为揭示雪茄烟叶工业发酵过程中细菌群落多样性和结构变化的规律,采集雪茄茄芯烟叶混配堆积发酵过程中的样品,采用Illumina MiSeq高通量测序技术分析细菌群落多样性和结构.结果表明:①在雪茄烟叶发酵过程中,与混配发酵前相比,混配发酵完成后样品细菌群落的OTU数量和物种丰富度降低,物种多样性提高;②优势菌门由厚壁菌门(Firmicutes)转变为变形菌门(Proteobacteria);优势菌属由海洋芽胞杆菌属(Oceanobacillus)、丛毛单胞菌属(Comamonas)、芽胞杆菌属(Bacillus)转变为丛毛单胞菌属(Comamonas)、鞘氨醇杆菌属(Sphingobacterium)、Georgenia、类芽胞杆菌属(Paenibacillus);③10个菌属的平均相对丰度显著增加,分别为鞘氨醇杆菌属(Sphingobacterium)、Georgenia、类芽胞杆菌属(Paenibacillus)、细粒类极小单胞菌属(Parapusillimonas)、假单胞菌属(Pseudomonas)、DSSF69、溶杆菌属(Lysobacter)、假黄色单胞菌属(Pseudoxanthomonas)、短芽胞杆菌属(Brevibacillus)和极小单胞菌属(Pusillimonas).其中鞘氨醇杆菌属(Sphingobacterium)、Georgenia和类芽胞杆菌属(Paenibacillus)为混配发酵完成后的优势菌属;7个菌属的平均相对丰度显著降低,分别为Terribacillus、克雷伯氏菌属(Klebsiella)、Atopococcus、棒杆菌属(Corynebacterium)、unclassified_f_Carnobacteriaceae、Aerococcus和Atopostipes.因此,雪茄烟叶工业发酵过程中的细菌群落多样性提高,群落结构变化显著.

以CX-014雪茄茄衣晾制结束后的烟叶为材料,对其进行微生物分离纯化,从中筛选出了一株产酶丰富且酶活均处于中高水平的菌株H1,通过形态学和分子生物学方法对该菌株进行了鉴定,并探究了在雪茄烟叶发酵过程中添加外源苯丙氨酸和葡萄糖对该菌株降解烟叶中大分子物质、增加雪茄烟叶香气成分的促进作用.结果表明:H1菌株被鉴定为贝莱斯芽孢杆菌(Bacillus velezensis);在添加外源营养物的条件下经菌株H1发酵后的雪茄烟叶中果胶降解率达到34.93％,淀粉、木质素、蛋白质和纤维素等大分子物质的含量也明显降低;当接菌量为2.4×107 CFU/g,并添加0.25％苯丙氨酸和0.5％葡萄糖的外源物质的情况下,雪茄烟叶在37℃下发酵6 d后,烟叶中新增了异佛尔酮和2,5-十八碳二炔酸甲酯等香气物质,且β-大马酮、2-仲-丁基环戊酮等的含量明显增多,香气得到明显改善.同时,菌株H1的产香发酵成本低,操作工艺简单,可以在工业生产中推广,以提高烟叶品质.

为降低茄芯烟叶中木质素含量、改善烟叶品质,从烟叶提取液中筛选出一株具有木质素降解能力的菌株,鉴定为巨大芽孢杆菌(Bacillus megaterium).其中木质素过氧化物酶活性达到19.03 U/L,锰过氧化物酶活性达到4.35 U/L.使用该菌株进行茄芯烟叶发酵,通过单因素试验及中心组合响应面法优化,得到最佳工艺条件:接种量7％、发酵温度37℃、起始含水量25％、发酵时间6d.在此最佳条件下,茄芯烟叶木质素降解率达到23.02％,且发酵后中性香气总量较发酵前提高了30.22％.

为研究混菌发酵对雪茄烟叶品质的影响,利用从烟叶表面筛选出来的菌株C1与酵母菌作为复合微生物添加到雪茄烟叶发酵中,探究其对雪茄烟叶化学成分、香气品质的影响.结果表明,在单菌发酵后烟叶中还原糖、总糖含量范围分别在0.28％-0.32％、0.70％-0.83％,而在混菌发酵后分别在0.21％～0.26％、0.60％左右;与单独添加菌株C1发酵后相比,添加菌株C1和汉逊酵母发酵后的雪茄烟叶香气物质总量提高了 56.40％,说明加入菌株C1和酵母菌对雪茄烟叶进行混菌发酵,更有助于雪茄烟叶香气品质的改善,降低杂气.

在湖北恩施的连作土壤中分离出一种能够高效降解烟碱的细菌P3a,从形态结构特征、16S rDNA序列分析以及建构系统发育树等方面对其进行鉴定,结果表明P3a为节杆菌(Arthrobacter sp.).该菌株能在pH 7.0、温度37℃、230 r/min的条件下,36 h内对2 g/L烟碱的降解率为99.06％.并将菌株P3a应用到新鲜的烟叶中,发现它可以减少1 kg新鲜烟叶中55.11％的烟碱.鉴于其对烟碱的降解能力很强,可高效地应用于环境和烟草废弃物中烟碱降解.

不同品种雪茄烟叶晾制后叶色存在显著差异.本研究以晾制后的雪茄烟品种楚雪10号(CX-010)和楚雪14号(CX-014)烟叶为材料,运用液相色谱-串联质谱(LC-MS/MS)联用技术开展非靶向代谢组学分析,探索两品种烟叶在晾制后代谢物的相对含量差异及其与叶色的关系.结果表明,两个品种晾制后的烟叶代谢物相对含量存在显著差异.与CX-014相比,CX-010烟叶中游离氨基酸与生物碱相对含量总体较低,脂质相对含量总体较高,相差最大的32种差异代谢物包含2种氨基酸、10种酚酸、3种酰胺等物质.结合KEGG代谢通路分析,发现精氨酸和脯氨酸代谢通路中脯氨酸生物合成途径在两品种间差异显著.差异代谢物的生理功能分析显示,两品种烟叶晾制后的叶色差异主要与脯氨酸、水杨酸、黄酮类和生物碱的含量差异有关.

为明确不同雪茄品种生长发育对光强的响应差异,在室内模拟条件下,以雪茄品种BES NO H382(简称H382)和古引4号为材料,设置高光强[T200,光照强度为200±12μmol/(m2·s)]和低光强[T100,光照强度为100±12μmol/(m2·s)]两个光强处理,分析了H382和古引4号在叶片形态、生物量、光合特性等方面对光强的响应差异.结果表明,与高光强处理相比,低光强处理下,H382和古引4号叶长分别增加19.8％、8.6％,叶宽分别增加18.1％、9.3％,叶面积分别增加42.1％、15.6％,H382叶片大小对光强变化的响应更敏感.同时,低光强处理下,H382和古引4号的生物量分别降低20.8％、41.8％,叶脉密度分别降低12.4％、9.4％,叶片厚度分别降低15.7％、19.7％,古引4号的生物量和叶片厚度对光强变化的响应更敏感.低光强处理下H382的表观量子效率升高34.4％,最大净光合速率和光补偿点分别降低21.9％、38.0％,而古引4号无显著变化.综上,H382和古引4号均对光强变化有一定的响应,可望通过遮荫等栽培措施调控烟叶生长.

为探究雪茄烟叶自然晾制过程中青色斑点(简称"青斑")形成的生理机制及代谢差异,提高茄衣烟叶外观品质,改进晾制技术,以雪茄烟'楚雪10号'品种为试验材料,利用高压液相色谱串联质谱(HPLC-MS/MS)非靶向代谢组学技术,分析烟叶"青斑"和正常两组织部位的代谢差异.研究结果表明:"青斑"部位叶绿素和绿原酸含量显著高于正常部位,大量未分解的叶绿素和绿原酸在晾制末期沉积是"青斑"形成的生理基础;"青斑"和正常两组织部位间,酪氨酸代谢、苯丙氨酸代谢和类黄酮生物合成途径具有显著差异;绿原酸和二氢玉米素可作为"青斑"形成过程的化学标记物,晾制过程中可检测其含量高低来监测是否会出现"青斑";烟叶变黄期晾房湿度骤降且波动过大,可能是形成"青斑"的外部环境因素,建议采用湿度可控晾房晾制并适当提高变黄期湿度.

Background: Artemisia hedinii is a well-known traditional Chinese medicine. It can be used to extract dihydroartemisinin (DHA). Objective: The purpose of this study was to explore the optimal conditions for the homogenate extraction of DHA from A. hedinii and the antifungal activity of DHA. Methods: In this study, single-factor experiments and the response surface method were used to determine the optimal extraction conditions of crude extract and DHA. The method of spore germination was used to study the antifungal activity of DHA on Alternaria alternata. Results: The optimal conditions were found as follows: ratio of liquid to material 22 mL/g; extraction time 60 s; and soaking time 34 min. Under these conditions, extraction yield of DHA was (1.76 +/- 0.04%). When the concentrations of crude extract were 0.5 and 8 mg/mL, the spore germination inhibition rates of A. alternata were (17.00 +/- 2.05%) and (92.56 +/- 2.01%), which were 3.34 and 1.15 times that of the DHA standard, respectively. Conclusions: Homogenate extraction technology is a fast and efficient method for extracting DHA from A. hedinii. The crude extract has significant antifungal activity against A. alternata and is inexpensive, providing possible DHA usage in the prevention and treatment of plant pathogenic fungi. Highlights: The optimum conditions of the extraction of DHA from A. hedinii by homogenate extraction were obtained. DHA has antifungal activity against A. alternata. Compared with pure DHA, the crude extract has stronger antifungal activity against A. alternata.

采用闪式提取法提取箬竹叶,并通过菌丝生长速率法对箬竹叶粗提物及其萃取相进行抗真菌活性测定.结果表明:箬竹叶粗提物对深绿木霉和烟草链格孢菌菌丝生长的EC 50值分别为2.33 mg·mL-1、3.67 mg·mL-1.在粗提物EC 50浓度下,各萃取相中,正丁醇相对深绿木霉的抗菌活性最高,菌丝生长抑制率为44.23％;乙酸乙酯相对烟草链格孢菌的抗菌活性最高,菌丝生长抑制率为54.39％.

The extraction of Artemisia hedinii Ostenf. et Pauls(A. hedinii) were rough and further isolated by chromatographic separation technology, and the antifungal activity of the component was evaluated by the the spore germination method. Furthermore, the composition of antimicrobial components was analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS). The results showed that 13 kinds of components(Fr.1-Fr.13) were rough isolated from the extraction of A. hedinii,in which the component Fr.5 showed the best antifungal activity. Four kinds of components (Fr.5.1-Fr.5.4) were obtained by the further separation of the component Fr.5. The EC50of A. hedinii crude extraction on Alternaria alternate (A.alternate) spores was 5.04 g/L, and that of the component Fr.5.2 was 0.57 g/L. The HPLC-MS results showed that the components of Fr.5.2 mainly contained deoxy double hydroxyl artemether (17 %),deoxy dihydroartemisinin (43.65 %) and deoxyartemisinin (16.15 %). The content of dihydroartemisinin of component Fr.5.2 was calculated as 40.95 % by using dihydroartemisinin as the standard. The EC50of dihydroartemisinin on A.alternate was 2.70 g/L, indicating that the major antifungal matter in component Fr.5.2 was artemisinin derivatives.

前期研究从臭蒿中提取分离出臭蒿抗菌流分Fr.5.2主要成分为青蒿素衍生物.为了研究青蒿素衍生物的抗菌机理,测试Fr.5.2和双氢青蒿素(Dihydroartemisinin,DHA)对烟草链格孢菌的抗菌活性、细胞膜完整度、胞内物质的泄露、菌体和孢子的影响.结果发现:Fr.5.2对烟草链格孢菌最小抑菌浓度(minimum inhibitoryconcentration,MIC)为1.25 mg/mL,最小杀菌浓度(minimum bactericidal concentration,MBC)为2.5 mg/mL,DHA对烟草链格孢菌的MIC为10 mg/mL,MBC为10 mg/mL.0.625 mg/mL Fr.5.2和2.5 mg/mL DHA作用于烟草链格孢菌,在0～3h,细胞膜完整度不断下降,胞内核酸大分子不断流失;SEM观察发现菌丝体细胞膜细小、有空洞,孢子表面也有凹陷;Fr.5.2和DHA还可以抑制麦角甾醇的合成,与空白组相比,麦角甾醇的含量分别降低29.77％,28.99％,孢子平均密度提高,但是萌发率降低.这些实验结果表明,青蒿素衍生物能够抑制真菌麦角甾醇的合成,破坏细胞膜,阻碍真菌细胞的新陈代谢,破坏真菌孢子,能有效的抑制、杀死真菌.

2016年6月～8月,在湖北省房县土城镇进行了防治烟草赤星病的生防菌剂田间对比试验,结果表明:5种参试生防菌剂对烟草赤星病均有一定的防治效果.效果最好的为四霉素,相对防效达到63.99％,研制的拮抗菌中,以X10效果最好,达到58.18％,其次是X12,相对防效为51.05％;相对于化学药剂,5种生防菌剂对烟草赤星病的防治效果均好于药剂对照,其中X10、四霉素防效显著高于药剂对照.

[Objective]The study was conducted to survey the soil nutrients that affected occurrence of tobacco bacte-rial wilt in Qingjiang River Basin,and provide theoretical basis for the rational prevention and control of tobacco bacterial wilt in the area.[Method]The soil samples from two kinds of fields(bacterial wilt field and non-bacterial wilt field)were collected during full outbreak period of tobacco bacterial wilt(97 d after transplanting)in Xuan'en,Enshi,Hubei Province in 2016.The contents of nutrition elements in rhizosphere soil were determined,then the relationship between incidence of tobacco bacterial wilt disease,disease index and nutrient elements in rhizosphere soil was researched using a variety of statistical analysis methods.[Result]t test showed that,among the 16 soil nutrient elements indexes,10 indexes presen-ted significant difference(P<0.05)or extremely significant difference(P<0.01)in two kinds of fields.The results of canoni-cal correlation analysis indicated that increasing of pH and contents of cation exchange capacity,exchangeable calcium content,and reducing the contents of soil organic matter,organic nitrogen,organic carbon,effective iron could reduce the morbidity and disease index of tobacco bacterial wilt within certain extent.Group analysis revealed that the appropria-te soil nutrient condition for inhibiting the disease were pH at 5.0-7.0;the organic matter content 1.5%-2.5%,the contents of cation exchange capacity,exchangeable calcium and calcium/magnesium ratio were higher than 20.0 cmol/kg,1000 mg/kg and 15 respectively,and contents of organic nitrogen,organic carbon and effective iron lower than 0.15%,1.0% and 50 mg/kg respectively.[Conclusion]The occurrence of tobacco bacterial wilt is closely bound up with soil nutrients in Qingjiang River Basin.

In order to study the suitable tillage for flue-cured tobacco,this test using Nicotiana tabacum Yunyan 87 as the research materials,set up no-till,rotary tillage (20 cm),tillage (20 cm) and tillage (40 cm) to study the change of soil and tobacco leaf.Results showed that in January to March,the order of temperature about 15 cm soil is tillage (40 cm) ＞ tillage (20 cm) ＞ rotary tillage (20 cm) ＞ no-till.Respectively,the temperature of tillage (40 cm) is higher than no-till and shallow tillage average 0.75 ℃,0.71 ℃ and 0.52 ℃;The activities of enzyme,microbial biomass and microbial activity are characterized,which by tillage(40 cm) is higher than other processing,and no-till was the worst.The total bacteria about tillage(40 cm) is more than three times of the no-till.The production output and superior rate of the tillage (40 cm) are the highest,compared with no-till,increased 383.81 kg/hm2,12 415.19 yuan/hm2 and 8％ respectively.In conclusion,under the condition of the experiment,with the method of the tillage(40 cm),it can help to improve the soil temperature,improve the environment of soil and promote root activity and improve the production of tobacco leaf,can be used as a suitable model for farming on the tobacco leaf production areas in our province.

In recent years,the soils of planting tobacco are facing a series of problems such as acidification and reduction of organic matter content,which affects the yield and quality of tobacco leaves.The biochar has a high carbon content,large specific surface area,alkaline and other characteristics,it has obvious effect on improving the soil pH and nutrient availability.This paper reviews the properties of biochar,its effects on soil improvement,nutrient enhancement and soil carbon sequestration,so as to provide scientific basis for application and popularization in tobacco production.

The burley tobacco variety of TN90LC with low content of tobacco–specific nitrosamines(TSNAs) and N'?nitrosonornicotine (NNN) was introduced from America. The results of experiment and evaluation indicated that the content of TSNAs, NNN and the nornicotine conversion of TN90LC were significantly lower than those of Eyan 1 (control variety). The average yield of TN90LC was 2638.60 kg/hm2, and the percentage of the sum of first grade tobacco and middle grade tobacco reached 86.06%. The resistant results showed that TN90LC has moderate to normal resistance to black shank, and normal resistance to TMV, moderate resistance to root knot nematode disease. The appearance quality and sensory quality of TN90LC were both better than those of the control, the content of main chemical components was appropriate, and the harmony of chemical components was also better. By comprehensive analysis, the TN90LC was an excellent burley tobacco variety which is compatible in several important traits, including less–harmful, fine quality, proper yield and enough resistibility to main diseases.

To gain knowledge on the utilization of flue-cured tobacco hybrids in Zimbabwe and to take it as a reference for improving the ability of flue-cured tobacco breeding in China, the concept and strategy of flue-cured tobacco breeding, leaf research work and general program of hybrid utilization, the regional test, popularization and application of hybrids in Zimbabwe were introduced and analyzed on the basis of on-site investigation and research. In Zimbabwe, the flue-cured tobacco varieties bred in recent 20 years were all male sterile hybrids. Hybrid varieties had the advantage in speeding up breeding of new multi disease-resistant varieties. However, multi-resistant parents were the basis for the aggregation of resistances to more diseases in hybrids. Therefore, the breeding of superior parents with multi-resistant traits was the key of hybrid utilization, and effective resistant resources to most of major diseases in Zimbabwe tobacco production were screened out from germplasm resources. The resistance genes were successfully transferred into flue-cured tobacco cultivars, and a number of quality flue-cured tobacco parent materials with resistances to several diseases were bred, which built a foundation for the breeding of multi-resistant hybrids. Comparing with Zimbabwe practice, the number of quality multi-resistant hybrids independently bred and popularized in flue-cured tobacco production was less in China; the fundamental cause was the serious lack of quality parent materials with resistances to several diseases. Therefore, the research and utilization of disease-resistant germplasm in flue-cured tobacco breeding should be strengthened in China. At the same time, the breeding of quality multi-resistant parent materials should also be valued for making full use of hybrids.

概述了国内外应用无致病力青枯菌菌株、芽孢杆菌、链霉菌、假单胞杆菌、菌根真菌、噬菌体、转基因植物对烟草青枯病进行生物防治的研究进展,指出了目前该方面研究中仍存在的问题并展望了应用前景.

The status of flue-cured tobacco breeding and promotion in South Africa was introduced, including tobacco breeding sector, main target traits, breeding research methods, new varieties regional test, application of flue-cured tobacco varieties, and so on. Compared with the South African, in China cultivated varieties is relatively simple on flue-cured tobacco production, and the number of high quality and multi resistant hybrids is limited, especially the lack of multi-diseases-resistant and excellent pure line or type varieties is serious. Therefore, regional characteristic varieties should be targeted to develop according to the development requirements of flue-cured tobacco production areas in China. At the same time, it is necessary to pay attention to the development of excellent and multi resistant parent materials to make full use of hybrids.