been successfully validated for the rapid detection of the pathogen within plant tissues, including leaves and fruits, providing an efficient and practical solution for real-time field detection of the tangerine pathotype of A. alternata.