Identification of Pseudocercospora mori as the causal agent of grey leaf spot disease in mulberry (Morus atropurpurea) from various localities in Guangdong Province, China

During periods of high temperature and humidity, mulberry trees become susceptible to fungal leaf spot disease, which can significantly reduce both the yield and quality of their leaves. In this study, we collected samples of mulberry leaf spot disease from six regions of Guangdong province of China. The disease samples were studied using traditional morphological methods, high-throughput sequencing technology, molecular phylogenetic analysis, and pathogenicity tests. The observed morphological features of the pathogen were consistent with those of <i>Pseudocercospora</i>. High-throughput sequencing results revealed the presence of multiple fungal species in the samples, with <i>Pseudocercospora</i> spp. comprising the highest proportion. The complete rDNA and mitochondrial genome sequences of <i>Pseudocercospora</i> spp. were assembled. Based on the sequencing data, primers were designed to amplify and sequence barcode gene regions, including <i>ITS</i>, <i>Cyt b</i>, and <i>COI</i>. Phylogenetic analyses consistently placed the pathogen within the family Mycosphaerellaceae. ITS-based identification confirmed the pathogen as a member of the genus <i>Pseudocercospora</i>, while the <i>Cyt b</i> and <i>COI</i> sequences indicated a relatively distant relationship with the closely related genus <i>Cercospora</i>, thereby supporting the morphological classification of the pathogen at the molecular level. In addition, pathogenicity validation identified <i>Pseudocercospora mori</i> as a primary causal pathogen of leaf spot disease in mulberry. PCR primers specifically designed based on the rDNA sequence of <i>Pseudocercospora mori</i> achieved a detection sensitivity as low as 3 × 10⁻² ng/μL. In conclusion, based on morphological and molecular phylogenetic evidence, we identified <i>Pseudocercospora mori</i> as the causal pathogen of mulberry leaf spot disease. This study provides useful data for practical management of mulberry leaf spot disease at the field level, aiding in the sustainable development of sericulture.