赤星病是烟草上的重要叶部病害,每年给烟草生产造成巨大损失,培育抗赤星病品种是防治烟草赤星病为害的根本措施,而对赤星病抗性关联遗传位点的发掘、鉴定和利用是培育抗赤星病烟草品种的关键基础.本研究利用抗赤星病的烟草材料'Beinhart 1000-1'和加工品质好但不抗赤星病的烟草品种'K326',通过种间杂交和F1代自交分离,创建了'Beinhart 1000-1'和'K326'的F2代杂交分离群体.随后,通过赤星病抗病性鉴定,本研究在F2代分离群体中选择了 19株抗赤星病材料和19株感赤星病材料,分别进行了基因组DNA提取,并通过BSA法进行了基因组重测序.在重测序数据分析基础上结合表型鉴定结果,本研究鉴定出差异SNP位点约170万个,进行烟草赤星病抗性关联SNP位点分析,找到赤星病抗性相关基因10个,促进了烟草抗赤星病分子育种的发展.

Rebuilding soil healthy microbiota is very important for preventing bacterial wilt. A 3-year-long field trial was conducted in China as follows: T1 (conventional fertilization), T2 (T1 + liming), T3 (T1 + bioorganic fertilizer), and T4 (T2 + bioorganic fertilizer). Fluorescence quantitative PCR and high-throughput sequencing were employed to study the dynamics of Ralstonia solanacearum population, microbial community, and network organizations between bacteria and quality-related variables. After 3 years of bioremediation, the control efficacy of tobacco bacterial wilt reached 61.30% and the occurrence delayed by approximately 40 days in T4, which had the highest tobacco yield and output value. The pathogen population of T4 remained below 10 6 copies/g soil during the entire growth period. Role-shifts prevailed among the network members. Microbes were unipathically associated with variables in T1 but multiplex in T4. In conclusion, soil bioremediation rebuilds a healthy soil microbiota and forms a more interactive and relevant micro-system, thus effectively controlling tobacco bacterial wilt. Key points • This is the first time to effectively bio-control tobacco bacterial wilt in practical production in China, as well as to high-efficiently use the organic waste, thus promoting the organic cycling of the environment. • Soil bioremediation can effectively control soil-borne disease by rebuilding soil healthy microbiota and reducing abundance of pathogenic bacteria, thereby to prevent the soil borne disease occurrence. • After the soil remediated, microbes associated with soil and tobacco characteristics changed from unipathical to multiplex, and the keystone species play different roles compared with the original soil, thus signifying the complexity of multi-species interactions and achieving a closely relevant micro-system, which was ecologically meaningful to the environment.

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.

植物的根从土壤中吸收水分和养分,并将其运输至地上部供植物生长发育所需.内皮层是包围根部维管系统的最内层的细胞层,其分化而产生凯氏带和木栓层2个重要的质外体屏障.其中,木栓层近年被发现其在植物水分、养分根部径向运输的意义甚至不亚于凯氏带.内皮层分化在植物响应广泛的非生物胁迫和生物胁迫的过程中也起到至关重要的作用,特别内皮层中的凯氏带和木栓层通过自身的改变来响应环境变化,以提高对环境的适应力.因此,本文综述了近年来内皮层分化可塑性的最新研究进展,重点阐述了凯氏带和木栓层与环境胁迫、矿质元素吸收,以及生物胁迫的关系,以期为内皮层的可塑性在植物生理功能调控的理论与实践中提供参考.

Tobacco (Nicotiana tabacum) is an important economic crop and widely cultivated in rural areas in south of China. A previously uncharacterized disease was observed on field-grown tobacco during 2020 and 2021 around Tongren city, Guizhou province of China (27°59'25.73" N, 108°7'2.43" E). The disease mainly occurred from fast growing period (about 13-16 leaves) to leaf maturity stage. In severely diseased areas, the incidence rate was between 20%-100%. Symptoms first began as yellow-brown necrotic spots on leaves, then merged into larger irregular necrotic spots surrounded by chlorotic halos. Similar lesions were also found on the stems. Ten symptomatic leaf and stem samples were collected from the different infected plants for pathogen isolation. The small pieces of discolored tissues were surface-disinfected with 2% sodium hypochlorite for 3 min and 75% ethanol for 30 s, rinsed three times with sterile water, and blotted on sterile filter paper, placed on potato dextrose agar thenincubated at 28°C in the dark for 3-4 days. The obtained isolates were purified through single-spore culture. Colonies were initially white and fluffy in appearance, later turning gray. Hyphae were smooth, branched, septa, transparent or light brown. Spores were solitary, oblate or nearly spherical, dark brown to black, smooth, 14.3 to 16.1μm × 11.8 to 15.2 μm in diameter. DNA of fungal isolates were extracted using Fungi Genomic DNA Extraction Kit (Solarbio, Beijing, China), the internal transcribed spacer (ITS) of the ribosomal DNA, β-tubulin (TUB2) gene and translation elongation factor 1-alpha (TEF1-α) were amplified with primers ITS1/ITS4, βt2a/βt2b and EF1-1728F/EF1-986R, respectively. The resulting ITS, TUB2 and TEF1-α sequences were deposited at GenBank, NCBI under accessions MZ882151, MZ927749, MZ927747, respectively. The sequence identity of ITS, TUB2 and TEF1-α with those of Nigrospora oryzae strains HBN (KU254608), HGUP191068 (MZ724102) and LC7307 (KY019409) were 99.64%, 99.29% and 99.65%, respectively. Based on morphological features and phylogenetic analysis, the pathogen was identified as N. oryzae (Wang et al. 2017). Pathogenicity tests were conducted by placing agar plugs-containing fungal mycelia and agar blocks (control) on leaves of tobacco plants grown at 28°C with 60% humidity in greenhouse. Symptoms appeared on the pathogen inoculated leaves seven days after inoculation, whereas the control treatment remained symptomless. The pathogens were reisolated from diseased leaves and identified as N. oryzae based on morphological, molecular and phylogenetic analysis, which were fulfilling Koch's postulates. This pathogen was recently identified from watermelon and kiwifruit in the Guizhou (Far and Rossman, 2021). To our knowledge, this is the first report of leaf spot caused by N. oryzae on Nicotiana tabacum in China.

本研究采用2000-2015年气象站点数据,分析贵州不同烟区主要气象因子的时间、空间动态特征,结合烤烟种植适宜条件与省外邻近烟区进行比较,解析贵州烟区气象条件的生态适宜性,阐明贵州烟区大田不同生育期的主要气象资源匹配、限制问题,并提出相应的农艺解决措施.结果表明:(1)贵州烟区对比邻近烟区日均气温总体呈现先升高后下降的趋势,日均气温变异系数在前期和后期较高,中期相对稳定;黔西地区日均气温未满足烤烟生育期内对热量条件的要求;(2)贵州烟区及对比烟区降水基本集中在年积日(day of the year,DOY)121～271的时间范围内,该时期降水量变异系数也相对较低且变幅较小,降水总量为838.51 mm,满足了烤烟不同生育期对降水的需求;(3)贵州烟区日照时数在DOY 1～181和DOY 271～361时间范围内明显偏低,变异系数偏大;在DOY 182～270的时间范围内日照时数及其变异系数与对比烟区基本吻合;(4)与对比烟区相比,相同时间贵州烟区相对湿度偏高,尤其以DOY 1～166和DOY 286～361时间段内差异显著;(5)基于气候资源匹配下烤烟生育期时间优化研究后,黔北地区4月20日左右、黔东地区4月26日左右、黔南和黔中地区4月21-26日左右、黔西地区5月1日左右是当地烤烟适宜移栽期.综上,依据区域内不同地区主要气象指标的动态特征,结合地膜覆盖、群体结构调整等栽培措施,将烤烟移栽期适当前移有利于烤烟的优质生产,本研究为合理确定不同地区烤烟适宜移栽期提供了理论依据.

[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.

Our previous study identified that Bacillus siamensis strain LZ88 has significant antagonistic activity against a variety of pathogenic fungi, especially against Alternaria alternata, which causes tobacco brown spot. In the present study, the non-volatile compounds and volatile organic compounds (VOCs) produced by LZ88 were identified and tested for their antifungal activities. The non-volatile compounds had significant inhibitory effects on both mycelial growth and spore germination of A. alternata, with IC50 values of 24.25 mu g/mL and 29.04 mu g/ mL, respectively. HPLC-DAD-ESI-MS/MS analysis revealed that the non-volatile compounds which showed antifungal activities were iturins and macrolactin. HS-SPME-GC-MS identified ten VOCs from strain LZ88, including 8 ketones and 2 acids. Among the VOCs tested, 2-methylbutanoic acid and 3-methylbutanoic acid exhibited significant antifungal activity against A. alternata. Both 2-methylbutanoic acid and 3-methylbutanoic acid could inhibit mycelium growth of A. alternata with IC50 values of 83.10 mg/mL and 104.19 mg/mL, whereas inhibited spore germination with IC50 values of 139.63 mg/mL and 88.07 mg/mL, respectively. Furthermore, when exposed to non-volatile compounds or VOCs, the hyphae and spores of A. alternata were deformed and collapsed, and consequent hyphae without any attached conidia appeared ruptured, shrunken, and twisted. Additionally, the genetic mechanisms were analyzed by whole genome sequencing, and the gene clusters responsible for antibacterial metabolites were mined. These results indicate that strain LZ88 possessed a remarkable potential as alternative agrochemicals toward A. alternata.

采集贵州烟区耕层土壤样品与剖面土壤样品,对土壤大量和微量养分元素含量的状况及其空间与垂直分布规律进行了分析,并对碳、氮、磷、钾的生态化学计量学特征进行了研究.结果表明:贵州植烟土壤养分存在表层聚集的现象,耕层土壤氮、磷含量较高;土壤微量元素含量的空间变异程度大于大量元素;部分土壤需要补充钾、钙、镁;耕层土壤的C:N较高,C:P和N:P较低,且氮是土壤的主要限制因子.因此,贵州烟区在施肥时应重点关注氮、钾和中量元素.

AbstractBackgroundTraditional abamectin (Abm) formulations have several shortcomings, such as low water solubility, burst release behavior, poor photostability, and short persistence periods, which decrease their pesticidal activity and the risks they pose to the environment. Nanomaterial‐based pesticide delivery systems (PDSs) provide new strategies for the efficient and safe application of pesticides. Here, we developed Abm‐loaded graphene oxide (Abm/GO) as a PDS for the sustained release of Abm, which shows enhanced control efficacy against Plutella xylostella.ResultsThe hydrophobic Abm molecule was effectively loaded on GO nanocarrier by a physisorption method, which formed a uniform and stable Abm/GO nanoformulation. GO possesses high adsorption capacity and can effectively load Abm. The Abm/GO nanoformulation shows enhanced water dispersion stability and can remain stable during a 2‐year storage period in contrast to the water‐insoluble Abm. In addition, the Abm/GO nanoformulation exhibits sustained pesticide release behavior and possesses significantly improved anti‐ultraviolet properties. Thus, the Abm/GO nanoformulation shows superior pesticidal activity compared with Abm. Abm/GO showed negligible toxicity to maize seedlings, and its GO nanocarrier can reduce the cytotoxicity of Abm to A549 cells.ConclusionGO‐based PDSs can effectively overcome the disadvantages of traditional pesticides, such as their insolubility, burst release behavior, instability, and short persistence period. GO shows much future promise in agriculture in light of its industrialization potential. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

[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.

Tobacco brown spot disease, caused by Alternaria alternata, is responsible for causing significant losses in tobacco production in China. The use of biocontrol microorganisms to prevent and control this disease represent an important, environment-friendly, management strategy. In the present study, 17 bacterial strains with antimicrobial activity were isolated from 40 rhizosphere soil samples and evaluated for antifungal activity to identify an effective biocontrol agent for the control of tobacco brown spot disease in tobacco. Among the isolated strains, Bacillus siamensis, strain LZ88, exhibited the strongest antifungal activity with an inhibition rate of 81.96%. We confirmed that strain LZ88 controlled both mycelial growth and spore production in A. alternata by secreting protein-like antifungal metabolites and through the release of volatile organic compounds. Strain LZ88 also had the ability to induce plant basal immunity as evidenced by increased expression of the defense-related enzymes, peroxidase (POD) and polyphenol oxidase (PPO), in tobacco leaves. Results of greenhouse experiments indicated that LZ88 could effectively reduce the occurrence and degree of severity of tobacco brown spot disease. To the best of our knowledge, this is the first report regarding the use of B. siamensis to control brown spot disease in tobacco.

为实现对烟草叶片中烟草赤星病菌的SYBR Green Ⅰ实时定量PCR(q-PCR)快速检测,根据链格孢属ITS基因序列差异位点,设计了1对特异性引物.通过构建含有目的片段的重组质粒,建立了标准曲线,构建了q-PCR快速定量检测方法并对感病叶片进行检测,灵敏度达到1.0×10-3 ng/μL.实验样品验证表明,该方法可以对烟草赤星病菌进行快速检测和定量.

为明确贵州烟草弯孢菌叶斑病病原菌种类及生物学特性,分别采用组织分离法和离体叶片接种法对其病原菌进行分离和致病性测定,使用形态特征结合分子生物学方法对病原菌进行分类鉴定,并对其生物学特性进行研究.结果表明,该病原菌在以ITS序列构建的发育树中与棒状弯孢(Curvularia clavata)聚为一支,且支持率为100％,结合形态特征将其鉴定为棒状弯孢.生物学特性研究表明,病原菌菌丝生长最适温度为28℃,最适pH为8,在燕麦琼脂(OA)培养基上生长最快,以可溶性淀粉为碳源、牛肉浸粉为氮源时对菌丝生长最有利;菌丝致死温度为53℃,10 min,光照对菌丝生长无显著影响.研究结果可为烟草弯孢菌叶斑病防治提供理论依据.

为明确贵州烤烟棒孢霉叶斑病病原菌种类及生物学特性,本研究采用常规组织分离法对贵州省采集的烟叶进行病原菌分离与纯化,根据柯赫氏法则进行致病性测定,得到两个代表菌株YC1002和YC1104,通过形态学特征及ITS序列将其鉴定为多主棒孢霉(Corynespora cassiicola).生物学特性研究结果表明:病原菌在胡萝卜琼脂培养基(CA)上生长最快,在燕麦琼脂培养基(OA)上产孢量最多,最适生长温度为30℃,最适宜pH 6,菌丝致死温度为52℃ 10 min,光照对菌落生长影响不大.最适碳源为麦芽糖和可溶性淀粉,最适氮源为甘氨酸和苏氨酸.研究结果可为生产上烤烟棒孢霉叶斑病的有效防治提供一定的理论依据.

为了解NtMYB4a基因的表达是否与烟草多酚化合物的合成有关,本研究以NtMYB4a过表达和CRISPR/Cas9敲除植株及其野生型植株为试验材料,采用实时荧光定量PCR技术分析NtMYB4a和多酚物质合成相关酶基因PAL、C4H、4CL、HCT的表达,使用HPLC方法测定烟叶中绿原酸、芦丁等8种多酚物质的含量,并进行相关性分析.结果 表明,过表达NtMY B4a植株中,除花中的C4H外,茎、叶、花中PAL、C4H、4CL和HCT的表达量均极显著高于野生型,烟株茎、叶、花中绿原酸含量也显著增加;敲除NtMYB4a使烟株花、茎和叶中PAL、C4H、4CL和HCT的表达均下调,其中茎中PAL、HCT的表达极显著下调,使叶和花中的绿原酸、芦丁、槲皮素、莨菪亭含量均显著降低,茎中芦丁含量则显著增加,而山奈酚、芥子酸、对香豆酸和咖啡酸含量无显著性变化;NtMYB4a的表达量与绿原酸含量呈极显著正相关关系,与芦丁、莨菪亭、山奈酚、芥子酸、槲皮素、对香豆酸、咖啡酸含量无显著相关性.因此,NtMYB4基因对烟草绿原酸合成相关酶基因表达及绿原酸合成具有调控作用.本研究结果为进一步阐明烟叶中绿原酸形成的分子机制提供理论依据.

为评价不同烤烟品种的生态适应性和筛选适宜贵州毕节烟区种植的烤烟品种,以云烟87为对照,对贵烟202、湘烟5号、云烟116、CC297和CC27等5个品种的农艺性状、田间自然发病率、经济性状和感官质量进行分析,结合一年两点区域试验分析和综合指数法对各参试品种进行综合性状评价.结果表明,云烟116在两个试验点表现均优于对照,适应环境能力强,属广适性品种;CC27在黔西试验点表现优于对照,但是在七星关试验点表现较差,贵烟202和湘烟5号在黔西试验点表现良好,在七星关试验点表现与对照相当;CC297的均价、上等烟率、一年两点区域试验分析和综合指数评价均差于对照.云烟116的农艺性状、田间自然发病率、经济性状和感官质量等综合表现优于对照,适宜在毕节烟区种植.

Habrobracon hebetor (Say) is an important biological control agent for lepidopteran pests of stored products. In this study, the age-specific functional response, paralysis rate, and parasitism rate of H. hebetor under different host deprivation treatments (PC: without host deprivation, used as the control, P1d: host deprivation, but the host was removed after 1 d contact, and PW: host deprivation from beginning) were evaluated at different larval densities (5, 10, 20, 40, and 80) of the Ephestia elutella (Hübner) at 28 ± 1°C, 75 ± 5% RH and 16:8 h L:D. Ages of parasitoid females used were 2, 5, 10, and 20 d old. The logistic regression results indicated that the functional response of H. hebetor females under different host deprivation treatments was type II. The longest handling time was observed in 20-d old females, while the shortest handling time and highest maximum attack rate (T/Th) were estimated at the age of 2 d in all treatments. The paralysis and parasitism rates of H. hebetor were the highest at 2, 5, and 10-d old in all treatments. The results of this study suggest that H. hebetor females up to 10-d old can be used as an efficient biological control agent against E. elutella. The data of this study can also be used to predict the efficacy of different aged H. hebetor females in controlling E. elutella populations.