First Report of Leaf Spot Caused by <i>Penicillium koreense</i> on Tobacco in China

Tobacco (Nicotiana tabacum L.) is an important economic crop in China and a key economic source for the Guizhou Province (Chen et al. 2020). In July 2024, leaves of tobacco cv. Yunyan 87 in Dafang County (27.09° N, 105.36° E), Bijie City, Guizhou Province, China, developed gray necrosis and yellow-white spots. About 70% of the tobacco plants in a 2-ha field were infected. The spot spread to entire leaves under high humidity, losing baking value. Symptomatic leaves were cut and placed on potato dextrose agar (PDA) medium and incubated at 28°C in the dark for 2 days. A total of nine strains were isolated. Six of the strains had consistent morphology, while the other three had varying morphologies. Pathogenicity tests of all nine strains showed that three were non - pathogenic and six were pathogenic, producing similar lesion phenotypes. Strain MDG16 (pathogenic) was randomly selected for molecular identification. Colonies had dense texture, dark cyan center, white edges, light cyan reverse, and abundant spores. Conidiophores broom-shaped with secondary/tertiary stems; ellipsoidal conidia (2.52-3.24×3.60-3.98μm), resembling Penicillium sp. (Feng et al. 2021). PCR amplification and sequence analysis were conducted using universal primers ITS1/ITS4, bt2a/bt2b (Liu et al. 1999) and RPB2-5F/RPB2-7CR (Glass et al. 1995). The sequence of the ITS1-5.8s-ITS2 region of rDNA from the strain MDG16 (GenBank accession no. OR366345) was identical to the sequences of several P. janthinellum accession KX928777, MT447471, OP797635, KM268685 (99.15% similarity) , The sequence of the RPB2 region of rDNA from the strain MDG16 (GenBank accession no. PX247014) was identical to the sequences of several P. korennse accession MN969159, MK450842, ON113877, (97.85% similarity), The sequence of the BenA region of rDNA from the strain MDG16 (GenBank accession no. PX247015) was identical to the sequences of P. koreense GenBank accessions MK451100, MT732925, KM000844, (97.93% similarity). Maximum-likelihood (ML) analysis was carried out using the ITS, RPB2 and BenA sequence (Dos Reis et al. 2011). Morphological and molecular results confirmed this strain as Penicillium koreense. For the pathogenicity test, tobacco seedlings at the six-leaf stage (cv. Yunyan 87) were used. Six tobacco seedlings were inoculated with 50 μl of spore suspension (1×105 spores/mL) on healthy leaves. Another six tobacco seedlings inoculated with 50 μl sterile water on the leaves served as the control. After inoculation, all plants were incubated in a growth chamber at 22 ± 3°C and 80% relative humidity. Five days after inoculation, typical symptoms were observed on the treatment of tobacco leaves, whereas no symptoms occurred on the control leaves. Koch’s postulates were fulfilled by re-isolation of the pathogen from the diseased leaves and identification using the ITS, RPB2, and BenA gene sequence. Penicillium sp. has been reported to cause apple blue mold rot in China (Jiang et al. 2013) and citrus blue mold in China (Mo et al. 2025). However, to the best of our knowledge, this is the first report of P. korennse causing tobacco leaf spot in a commercial tobacco field in China. Although the disease has not yet broken out on a large scale in commercial tobacco fields in Guizhou, it still has the potential to cause more serious losses. These observations underscore the imperative for researchers and agriculturalists to devise efficient management strategies for controlling P. korennse infections in tobacco leaf.