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.

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.

为筛选能有效抑制烟草叶斑病新病原Stagonosporopsis vannaccii的药剂,采用菌丝生长速率法测定了啶酰菌胺等8种杀菌剂对广西贺州分离菌株GHZS80、GHZS21、GHFC63、GHFC54菌丝生长的抑制作用,选择毒力较高的4种杀菌剂开展田间试验,验证其实际防病效果.室内毒力测定结果表明,啶酰菌胺和氟吡菌酰胺的毒力最高,对4株供试菌的平均EC50分别为0.017、0.099 mg/L,其次是咪鲜胺(0.349 mg/L)和苯醚甲环唑(0.978 mg/L).田间试验结果表明,以50％啶酰菌胺水分散粒剂355g(每公顷有效成分用量)或41.7％氟吡菌酰胺悬浮剂65 g喷施3次对该病的田间防效较高,末次药后7 d的防效分别为71.11％、68.67％,显著高于其他田间施药处理.本研究结果可为防控由S.vannaccii引起的烟草叶斑病提供用药参考.

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.

Tobacco (Nicotiana tabacum) is one of the most important industrial crops in the world. Its leaves are the main raw material for cigarettes, but they are often threatened by fungal pathogens in the production process (Wang et al. 2022). From May to June 2022, a disease of tobacco (cv K326) (15% of plants) in a 0.3-ha field in Jingxi of Guangxi Province showed symptoms of local necrosis and perforation of middle and basal leaves (Fig S1). Pieces of leaf tissue (3 × 3 mm) were excised from the edge of the necrotic lesion of each plant, treated with 75% ethanol for 10 s, soaked in 2% NaClO solution for 1-2 min, rinsed with sterile water for three times, and then plated on potato dextrose agar（PDA）medium and incubated at 28°C. Isolate TJYA13 was used for subsequent studies. After 8 days, the colony margin was yellowish brown and irregular, the center was black and plicated. The isolate TJYA13 was incubated on oatmeal agar medium at 28°C for 4 days, and many pseudothecia were observed embedded on the surface of the medium. Pseudothecium was globose or subglobose, dark brown, and size was 184.7-304.7 µm × 187.5-340.5 µm (n=20). Ascospores were usually wrapped by the saccate ascus in pseudothecium, cylindrical or ellipsoidal, with 5-6 transverse septa, and size was 12.2-18.5 µm × 35.6-51.8 µm (n=80). The morphological characteristics of ascospores were consistent with a Leptosphaerulina species (Hou et al. 2020). For accurate identification, the genomic DNA of isolate TJYA13 was extracted with Ezup Column Fungi Genomic DNA Purification Kit (Sangon, Shanghai, China). The ITS region, 28s ribosomal RNA (LSU), β-tubulin (TUB), and RNA polymerase II second largest subunit (RPB2) were amplified with primers ITS1/ITS4 (Gardes and Bruns 1993; White et al. 1990), LROR/LR7 (Rehner and Samuels 1994), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), and RPB2-5F2/fRPB2-7cR (Liu et al. 1999), respectively and sequenced at Sangon Biotech (Sichuan, China). The sequences were deposited in GenBank (accession nos. OP926927, OP926933, OP939419, OP939422). The phylogenetic analysis grouped the isolate TJYA13 within the L. americana clade (Fig S2) (Hou et al. 2020). Pathogenicity of the isolate TJYA13 was verified on four healthy tobacco plants (cv K326). The mycelial plugs were inoculated on leaves sterilized with 75% ethanol, and control plants were inoculated with sterile PDA plugs. Plants were incubated at 28 ℃ and 78% humidity. After 10 days, the leaves inoculated with mycelial plugs had symptoms similar to those in the field, but there were no symptoms on the control leaves. L. americana were reisolated from the leaves inoculated with the mycelial plugs. To the best of our knowledge, this is the first report of L. americana causing holing disease on tobacco in China. This disease may reduce yields and lower quality of flue-cured tobacco leaf. Therefore, the emergence of tobacco holing disease should be noted to prevent potential damage to tobacco production in Guangxi. Reference 1. Hou L. W., et al. 2020. Stud. Mycol. 96: 309-396 2. Liu, Y. J., et al. 1999. Mol. Biol. Evol. 16:1799. 3. Rehner, S. A., and Samuels, G. J. 1994. Mycol. Res. 98:625. 4. Wang H. et al. 2022. Microorganisms. 10: 1890. 5. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. 6. Woudenberg, J. H. C., et al. 2009. Persoonia 22:56. The author(s) declare no conflict of interest. Funding: Funding was provided by Guangxi Zhuang Autonomous Region Tobacco Monopoly Bureau (grant no. 202,145,000,024,006). Tobacco (Nicotiana tabacum) is one of the most important industrial crops in the world. Its leaves are the main raw material for cigarettes, but they are often threatened by fungal pathogens in the production process (Wang et al. 2022). From May to June 2022, a disease of tobacco (cv K326) (15% of plants) in a 0.3-ha field in Jingxi of Guangxi Province showed symptoms of local necrosis and perforation of middle and basal leaves (Fig S1). Pieces of leaf tissue (3 × 3 mm) were excised from the edge of the necrotic lesion of each plant, treated with 75% ethanol for 10 s, soaked in 2% NaClO solution for 1-2 min, rinsed with sterile water for three times, and then plated on potato dextrose agar（PDA）medium and incubated at 28°C. Isolate TJYA13 was used for subsequent studies. After 8 days, the colony margin was yellowish brown and irregular, the center was black and plicated. The isolate TJYA13 was incubated on oatmeal agar medium at 28°C for 4 days, and many pseudothecia were observed embedded on the surface of the medium. Pseudothecium was globose or subglobose, dark brown, and size was 184.7-304.7 µm × 187.5-340.5 µm (n=20). Ascospores were usually wrapped by the saccate ascus in pseudothecium, cylindrical or ellipsoidal, with 5-6 transverse septa, and size was 12.2-18.5 µm × 35.6-51.8 µm (n=80). The morphological characteristics of ascospores were consistent with a Leptosphaerulina species (Hou et al. 2020). For accurate identification, the genomic DNA of isolate TJYA13 was extracted with Ezup Column Fungi Genomic DNA Purification Kit (Sangon, Shanghai, China). The ITS region, 28s ribosomal RNA (LSU), β-tubulin (TUB), and RNA polymerase II second largest subunit (RPB2) were amplified with primers ITS1/ITS4 (Gardes and Bruns 1993; White et al. 1990), LROR/LR7 (Rehner and Samuels 1994), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), and RPB2-5F2/fRPB2-7cR (Liu et al. 1999), respectively and sequenced at Sangon Biotech (Sichuan, China). The sequences were deposited in GenBank (accession nos. OP926927, OP926933, OP939419, OP939422). The phylogenetic analysis grouped the isolate TJYA13 within the L. americana clade (Fig S2) (Hou et al. 2020). Pathogenicity of the isolate TJYA13 was verified on four healthy tobacco plants (cv K326). The mycelial plugs were inoculated on leaves sterilized with 75% ethanol, and control plants were inoculated with sterile PDA plugs. Plants were incubated at 28 ℃ and 78% humidity. After 10 days, the leaves inoculated with mycelial plugs had symptoms similar to those in the field, but there were no symptoms on the control leaves. L. americana were reisolated from the leaves inoculated with the mycelial plugs. To the best of our knowledge, this is the first report of L. americana causing holing disease on tobacco in China. This disease may reduce yields and lower quality of flue-cured tobacco leaf. Therefore, the emergence of tobacco holing disease should be noted to prevent potential damage to tobacco production in Guangxi.

烟草棒孢霉叶斑病是影响烟草生产的主要叶部病害之一,为明确广西烟区该病害的病原菌及其毒素亚型,本研究对采自广西烟区的烟草叶斑病病原菌采用形态学、致病性、分子生物学进行鉴定.并利用毒素蛋白基因的特异性引物和对多种寄主植物的致病性进行毒素亚型测定.结果显示,广西烟草棒孢霉叶斑病菌为多主棒孢霉(Corynespora cassiicola),并首次鉴定、分析广西烟草棒孢霉病菌的毒素蛋白亚型,其中广西烟草棒孢霉病菌15个菌株中,14个菌株的毒素蛋白亚型为Cas0,1个菌株的毒素蛋白亚型为Cas7.结果表明,Cas0基因亚型较多,在靖西市同德乡首次发现烟草棒孢霉病菌中的Cas7毒素蛋白基因.本研究明确了广西烟草棒孢霉叶斑病病菌和病菌群体毒素亚型,可为防治烟草棒孢霉叶斑病提供基础依据.

In order to effectively control the tobacco Corynespora leaf spot, the biocontrol strain YC2140 screened in the previous work of our laboratory was identified by Biolog automatic microbial identification systemlog automatic microbial identification system 16S rRNA and gyr B sequence molecular identification. Five fungicides commonly used in production and YC2140 were selected for pot and field control experiments. Results showed that the YC2140 strain was identified as Pseudomonas fluorescens by molecular method and Biolog identification system. The pot experiment results showed that under the recommended dosage（449.78 mL/hm~2 or 449.78 g/hm~2）, the control efficacy on Corynespora leaf spot from high to low was fluazinam 500 g/L SC（the control efficacy was78.77%）, prochloraz 450 g/L EW（77.85%）, tebuconazole 430 g/L SC（67.12%）, 1 × 10~8 cfu/mL YC2140 fermentation broth（61.64%） and boscalid 50% WG（46.12%）. The field trial results under the same dosage showed that the control efficacy from high to low was prochloraz 450 g/L EW（43.33%）,fluazinam 500 g/L SC（40.97%）, tebuconazole 430 g/L SC（27.94%）, 1 × 10~8 cfu/mL YC2140fermentation broth（26.15%）, 70% mancozeb WP（dosage 2998.50 g/hm~2, control efficacy 21.99%）. The results of this study can provide reference for the screening of fungicides for the control of tobacco Corynespora leaf spot disease.

为明确广西壮族自治区贺州市钟山县发生的真菌性叶斑病害的病原菌种类和生物学特性,分别采用组织分离法和活体伤口接种法对病原菌进行分离和致病性测定,利用 PCR 扩增病原菌核糖体内转录间隔区(ITS)、肌动蛋白(ACT)和几丁质合成酶(CHS)进行分子鉴定,结合形态学特征对病原菌种类进行鉴定,采用菌丝生长速率法测定病原菌生物学特性.聚类分析结果表明,病原菌与平头炭疽(Collettrichum truncatum)聚为一支,且支持率为100%,结合形态学特征,将病原菌鉴定为平头炭疽(C.truncatum),这是平头炭疽寄生烟草引起烟草炭疽病的首次报道.病原菌生物学特性测定结果表明,病原菌对环境有较强的适应性,最适培养基为营养琼脂(NA),最适温度30℃,最适光照条件为全黑暗,最适碳源为淀粉,最适氮源为蛋白胨,pH为7 时病原菌生长最快,病原菌致死温度为53℃、10 min.研究结果为病害的及时准确防治和深入研究提供了理论依据.

2022 年 5 月中下旬,长顺县生联村的水稻秧苗感病,秧苗整体矮小、叶片黄化,经实验室鉴定,该病为水稻霜霉病.本文综合叙述了霜霉病的发生特点,并指出了影响该病发生的因素,同时提出了防治建议,为长顺县水稻病害绿色防控提供参考,助力水稻产业的可持续健康发展.

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.

[目的]明确广西贺州烟区一种烟草真菌性叶斑病害的病原菌种类和生物学特性,为病害的及时防控和后续研究提供理论参考.[方法]采用组织分离法对从广西贺州烟区采集的具明显叶斑病症状的烟草叶片组织进行病原菌分离纯化,利用柯赫氏法则验证其致病性;通过形态学结合多基因(ITS、LSU、TUB2、RPB2)序列联合分析对病原菌进行种类鉴定;应用十字交叉法测定病原菌的生物学特性.[结果]经分离纯化共获得7株菌株,通过柯赫氏法则证明7株菌株均为烟草品种K326的叶斑病病原菌,7株菌株间的致病力无明显差异.基于ITS、LSU、RPB2和TUB2构建的系统发育进化树显示,7株菌株均与菌株Stagonosporopsis vannaccii LFN0148和S.vannaccii YTH-12聚为一支,且支持率为100％.生物学特性测定结果表明,7株菌株菌丝生长的最适温度为25～28℃,最适碳源为可溶性淀粉,最适氮源为蛋白胨和牛肉浸粉,最适pH为6～9;适宜培养基为马铃薯葡萄糖培养基(PDA)、燕麦培养基(OA)和南瓜汁琼脂培养基(PA),在全光照条件下菌丝生长最快,最低致死温度为50℃水浴10 min.[结论]引起广西贺州烟区烟草叶斑病的病原菌为S.vannaccii,该病原菌喜好在弱酸性至弱碱性、光照充足且营养丰富的环境条件下生长.在烟草生产过程中可通过控制烟草种植密度、加强田间管理以及采取化学、生物防治等方法对由S.vannaccii引起的烟草叶斑病进行综合防控.

广西烤烟生产因受到普通花叶病毒(TMV)感染的影响,产量和品质均遭受严重损失.为了掌握广西烟区TMV的多样性,解释其发生规律,本研究在广西壮族自治区贺州市、百色市和河池市的8个地点收集了17个仅感染TMV的烟叶样品.经过总RNA提取和逆转录得到对应的cDNA,针对TMV外壳蛋白区域设计了1对引物,经PCR扩增获得广西烟区17个TMV样品的外壳蛋白完整序列.基于TMV外壳蛋白核苷酸序列的系统发育分析结果表明,广西烟区的TMV存在着不同于我国其他地区的独特类群,在广西不同地区之间,贺州的TMV具有很近的亲缘性,来源相对单一,而百色的TMV则具有很高的多样性,体现了TMV传播途径的差异.研究结果可为广西烟区不同地区TMV的绿色防控提供参考.

[目的]明确贵州烤烟蛙眼病病原菌种类及生物学特性,为烤烟蛙眼病的综合防治提供科学依据.[方法]以采集自贵州烟区的烤烟蛙眼病病叶为材料,采用常规组织分离法和离体叶片接种法进行病原菌分离及致病性测定,利用形态学特征和分子生物学方法对病原菌进行种类鉴定,并对典型菌株进行生物学特性测定.[结果]从采集的病叶样本中共分离获得11株菌株,选取具有代表性的3株菌株YC1110、YC1111和YC1112接种到健康烤烟叶片上,接种6 d后出现典型症状,与田间症状一致,证明3株菌株为烤烟蛙眼病病原菌.ITS序列及系统发育分析表明,供试3株菌株与烟草尾孢(Cercospora nicotianae)的遗传关系很近,且菌落及孢子等形态特征亦符合烟草尾孢.生物学特性测定结果显示,病原菌在25℃、pH 7、光照条件为24 h全黑暗、碳氮源分别为果糖和牛肉浸粉、培养基为番茄琼脂培养基(TA2)时产孢量最多;在25℃、pH 7、碳氮源分别为甘露醇和蛋白胨、培养基为胡萝卜琼脂培养基(CA)和烟叶煎汁琼脂培养基(TA1)时生长最快.病原菌菌丝致死温度为54℃水浴10 min.[结论]引起贵州烤烟蛙眼病的病原菌为烟草尾孢.

Passion fruit (Passiflora edulis Sims) is a widely cultivated dicotyledonous perennial plant with woody vines (Asande et al. 2020). In November 2020, leaf blight was observed on leaves of P. edulis (cultivar: 'Panama Red') newly planted in Wangyou, Huishui county, Guizhou province, China (25°82'57" N, 106°50'49" E). The leaf blight occurred on both young and old leaves, starting from the margins, and then extended to the entire leaves. The color of the affected tissue was brown with a yellow hallo in the early period, and then gradually turned to grey. The disease incidence was 60%-70% on a 0.08-ha field. Following isolation of the potential pathogen from 12 diseased leaves, nine isolates were obtained. The colonies were white with a regular round shape at the early stage and became black with fluffy hyphae after eight days on potato dextrose agar (PDA) medium, incubated at 25°C in the dark for 10 days. The single cell conidia were solitary, spherical or slightly ellipsoidal, black, shiny, smooth, aseptate, spherical, and 8.1-13.5 μm (n=50) in diameter. Conidiophores (5.2-9.9 × 4.4-7.2 μm) were mostly reduced to conidiogenous cells and aggregated in clusters on hyphae. Conidiogenous cells were hyaline to pale brown or black, globose to ampulliform or clavate. Morphological characteristics of the isolates matched the description of the genus Nigrospora Mei Wang & L. Cai (Wang et al. 2017). For molecular identification, DNA was extracted, and PCRs were performed with primers ITS1/ITS4 for the ITS region (White et al. 1990), primers Bt2a/Bt2b for the β-tubulin gene (TUB) (Glass and Donaldson 1995), and primers EF1-728F/EF1-986R for the translation elongation factor 1-alpha gene (EF1-α) (Carbone and Kohn 1999). Representative sequences of the ITS region, EF1-α, and TUB sequences (from isolate WYR007) were deposited in GenBank (accession numbers: MW561355; MZ053463; MZ032030) and are included in the supplementary materials. BLAST analysis against sequences from previously published studies showed 99.58% (ITS region), 99.54% (EF1-α), and 99.45% (TUB) identity to Nigrospora sphaerica sequences (accession numbers: MN215808.1; MN864137.1; KY019606.1). In addition, homology was confirmed with a phylogenetic tree using concatenated sequences from ITS, EF1-α and TUB constructed with MEGA 7 for which the maximum likelihood method was used with 1,000 bootstrapping iterations. To complete Koch's postulates, conidia suspensions of isolate WYR007 (prepared from 1-month-old colonies in 0.05% Tween 20 buffer and adjusted to a concentration of 1 × 103 conidia/mL) were sprayed on 15 leaves (200 μL per leaf) of 5 one-year-old healthy P. edulis plants (cultivar: 'Panama Red'). The same number of leaves from control group plants was only treated with 0.05% Tween buffer. All plants were incubated at 26°C ± 2°C under a 16 h/8 h photoperiod and 70%-75% relative humidity (RH) after inoculation. After 14 days, symptomatic blight appeared on all inoculated leaves. In contrast, no symptoms appeared on leaves in the control group. The disease assays were repeated three times. Pure cultures were re-isolated from diseased leaves and confirmed to be N. sphaerica based on the morphological and molecular methods mentioned above (ITS region, the TUB, and the EF1-α sequences). To our knowledge, this study is the first report of N. sphaerica as a pathogen on P. edulis causing leaf blight. The identification of the pathogen could provide relevant background for its future management.s Sims) is a widely cultivated dicotyledonous perennial plant with woody vines (Asande et al. 2020). In November 2020, leaf blight was observed on leaves of P. edulis (cultivar: 'Panama Red') newly planted in Wangyou, Huishui county, Guizhou province, China (25°82'57" N, 106°50'49" E). The leaf blight occurred on both young and old leaves, starting from the margins, and then extended to the entire leaves. The color of the affected tissue was brown with a yellow hallo in the early period, and then gradually turned to grey. The disease incidence was 60%-70% on a 0.08-ha field. Following isolation of the potential pathogen from 12 diseased leaves, nine isolates were obtained. The colonies were white with a regular round shape at the early stage and became black with fluffy hyphae after eight days on potato dextrose agar (PDA) medium, incubated at 25°C in the dark for 10 days. The single cell conidia were solitary, spherical or slightly ellipsoidal, black, shiny, smooth, aseptate, spherical, and 8.1-13.5 μm (n=50) in diameter. Conidiophores (5.2-9.9 × 4.4-7.2 μm) were mostly reduced to conidiogenous cells and aggregated in clusters on hyphae. Conidiogenous cells were hyaline to pale brown or black, globose to ampulliform or clavate. Morphological characteristics of the isolates matched the description of the genus Nigrospora Mei Wang & L. Cai (Wang et al. 2017). For molecular identification, DNA was extracted, and PCRs were performed with primers ITS1/ITS4 for the ITS region (White et al. 1990), primers Bt2a/Bt2b for the β-tubulin gene (TUB) (Glass and Donaldson 1995), and primers EF1-728F/EF1-986R for the translation elongation factor 1-alpha gene (EF1-α) (Carbone and Kohn 1999). Representative sequences of the ITS region, EF1-α, and TUB sequences (from isolate WYR007) were deposited in GenBank (accession numbers: MW561355; MZ053463; MZ032030) and are included in the supplementary materials. BLAST analysis against sequences from previously published studies showed 99.58% (ITS region), 99.54% (EF1-α), and 99.45% (TUB) identity to Nigrospora sphaerica sequences (accession numbers: MN215808.1; MN864137.1; KY019606.1). In addition, homology was confirmed with a phylogenetic tree using concatenated sequences from ITS, EF1-α and TUB constructed with MEGA 7 for which the maximum likelihood method was used with 1,000 bootstrapping iterations. To complete Koch's postulates, conidia suspensions of isolate WYR007 (prepared from 1-month-old colonies in 0.05% Tween 20 buffer and adjusted to a concentration of 1 × 103 conidia/mL) were sprayed on 15 leaves (200 μL per leaf) of 5 one-year-old healthy P. edulis plants (cultivar: 'Panama Red'). The same number of leaves from control group plants was only treated with 0.05% Tween buffer. All plants were incubated at 26°C ± 2°C under a 16 h/8 h photoperiod and 70%-75% relative humidity (RH) after inoculation. After 14 days, symptomatic blight appeared on all inoculated leaves. In contrast, no symptoms appeared on leaves in the control group. The disease assays were repeated three times. Pure cultures were re-isolated from diseased leaves and confirmed to be N. sphaerica based on the morphological and molecular methods mentioned above (ITS region, the TUB, and the EF1-α sequences). To our knowledge, this study is the first report of N. sphaerica as a pathogen on P. edulis causing leaf blight. The identification of the pathogen could provide relevant background for its future management.

烟草青枯病与黑胫病为广西烟区2种重要土传病害.为了解2种病害在广西主要烟区混合发生情况,规范生产上病害药剂防治方法.2021年5～6月对广西百色市、贺州市和河池市等3市主要烟区的烟草青枯病和黑胫病发生危害进行了较为全面的调查,并对具有典型青枯病和黑胫病症状的烟株进行病原菌分离,烟草青枯菌采用鉴别培养基、多重菌落PCR扩增16S rDNA、speI和rpsS基因部分序列并测序相结合的方法鉴定;烟草黑胫病菌采用形态特征与ITS序列测定相结合的方法鉴定.从12个地点采集发生烟草青枯病、黑胫病标本116份,分离到烟草青枯病菌(Ralstonia solanacearum)103株,烟草黑胫病菌(Phytophthora nicotianae)13株.结果显示,烟草青枯病在广西百色市、贺州市和河池市等3市主要植烟区普遍发生,其中靖西市新甲乡、地州镇和鲁利镇,钟山县公安镇,富川县福利镇和葛坡镇等地2种病害混合发生情况最为普遍.结果表明,烟草青枯病与黑胫病在广西主要烤烟种植区广泛流行,且2种病原菌常混合侵染烤烟.

[目的]近年来由多主棒孢霉病菌引起的烟草棒孢霉叶斑病在广西烟区危害日益加重.为筛选出防治烟草棒孢霉叶斑病的优良杀菌剂,并针对棒孢霉病原菌对甲氧基丙烯酸酯类(QoIs)杀菌剂的耐药性进行初步探究.[方法]采用菌丝生长速率法测定10种杀菌剂对来自广西贺州、百色、河池3个烟区8株烟草棒孢霉病菌菌株的生物活性;扩增并分析了多主棒孢霉细胞色素b基因(Cytb)部分序列,以期探寻该病原菌对QoIs类的2种杀菌剂敏感性下降的分子机制.[结果]供试杀菌剂对烟草棒孢霉病菌菌丝生长均有不同程度的抑制,杀菌剂效果比较好的是咪鲜胺、氟啶胺和吡噻菌胺,EC40值分别为(0.049±0.027)、(0.063±0.045)、(0.286±0.077)mg/L.[结论]广西烟草棒孢霉叶斑病防治中杀菌剂应优先选用咪鲜胺、氟啶胺.广西烟区烟草棒孢霉病菌群体对QoIs类杀菌剂产生很高的耐药性,因此田间生产中不推荐再使用该类杀菌剂防治烟草棒孢霉叶斑病.且经试验发现多主棒孢霉抗性菌株与抗性相关的位点未出现Cytb基因突变.上述结果可为烟草棒孢霉叶斑病的有效防治提供依据,同时可为探寻烟草棒孢霉病菌对甲氧基丙烯酸酯类(QoIs)杀菌剂敏感性下降的分子机制提供参考.

为明确烟草棒孢霉叶斑病病原菌产孢的最适条件,采用制作孢子悬浮液于显微镜下计数的方法,研究不同温度、pH值、诱孢方式以及碳氮源等因素对病原菌产孢量的影响.研究结果表明,病原菌在不同的碳氮源中,以果糖和硝酸钠的产孢量最高;在PSA培养基上,病原菌产孢量最多的条件是:温度30℃,pH值7,诱孢方式为将健康离体烟叶洗净,剪成2 cm×2 cm长的方块,121℃高压灭菌20 min放凉后,置于接种的菌饼表面,12 h近紫外光照射/12 h黑暗交替培养.在PDA培养基上,病原菌产孢量最多的条件是:温度28℃,pH值6,诱孢方式为将健康离体烟叶洗净,剪成2 cm×2 cm长的方块,121℃高压灭菌20 min放凉后,置于接种的菌饼表面,12 h光照培养/12 h黑暗交替培养;12 h近紫外光照射/12 h黑暗交替培养.大量的产孢是病原菌试验研究的基础,本研究明确了烟草棒孢霉叶斑病病原菌产孢的最适条件,可为烟草棒孢霉叶斑病的致病机制等试验研究提供相关理论依据.

烟草专业是面向行业培养从事烟草科技创新和生产实践的高素质应用型人才的本科专业,"烟草病虫害"课程实习是烟草专业本科生培养过程中实现病虫害防控生产实践同植物保护基本知识相结合的重要环节,深化本门课程的产教融合是锻炼学生创新实践能力的主要途径之一.在产教融合的背景下,对烟草病虫害实习地点选择、实习内容和开展方式、考核形态与课程思政等方面进行了探讨和建议,以期培养高素质的符合烟草行业需求的应用型人才.

[目的]明确贵州省烟草附球菌叶斑病病原菌种类及生物学特性,为烟草附球菌叶斑病的防控提供理论依据.[方法]以采集自贵州省烟区的烟草附球菌叶斑病叶为材料,使用常规组织分离法和离体叶片接种法分别对其病原菌进行分离和Koch's法则验证,使用形态学特征结合核糖体内转录间隔区(ITS)、28S rRNA(LSU)、β-微管蛋白(tub2)和RNA聚合酶II第二大亚基(rpb2)部分序列的多核苷酸序列系统学分析方法对病原菌进行鉴定,并使用菌落生长法研究病原菌生物学特性.[结果]从烟草附球菌叶斑病叶片病斑上分离获得病原菌(标记为YC1105),经ITS、LSU、tub2和rpb24个核苷酸片段序列和系统发育分析,菌株YC1105与Epicoccum latusicollum聚为一支,且支持率为100％;在PDA培养基上菌丝为红色,分生孢子椭圆形,单胞,大小为4.35～6.44μm×2.10～3.27μm,分生孢子器椭圆形,无刚毛,NaOH颜色反应呈阳性,其特征与Epicoccum latusicollum相符,结合形态特征和分子生物学方法将病原菌鉴定为E.latusicollum.病原菌生物学特性测定结果表明,菌株YC1105菌丝生长最适培养基为胡萝卜琼脂培养基,在5和10℃生长缓慢,最适温度为28℃,最适碳源为麦芽糖,最适氮源为牛肉浸粉,尿素不利于菌丝生长;病原菌在pH 4～10范围内均能生长,最适pH为6,光照对菌丝生长影响不显著(P>0.05);菌丝致死温度为49℃,水浴10 min.[结论]明确引起贵州烟草附球菌叶斑病的病原菌为Epicoccum latusicollum,该病原菌适宜在弱酸性条件下生长,菌丝生长温度范围较宽.