First report of Pseudopithomyces palmicola causing leaf spot on tobacco in China

In July 2023, a new leaf spot disease emerged on tobacco leaves in Meitan County, Guizhou Province, China (27°20'18" - 28°12'30"N, 107°15'36" - 107°41'08"E, average altitude 972 meters). Initially, the symptoms showed raised yellow-brown spots; subsequently, the lesions expanded and became broken and perforated, leading to a significant loss of economic value, the prevalence rate exceeded 30%. For isolation, two tissue fragments (0.2 × 0.2 cm) of symptomatic leaves were sterilized in 75% ethanol for 30 s, 3% NaClO for 2 min, and were washed 3 times in sterilized distilled water, and were subsequently inoculated on potato dextrose agar (PDA), and incubated at 28°C for 9 days in the dark. The two strains CW16 and CW28 were isolated using the single hyphae method (Nouri et al. 2023). Both strains formed pale to yellow white colonies on PDA. Conidia had three constricted transverse septa and 1 to 2 longitudinal septa in the central cells, with thick and hyaline conidiophores and mostly globose, pale brown conidia with slightly constricted septa, their average size were measured as 13.4-22.4×8.358-13.347 μm (n = 50). Genomic DNA was extracted from the isolated strains CW16 and CW28. The internal transcribed spacer regions 1 and 2 as well as 5.8S nuclear ribosomal RNA (ITS), large subunit nrRNA (LSU), and partial DNA-directed RNA polymerase II second largest subunit (RPB2) genes were amplified using primers (Cui et al. 2023). The sequences had been deposited in GenBank under accession numbers ITS: PP024201, PP024205; LSU: PP024207, PP024209; RPB2: PP060480, PP060481. The sequences analysis revealed a high similarity of 99.74 to 100% between strains CW16 and CW28 with P. palmicola isolate KM42 (ITS OQ875842, LSU OQ875844, RPB2 OQ883943) in GenBank. Using BLAST for homology matching, two isolates (CW16, CW28) and with the sequences of the ten type isolates from GenBank, phylogenetic analysis was conducted using the Maximum Likelihood method in MEGA (11.0) software based on ITS, LSU and RPB2 sequences, which showed that strains CW16, CW28 clustered in the same score as the Pseudopithomyces palmicola, confirming the morphological and molecular characteristics identification. The pathogenicity tests were conducted on healthy tobacco plants with 4-5 leaves (Fig. S1B), the isolated strains, CW16 and CW28, were used to inoculate the healthy tobacco leaves, while blank PDA was used as a control. All plants were maintained in a greenhouse at 28°C with a relative humidity of 90%. After 9 days, necrotic spots were observed on all tobacco leaves inoculated with CW16 and CW28 fungal plugs, while the blank PDA-inoculated tobacco leaves showed no symptoms. Based on morphological and molecular characteristics, the same pathogen P. palmicola was identified from the inoculated leaves, fulfilling Koch's postulates. This study represents the first reported of tobacco leaf spot caused by P. palmicola in China and provides a theoretical basis for future prevention and control measures.