Analysis of Whole-Genome for Alternaria Species Identification

The genus <i>Alternaria</i>, functioning as a saprobe, endophyte, and plant pathogen, is widely distributed across various natural and human-impacted environments. Leaf spot and black spot diseases, caused by <i>Alternaria</i> species, are the most prevalent plant diseases within this genus, leading to significant reductions in crop yields and substantial economic losses. To facilitate the timely detection of <i>Alternaria</i> species during the early stages of infection, enable targeted treatments, and mitigate associated damages, we employed a species identification method based on Analysis of whole-GEnome (AGE). We downloaded 148 genomes, including 31 <i>Alternaria</i> species, from the NCBI GenBank database. Through bioinformatics analysis, we constructed a specific-target sequence library and selected a representative sequence per species. The specific target sequences of the seven exemplary <i>Alternaria</i> species were subsequently used for validation and rapid detection, utilizing Sanger sequencing and CRISPR-Cas12a technology, respectively. The results demonstrated that our method accurately identified the target species. Additionally, by combining Enzymatic Recombinase Amplification (ERA) with CRISPR-Cas12a, we achieved rapid and precise identification of genomic DNA samples, with a detection limit as low as 0.01 ng/µL within 30 min. Therefore, AGE proves to be a highly robust and efficient method for the detection of <i>Alternaria</i> species, offering broad potential for various applications.