[目的]筛选有效防控烟草弯抱叶斑病的杀菌剂.[方法]采用菌丝生长速率法,测定13种杀菌剂对烟草弯孢菌叶斑病菌的生物活性,并选出生物活性较强的几种杀菌剂开展田间防效试验.[结果]室内生物活性测定筛选出的活性较强的5种杀菌剂为450 g/L咪鲜胺水乳剂、125 g/L氟环唑悬浮剂、42.4％唑醚·氟酰胺乳油、250 g/L吡唑醚菌酯乳油、10％苯醚甲环唑水分散粒剂,EC50值分别为0.2997、0.4054、1.4757、3.5247、3.9322 mg/L,田间防效试验防治效果较好的杀菌剂为450g/L咪鲜胺水乳剂和125 g/L氟环唑悬浮剂,防效均在59％以上.[结论]综合室内生物活性测定和田间防效试验的结果,推荐450 g/L咪鲜胺水乳剂和125 g/L氟环唑悬浮剂2种杀菌剂作为烟草弯抱菌叶斑病的优选防治药剂.

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.

[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.

比较不同抗性烟草品种在赤星病胁迫下的光合生理响应差异可揭示赤星病和光合作用之间的关系,有助于进一步明确烟草赤星病的抗性机理。本实验采用盆栽方法,分析赤星病胁迫对2个烟草抗、感病品种JYH和CBH的光合作用、叶绿素荧光特性、光合色素、光合碳同化关键酶以及叶黄素循环的影响。结果显示,在5 d时,赤星病胁迫对JYH的一系列光合生理活动均未产生不利影响。赤星病胁迫明显抑制9d时的JYH和5～9 d时的CBH的叶绿素荧光参数、光合色素含量、光合碳同化关键酶活性及叶黄素循环功能,最终表现为光合作用下降。JYH的光合作用降低是由气孔限制因素引起,而非气孔限制因素是CBH的光合作用被抑制的主要原因。与CBH相比, JYH具有较高的PSII最大光化学效率（Fv/Fm）、PSII实际光化学效率（ΦPSII）、光化学淬灭系数（qp）、非光化学淬灭系数（NPQ）和叶绿素a、叶绿素a+b、类胡萝卜素含量以及核酮糖-1,5-二磷酸羧化/加氧酶（Rubisco）、景天庚酮糖-1,7-二磷酸酯酶（SBPase）、果糖-1,6-二磷酸酯酶（FBPase）活性,且其花药黄质（A）、玉米黄质（Z）、叶黄素循环库（A+V+Z）含量与叶黄素循环脱环氧化状态（A+Z）/（A+V+Z）也显著大于CBH。研究结果表明,赤星病胁迫对抗病品种光合生理活动的危害程度小于感病品种,有利于保持较高的光合作用强度,从而具有较强的抗病性。

Nutritional correlations between plants and pathogens can crucially affect disease severity. As an essential macronutrient, the availability of nitrogen (N) and the types of N content play a fundamental part not only in energy metabolism and protein synthesis but also in pathogenesis. However, a direct connection has not yet been established between differences in the level of resistance and N metabolism. Pertinently, former studies hold ammonia (NH3) accountable for the development of diseases in tobacco (Nicotiana tabacum L.) and in some post-harvest fruits. With a purpose of pinpointing the function of NH3 volatilization on Alternaria alternata (Fries) Keissl pathogenesis and its correlation with both N metabolism and resistance differences to Alternaria alternata infection in tobacco, leaf tissue of two tobacco cultivars with susceptibility (Changbohuang; CBH), or resistance (Jingyehuang; JYH) were analyzed apropos of ammonia compensation point, apoplastic NH4+ concentration, pH value as well as activities of key enzymes and N status. At the leaf age of 40 to 60 d, the susceptible cultivar had a significantly higher foliar apoplastic ammonium (NH4+) concentration, pH value and NH3 volatilization potential compared to the resistant one accompanied by a significant reduction in glutamine synthetase (GS), which in particular was a primary factor causing the NH3 volatilization. The NH4+ concentration in CBH was 1.44 times higher than that in JYH, and CBH had NH3 compensation points that were 7.09, 6.15 and 4.35-fold higher than those of JYH at 40, 50 and 60 d, respectively. Moreover, the glutamate dehydrogenase (GDH) activity had an upward tendency related to an increased NH4+ accumulation in both leaf tissues and apoplast but not with the NH3 compensation point. Collectively, our results strongly suggest that the accumulation of NH3 volatilization, rather than NH4+ and total N, was the primary factor inducing the Alternaria alternata infection in tobacco. Meanwhile, the susceptible cultivar was characterized by a higher N re-transfer ability of NH3 volatilization, in contrast to the disease–resistant cultivar, and had a stronger capability of N assimilation and reutilization. This study provides a deeper understanding of the pathogenicity mechanism induced by Alternaria alternata, which is useful for breeding Alternaria alternata-resistant varieties of tobacco, at the same time, our research is also conducive to control tobacco brown spot caused by Alternaria alternata in the field.

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.

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.

为进一步明确烟株内源激素与赤星病抗性的关系,以抗赤星病烟草品种净叶黄和感病品种长脖黄为研究对象,通过盆栽种植并接种赤星病菌,比较不同抗性品种内源激素含量对赤星病胁迫的响应差异.研究结果显示,在赤星病胁迫下,净叶黄、长脖黄的脱落酸(ABA)含量和乙烯(ET)释放量均呈上升趋势,而生长素(IAA)以及细胞分裂素(CTK)含量表现为降低.同时,赤星病胁迫导致净叶黄的赤霉素(GA)、茉莉酸(JA)含量增加,引起长脖黄的JA含量降低,GA含量则先增加后降低.与净叶黄相比,长脖黄的ABA含量增幅较小,而GA、IAA、CTK与JA含量降幅较大,并且ET释放量较高.综上,在赤星病胁迫下,内源激素的含量差异以及变化幅度可以反映不同品种的抗病能力.

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.

为探究分离自烟草根际的2株芽孢杆菌的种类及其抑菌促生特性,利用16S rDNA和gyrB基因序列分析法,将菌株YC2006和YC2008分别鉴定为枯草芽孢杆菌(Bacillus subtilis)和解淀粉芽孢杆菌(Bacillus amyloliquefaciens).菌株对烟草赤星病病原(Alternaria alternata)、烟草黑胫病病原(Phytophthora parasitica)和烟草立枯病(Rhizoctonia solani)病原均具有明显的抑制作用,对烟草立枯病病原的抑制率分别达到60.35％和68.08％.菌液浸种处理可显著提高烟草种子发芽势,在育苗基质中添加菌剂可显著提高烟苗的株高、茎围、最大叶面积、地上部鲜质量、地下部鲜质量、总根长和总根表面积.2株芽孢杆菌对烟草主要真菌病原具有拮抗活性,同时兼具促进种子萌发和烟苗生长的作用,具有良好的研究开发应用前景.

为烟田合理利用食蚜瘿蚊进行烟蚜生物防控提供依据,采用室内笼罩法和田间罩笼移笼法相结合,研究食蚜瘿蚊大田控制烟蚜种群虫口减退率及控害指数的效果.结果 表明:以益害比为1∶10和1∶15释放食蚜瘿蚊对烟蚜的控制效果最好,7d后虫口减退率可达95.44％和96.44％,显著高于其余处理;食蚜瘿蚊对烟蚜的田间防效以6月2日前后进行释放效果最好,控害指数为0.644 5;不同笼罩状态下田间烟蚜发生量总体趋势为天敌释放区罩笼组＞自然对照区自然组＞天敌释放区开放组.食蚜瘿蚊对大田烟蚜的防控以益害比为1∶15,在每年5月下旬至6月上旬连续释放2次的效果最佳.

In order to understand the relationship between soil bacterial community structure and tobacco bacterial wilt disease,the soil bacterial composition and diversity in tobacco bacterial wilt disease nursery were investigated by 454 pyrosequencing.The results indicated that a total of 1 963 effective reads were obtained from soil sample,which belonged to 14 phyla of bacteria,uncultured bacteria and unclassified bacteria.Proteobacteria,Acidobacteria,Chloroflexi,Acidobacteria,Planctomycetes,Actinobacteria and Gemmatimonadetes were dominant taxa,accounting for 29.91%,17.10%,13.73%,11.46%,7.08%,6.40% of the total bacterial groups in the soil,respectively.The lower abundance of Bacteroidetes and Actinobacteria might be associated with the occurrence of tobacco bacterial wilt disease.

In order to provide a basis for the reasonable application of insecticides and controlling M. per-sicae in Guizhou province,the resistance level of 34 M. persicae field populations to methomyl andλ-cyha-lothrin collected from nine districts was determined with dipping method. The results showed that com-pared with the susceptible strain, the average resistance ratio of aphids collected from nine districts to methomyl ranged from 2. 53 to 5. 13,and that toλ-cyhalothrin ranged from 3. 15 to 4. 44. For methomyl, the average resistance of aphids collected from Liupanshui developed to low level,and the average sensi-tivity of aphids collected from Bijie, Qiannan, Anshun, Tongren, Qiandongnan and Zunyi was at the de-creasing level. For λ-cyhalothrin,the average sensitivity of aphids in all nine districts was at the decrea-sing level. In all the 34 populations tested,the resistance to methomyl of aphids collected from Shuicheng, Bijie,Jinsha, Changshun, Guiding, Ziyun and Dejiang developed to low level and the resistance ratio ranged from 5. 20 to 8. 56. The resistance toλ-cyhalothrin of aphids collected from Ziyun,Panxian,Xingyi and Zhenyuan developed to low level and the resistance ratio ranged from 5 . 39 to 5 . 82 . The other strains were still sensitive or at the sensitivity-decreasing level to the tested insecticides.

In order to investigate the resistance level of Phytophthora parasitica var.nicotianae to metalaxyl in Guizhou Province,samples of tobacco black shank were collected in major tobacco growing areas in 12 cities and counties of the province,and 98 P .parasitica strains were isolated.The sensitivity of these strains to metalaxyl was determined by mycelial growth rate.The results showed that the sensitivity of P .parasitica from different districts differed significantly,with EC50 values ranged from 0.419 2 to 20.486 1 μg/mL and the average EC50 val-ue between 0.551 9 and 10.986 0 μg/mL.The lowest and the highest resistance level were 1.00 and 48.87,re-spectively.The average resistance level of P .parasitica from different districts was between 1.32 and 26.21.The resistant isolates existed in Zunyi,Xingren,Guiding,Hezhang and Xingyi regions.The resistance levels of some strains in Zunyi were high,but the other strains were at the middle resistance level.

为筛选对烟草花叶病毒（ tobacco mosaic virus ，TMV）具有拮抗活性的放线菌，以烤烟Coker 176、K326为材料，采用生物活性测试和半叶枯斑法，测定47株放线菌菌株发酵上清液抗TMV的活性，对具活性的菌株进行分子鉴定和系统发育分析。结果表明，6株放线菌的发酵上清液具有一定的抗TMV活性，其中菌株F187、F349、F417发酵上清液对TMV具有明显钝化作用，发酵上清液与TMV作用30 min后抑制率分别为57．18％、60．27％、70．19％；分子鉴定和系统发育分析显示菌株F187、F417、F349均属链霉菌；链霉菌F187、F349、F417的代谢产物对TMV有较高的抑制活性，值得在菌种鉴定、发酵条件优化、活性物质的分离与鉴定等方面进行深入研究。

Objectives] Tobacco bacterial wilt is one of the most serious soil-borne diseases for tobacco production.The biocontrol of tobacco bacterial wilt has become a hot topic recently.To investigate effect of specific bio-organic fertilizers on biocontrol of tobacco bacterial wilt, a two-year field experiment was conducted to investigate the biocontrol efficiency and soil microbial community.[Methods] In this experiment, two antagonistic strains ( L-25 and L-9 ) with strong inhibitory effects on Ralstonia solanacearum in vitro were isolated from rhizosphere soil in a severely wilt-diseased field.Two solid bio-organic fertilizers ( BOF) were secondly fermented by the two antagonists, strain L-25 and L-9.A two-year field experiment was conducted in Anhui Province.The biocontrol efficiency was investigated at 50 d and 105 d after tobacco transplanting.The effects of the bioorganic&nbsp;fertilizers on soil microbial community and tobacco yield were measured and analyzed.In order to reveal the biocontrol mechanism of the bioorganic fertilizers, the colony counting method, biolog phenotype analysis and denaturing gradient gel electrophoresis ( DGGE ) were used to analyze microbial population, functional and structural diversity of microorganisms, respectively in rhizosphere soil after the application of the bioorganic fertilizers.[Results]1) At 50 d after the transplanting, compared with the control, the control efficiencies of the BOF treatments reach to 82.2%and 96.2%for the first and second year, respectively, and to 75.2%for the first year and 95.4%for the second year at 105 d after the transplanting.2) The tobacco yields are increased to 2212.5 kg/ha and 1475.5 kg/ha in the first and second year in the BOF treatments which are about 2.4 times and 2.6 times of control, respectively.3) The populations of rhizosphere cultivated bacteria and actinomycetes of the BOF treatments are significantly higher than those of the control, while the counts of fungi of the BOF treatments are significantly lower than those of the control ( P <0.05 ) .In the first year, the populations of antagonists are increased by 241.8 times and 13.4 times of the control in 50 d and 105 d after the transplanting in the BOF treatments, while the populations of pathogen are 19.7% and 56.6% of the control, respectively.In the second year, the counts of antagonists are increased by 111.0 times and 26.7 times of the control in 50 d and 105 d after the transplanting in the BOF treatments.However, the pathogen counts in the BOF treatments are 9.1% and 31.4%of the control.4) The Shannon Index, Simpson Index and Mclntosh Index of soil microbial community of the BOF treatments are significantly higher than those of the control in both 50 d and 105 d after the transplanting in the two years.5) There are significant differences of soil microbial structural diversity between the BOF treatments and the control.The denaturing gradient gel electrophoresis ( DGGE) patterns of soil bacteria and fungi of the BOF treatments and the control treatment belong to two corresponding clusters,which suggests that bacterial species are increased and fungi species are decreased under the application of BOF.[Conclusions]The application of tobacco specific BOF in the severely wilt-diseased tobacco field can significantly reduce the wilt disease incidence and thus increase tobacco yield.Bioorganic fertilizer depresses the population of the pathogen and increases the counts of antagonists.Meanwhile, it can increase the functional diversity of the soil microbial community and improve the microbial structural diversity.Therefore, the soil micro-ecology keeps balanced.

Using 4 soil pretreatment method and 4 medium, we isolated 607 strains of actinomycetes from 29 tobacco rhizosphere soil samples collected from Yunnan, Guizhou and Henan. The protease, chitinase, cellulase and amylase activity of 42 representative strains were tested byclarity circle method on plate. Genes encoding typeⅠandⅡpolyketide synthases( PKS-Ⅰ, PKS-Ⅱ), nonribosomal peptide synthase(NRPS) were screened by PCR. Four isolates were preliminary classified by its 16S rRNA gene sequences. The 42 tested strains of actinomycetes showed different enzyme activities. There were four strains showed positive results for PKS(typeⅠandⅡ)and NRPS genes screening, the four strains belongs to Streptomyces according to 16S rRNA sequence analysis.

To prolong streptomycin sulfate effective time,streptomycin sulphate-loaded nanocapsule coated with chitosan and γ-polyglutamic acid was prepared and characterized.The composite nanocapsule was prepared using a simple,mild ionic-gelation method,and streptomycin sulphate was loaded during gelating.The effects of streptomycin sulfate dosage and chitosan/poly-γ-glutamic acid mass ratio on the process of encystation were tested.The results indicated that loading efficiency increased with increasing of streptomycin sulfate dosage,encapsulation efficiency increased and loading efficiency decreased when the mass ratio of chitosan and γ-polyglutamic acid decreased.When the dosage of streptomycin sulfate was 10 mg,and the mass ratio of chitosan to γ-polyglutamic acid was 20 ∶17,the highest encapsulation efficiency(85.2%) was gotten,and loading efficiency reached 46.8%.The Fourier transform infrared spectroscopy and differential scanning calorimetric analysis confirmed that streptomycin sulfate was loaded in nanocapsule,and the solid nanocapsule with average diameter 20-50 nm was observed under transmitted electronic microscopy.Sustained-releasing of streptomycin sulfate was observed under simulation releasing test.

为提高甲霜灵的抗病效果与减少农药残留,以壳聚糖包埋甲霜灵制备复合纳米粒子,然后将纳米溶液制备成壳聚糖纳米粒子/海藻酸钙膜,并对该膜的释放特征进行表征;将甲霜灵包埋膜分别在烟草移栽及移栽后10 d置于烟草根部,移栽后90d防治黑胫病的效果仍达77.8％,但其防治效果要低于对照甲霜灵·锰锌的防治效果.