First Report of Pestalotiopsis trachycarpicola Causing Leaf Spot on Tobacco in China

In June 2023, yellowish-brown leaf spots with elevated black small spots were observed on tobacco in Zheng'an County, Zunyi City, Guizhou Province, China (107°4′-107°41′E, 28°9′-28°51′N, average altitude 1200 meters). About 20% of tobacco plants in a 20-acre field showed significant growth suppression and yield reduction. The chemical balance of tobacco leaves was disrupted, leading to deteriorated leaf appearance quality, which affected the flavor characteristics and combustion performance of cigarettes, further reducing purchase prices and farmer income. The symptomatic tobacco leaves were collected to isolate the causal agent. Symptomatic tissues were surface-sterilized with 75% ethanol for 45 seconds, rinsed 3 to 4 times with sterile distilled water, and dried with sterile cotton, then transferred to the potato dextrose agar (PDA) plates, which were incubated at 26°C in the dark for 5 to 7 days (Fan et al. 2021). Three cultures were obtained with an isolation frequency of 16% by hyphal transfer method. The fungus produced white colonies on PDA and appeared reddish-brown on the underside of the plates after one week. The conidia with four septa resembled those of Pestalotiopsis trachycarpicola and were spindle-shaped, ranging from 3.16 to 6.39 × 15.22 to 24.01 μm (n = 50). The apical and basal cells were colorless and triangular, while the middle three septal cells were light brown to brown. One filamentous averaging 4.39 to 9.54 μm in length (n = 50) appendage at the apex, and 2 to 3 averaging 9.20 to 15.92 μm (n = 50) appendages at the base. Three loci—internal transcribed spacer (ITS), partial β-tubulin (TUB), and partial translation elongation factor 1-α (EF-1α)—were amplified and sequenced using specific primers for isolated Genomic DNA (Zheng et al. 2023). These sequences have been submitted to GenBank under accession numbers ITS: PP024198, PP648161, PP654438; TUB: PP054320, PP662648, PP662649; EF-1α: PP054321, PP662650, PP662651. The BLAST analysis revealed a sequence homology range of 98.01 to 100% of the strain SVG00116F (accessions: ITS ON238108, TUB OP895026, EF-1α OP895025; Araujo et al. 2023). A phylogenetic tree from the three loci ITS, TUB, and EF-1α was constructed using Maximum Likelihood (ML), and neighborhood connections were made with the sequences of the twelve types of isolates in GenBank, which showed that the three strains clustered in the same clade as Pestalotiopsis trachycarpicola, confirming morphological identification. Pathogenicity tests were conducted twice on healthy tobacco plants with 5-7 leaves. Wound inoculation involved applying 1–3 fungal mycelial plugs (5 mm diameter) per site across three flue-cured cultivars (Yunyan87, K326, Guiyan20). Fungal-free sterile PDA plugs served as controls. The plants were kept in a greenhouse at 28°C and 90% relative humidity. After 10 days, all tobacco except for blank PDA-inoculated tobacco leaves inoculated with all isolates showed necrotic spots, and the spores observed in the lesion were morphologically consistent with P. trachycarpicola. Based on morphological and molecular characteristics, the same pathogen P. trachycarpicola was re-isolated from the inoculated leaves, fulfilling Koch’s postulates. This is the first report of leaf spot caused by P. trachycarpicola in China. The disease spreads widely and causes a decrease in tobacco leaf yield; this first report provides a basis to prevent its future spread and curb further losses.