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.

First Report of Grey Spot on Tobacco caused by Alternaria alstroemeriae in China.

Tobacco (Nicotiana tabacum L.) is an important economic crop belonging to family Solanaceae and is widely cultivated in China (Basit 2021). From April to July in 2022, a foliar disease with symptoms similar to grey spot was extensively observed on tobacco in Guangxi Province (24°52' N, 111°23' E), China. Field surveys were conducted in 18 towns and the disease incidence was 0.89% to 6.95%. Symptomatic leaves displayed irregular, dark brown lesions surrounded by yellow halos and accompanied with black conidiomata in gray centers (Fig 1A-E). Symptomatic leaves were collected from 54 different tobacco plants. After surface sterilization (0.5 min in 75% ethanol and 1 min in 3% NaOCl, washed three times with sterilized distilled water), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on PDA and incubated at 25°C for 5 days (Fang 2007). Three single-spore isolates, GUCC BZ6-3, GUCC LJ3-4, and GUCC XH1-13 were obtained, which were identical in morphology and molecular analysis. Therefore, the representative isolate GUCC BZ6-3 was used for further study. The colonies on PDA were villiform, greyish (Fig 1F-G). Conidia were abundant, ovoid, with 2-6 transverse septa and 1-2 longitudinal septa 12.60 (9.43 to 14.76) × 4.30 (3.57 to 5.14) µm (n=50) (Fig 1H-S). The morphological features were consistent with Alternaria alstroemeriae E.G. Simmons & C.F. Hill (Simmons 2007; Nishikawa & Nakashima, 2013). The pathogen was confirmed to be A. alstroemeriae by amplification and sequencing of the ITS, GAPDH, LSU, TEF1, and RBP2 genes using primers ITS1/ITS4, gpd1/gpd2, LSU1Fd/LR5, EF1-728F/EF1-986R, and RPB2-5F2/fRPB2-7cR, respectively (Woudenberg 2013). The sequences of the PCR products were deposited in GenBank with accession numbers ON693856 (RBP2), ON714497 (ITS), ON694345 (GAPDH), ON931420 (TEF1) and ON714499 (LSU). BLAST searches of the obtained sequences revealed 99% (565/567 nucleotides), 99% (577/579 nucleotides), 99% (908/911 nucleotides), 99% (238/239 nucleotides), and 99% (751/753 nucleotides) homology with those of A. alstroemeriae in GenBank (MH863036, KP124154, MH874589, KP125072, and KP124765, respectively). Phylogenetic analyses of the sequence data consisted of Bayesian and Maximum likelihood analyses of the combined aligned dataset (MEGA 7.0 and PhyloSuite 1.2.2). The GUCC BZ6-3 in a well-supported cluster with A. alstroemeriae (Fig 2). The pathogen was thus identified as A. alstroemeriae based on morphological characterization and molecular analyses. The pathogenicity of GUCC BZ6-3 was tested through pot assay and carried out three times (Fang 2007). Ten healthy 30-day-old tobacco plants were inoculated by spraying a spore suspension (106 spores·ml-1) of strain GUCC BZ6-3 onto leaves until runoff, and the control leaves were sprayed with sterile water. The plants were maintained at 28°C with high relative humidity (95%) in a growth chamber. The symptoms developed on all inoculated leaves but not on the control. The lesions were first visible 48 h after inoculation, and typical lesions similar to those observed on field plants appeared after 7 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses from the infected leaves but not from the noninoculated leaves. Results of pathogenicity experiments fulfilled Koch's postulates. To our knowledge, this is the first report of grey spot disease on tobacco caused by A. alstroemeriae in China. Our findings would be of great importance for the diagnosis and control of the emerging grey spot on tobacco.

First Report of Colletotrichum truncatum Causing Anthracnose in Tobacco in China.

In July 2022, large spots were observed on the leaves of tobacco in Guangxi province, China, whose shape was round and elliptical or irregular. The margins of spots were brown or dark brown with a pale yellow centre and several small black fruiting bodies. The pathogen was isolated by tissue isolation. Diseased leaves collected were cut into small pieces, sterilized with 75% ethanol for 30s and 2% sodium hypochlorite (NaCIO) for 60s, and rinsed with sterile deionized water for three times. Each air-dried tissue segment was cultured on potato dextrose agar (PDA) and incubated at 28℃ for 5 to 7 days in the dark (Wang et al. 2022). A total of six isolates were isolated, with differences in colony shape, edge type and colony colour, and aerial mycelium morphology, with the colony shape round or subrounded, and the edge rounded crenate, dentate or sinuate. The color of the colony was initially light yellow, then gradually changed to yellow and dark yellow. After 3-4 days, white aerial mycelia gradually grew up, which was peony-like or covered the whole colony, thus the color of the colony appeared white, and then gradually changed to orange, gray or nearly black, and all six isolates rarely produced conidia, which was consistent with the description of previous reports(Mayonjo and Kapooria 2003, Feng et al. 2021, Xiao et al. 2018). Conidia were hyaline, aseptate, and falcate, with the size of 7.8 to 12.9 × 2.2 to 3.5 µm. For molecular identification, the colony PCR method was used to amplify the internal transcribed spacer(ITS), actin(ACT), chitin synthase(CHS), and beta-tubulin(TUB2) loci of the six isolates using primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-354R, and T1/Bt2b, respectively(Cheng et al. 2014). Partial sequences were amplified, sequenced, and uploaded to GenBank (GenBank accession Nos. OP484886，OP518265，OP518266，OP756065，OP756066, and OP756067 for ITS, OP620430 to OP620435 for ACT, OP620436 to OP620441 for CHS, and OP603924 to OP603929 for TUB2). These sequences had 99 to 100% similarity with C. truncatum isolates C-118(ITS), TM19(ACT), OCC69(CHS), and CBS 120709(TUB2) in GenBank. Homology matching was performed using BLAST and a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method using MEGA (7.0) software based on ITS, ACT, CHS, and TUB2 sequences, which showed that all six isolates clustered in the same score as the C. truncatum. A pathogenicity test was performed with healthy tobacco infected with mycelial plugs (about 5 mm in diameter) of six isolates of C. truncatum from a 5-day-old culture, while negative controls on the other leaves were inoculated with sterile PDA plugs. All plants were placed in a greenhouse at 25℃ to 30℃ with 90% relative humidity. The experiment was conducted three times. Five days later, all inoculated leaves had diseased spots, whereas no symptoms appeared on negative controls. The same pathogen, C. truncatum, was identified from the inoculated leaves on the basis of morphological and molecular charchseristics as described above, fulfilling Koch's postulates. In this study, it is the first time to report that the anthracnose on tobacco was caused by C. truncatum. Thus, this work provides a foundation for controlling tobacco anthracnose in the future.