Abstract In this study, a strain G05, which can efficiently convert the main ginsenoside Rb2 to the rare ginsenoside Rg3, was screened from the endophytic bacteria of dry Panax ginseng by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) analysis. The strain was identified as Leifsonia by morphological and molecular biological analysis and named Leifsonia sp.G05. The effect of medium, inoculum amount, transformation temperature, medium pH and substrate concentration on the conversion rate of ginsenoside Rb2 to Rg3 was investigated to obtained the optimum conditions for transformation and high conversion rate, as well as the transformation path of strain G05. The results showed that the strain G05 could convert 1 mg/mL ginsenoside Rb2 more efficiently when the medium was Luria-Bertani, the inoculum amount was 1%, the transformation temperature was 30°C, and the medium pH was 7. Under these conditions, the main ginsenoside Rb2 could be nearly completely converted to the rare ginsenoside Rg3 by strain G05 after 9 hours of fermentation, the conversion rate reached 98%, and the transformation path was Rb2→Rd→Rg3. The research results could improve the utilization rate of the main ginsenoside Rb2 to a certain extent, and provide safer and specific rare ginsenosides for the pharmaceutical and food preparation industries.

To isolate endophytic bacterium with the ability to specifically convert ginsenoside Rc from Panax quinquefolius. An endophytic bacterium G9y was isolated from Panax quinquefolius and indentified as Bacillus sp. based on 16s rDNA gene sequence. Ginsenoside Rc was effectively converted to Rd by G9y, which was confirmed by thin-layer chromatography and high performance liquid chromatography (HPLC) analysis. The biotransformation conditions were further optimized as follows: inoculum amount 5%, converting temperature 45 °C, medium beef extract peptone broth at pH of 7, and the time of Rc addition was 4 h after bacterium G9y growth, under which ginsenoside Rc was completely converted to Rd by bacterium G9y within 25 h after inoculation. A strain of G9y with the ability to convert ginsenoside Rc into Rd was screened from endophytic bacteria isolated from P. quinquefolius. The results provide a new microbial resource for preparing ginsenoside Rd via biotransformation, and explore a pathway for Rc utilization, which has great potential application value.

Panax notoginseng has long been used as a Chinese herb with high medicinal value. The endophytic bacteria in this medicinal plant have multiple biological functions. High-throughput sequencing is a rapidly evolving technique that helps profile the endophytic bacterial community structure of medicinal plants. However, few studies on the endophytic bacteria in P. notoginseng, particularly in dry P. notoginseng roots as a raw medicinal material, have been conducted. In this study, fresh P. notoginseng and dry P. notoginseng were analysed using high-throughput sequencing on an Illumina MiSeq platform to explore the diversity and functions of the endophytic bacteria in different parts of P. notoginseng. The results showed that a total of 201 operational taxonomic units were obtained from fresh P. notoginseng and dry P. notoginseng. The dominant phyla in the fresh and dry P. notoginseng were Proteobacteria (85.9%) and Firmicutes (99.9%), respectively, whereas the dominant genera in these samples were Enterobacter (84.4%) and Bacillus (99.6%), respectively. Fresh P. notoginseng exhibited a higher degree of endophytic bacterial diversity than dry P. notoginseng, but functional prediction of metabolism indicated that the relative abundance of the metabolic function of terpenoids and polyketides synthesis in the dry sample was higher than that in the fresh sample. Our study indicates significant differences in the diversity and metabolic function of the endophytic bacteria between fresh and dry P. notoginseng, providing useful information for the exploitation and utilization of endophytic bacteria resources from P. notoginseng.

寻找能够高效降解磺酰脲类除草剂苯磺隆的新型菌株,为生物降解苯磺隆提供微生物资源.对明党参内生菌进行分离、培养与纯化,从筛选得到的多株内生菌株出发,采用富集培养法筛选苯磺隆降解菌.采用紫外分光光度计法和高效液相色谱法测定苯磺隆的含量,计算苯磺隆的降解率.结果表明,从29株明党参内生细菌中筛选出8株能降解磺酰脲类除草剂苯磺隆的菌株,通过复筛得到高效降解苯磺隆菌株PMG3,其降解率为89.07％,并经鉴定为芽孢杆菌属.PMG3比以往筛选出的苯磺隆降解菌降解效率都要高,为利用中草药内生菌对受农药苯磺隆污染的土壤进行原位修复提供理论依据和现实意义.

为解决湘南烟区烤烟生产上存在的高温逼熟问题,采用大田试验研究了主要生态因素对烤烟高温逼熟现象发生的影响效应,并分析了烤烟高温逼熟现象形成的机制.结果表明:湘南烟区烤烟高温逼熟现象由综合生态因素引起;前期降雨量和后期高温(日最高温度≥38℃)天数与烟叶高温逼熟率呈显著正相关,相关系数分别为0.904和0.914;在湘南烟区气候条件下,前期降雨量过多,是导致高温逼熟现象发生的主要前提因素,成熟期高温(日最高温度≥38℃)天数过多是导致高温逼熟现象发生的主要胁迫因素;而成熟期高光强(日最高光强≥100000 Lx)天数过多是高温逼熟现象发生的次要因素.湘南烟区前期降雨过量,造成了烤烟根系发育不良,烟株抗逆性降低且难以适应后期高温,从而导致高温逼熟烟叶现象的发生.

目的 获得鲜三七和干三七菌群结构信息.方法 以鲜三七和干三七为研究材料,利用Illumina MiSeq平台,采用高通量测序并进行数据分析.结果 鲜三七和干三七共获得637个操作分类单元(OTUs),其中15个OTU为共有,鲜三七和干三七特有的OTU数量分别为484、123个.新鲜三七中占比较大的菌门为变形菌门(Proteobacteria,59.7％)、厚壁菌门(Firmicutes,40.1％);干三七中占比较高的菌门为厚壁菌门(Firmicutes,99.8％).鲜三七中占比较大的菌属为肠杆菌属(Enterobacter,37.4％)、嗜碱菌属(Alkaliphilus,11.5％);干三七中占比较大的菌属是芽孢杆菌属(Bacillus,54.6％),类芽孢杆菌(Paenibaciiius,44.9％).KEGG功能预测表明干三七的萜类化合物和聚酮类化合物代谢功能菌群的相对丰度高于鲜三七.结论 鲜三七菌群多样性高于干三七;干三七中的萜类化合物和聚酮类化合物代谢功能菌群丰富度高于鲜三七,这为三七发酵皂苷菌株筛选提供了重要参考.

针对降解高毒、高危害的尼古丁微生物种类较少的实际情况筛选出具有高尼古丁降解活性的菌株,探究其降解条件及降解特性,为尼古丁的生物降解提供新的微生物资源.从烟草种植大田土壤分离纯化得到一株具有高尼古丁降解能力的菌株,对其形态学、生理生化特征、16S rRNA基因序列同源性进行了分析鉴定,探究了不同条件下尼古丁降解特性并对其动态降解过程进行了HPLC分析.经鉴定命名为Stenotrophomonas sp.ZUC-3,适宜的发酵条件为:温度30℃、起始pH 7.0、接种量10％、培养转速180 r/min,此条件下尼古丁降解率可达到91.53％,HPLC分析显示了ZUC-3降解尼古丁过程的动态变化及其高效性.研究结果为尼古丁生物降解提供了新的微生物资源,对烟草行业可持续发展具有理论意义与实际应用价值.

研究郑州市栽培明党参内生菌资源状况,筛选出对金黄色葡萄球菌具有拮抗作用的内生菌.采用平板培养法从明党参中分离内生细菌,通过混菌法和牛津杯双层平板扩散法筛选出对金黄色葡萄球菌具有明显抑制作用的菌株,并通过培养特征、形态特征、生化特征、全自动微生物质谱检测、16S rDNA基因序列分析和系统进化树分析对菌株进行了分类学鉴定.在明党参中共分离到29株内生细菌,通过初筛和复筛后,筛选出对金黄色葡萄球菌具有明显抑制作用的4株菌株NMY1、NMJ10、NMG10、PMG3,其中NMJ10对金黄色葡萄球菌的抑菌圈直径达20.3 mm,经鉴定菌株NMJ10和菌株PMG3为芽胞杆菌属新菌种(Bacillus spp.),菌株NMY1为枯草芽胞杆菌(B.subtilis),菌株NMG10为假蕈状芽胞杆菌(B.pseudomycoides).明党参内生菌具有多样性,其中芽胞杆菌属为优势菌株,有拮抗金黄色葡萄球菌活性高的菌株存在,其代谢产物对金黄色葡萄球菌有较高抑制作用,为抗菌药源成分的开发提供了新的来源.

为探究成熟期烤烟对环境胁迫的响应规律,以云烟87为供试材料,采用盆栽试验,模拟湖南烟区烤烟生育后期的高温强光胁迫,分析不同光温胁迫对成熟期烤烟光合生理特性的影响.结果表明:①随着光照强度的变化,常温下烟株蒸腾速率、气孔导度和净光合速率的变化明显,在80 000 lux处达到最大值;高温下烟株蒸腾速率、气孔导度和净光合速率则明显降低.②整体上,同等光强下,高温处理的烟株蒸腾速率、气孔导度和净光合速率呈下降趋势,胞间CO2浓度升高.③高温强光胁迫下,成熟期烤烟的蒸腾速率、气孔导度和净光合速率均显著降低,胞间CO2浓度升高,光合能力下降.④相关性分析表明,烤烟净光合速率与气孔导度呈极显著正相关,蒸腾速率与净光合速率、气孔导度呈显著正相关.

采用大田控光试验,对生育后期大田烟株进行遮阴处理(自然光强的70％),探讨光照度对湘南烟草成熟特性的影响.结果表明,烟株生育后期适当遮阴可以促进烟叶色素的积累,延缓叶片失绿早衰.生育后期遮阴烟叶蒸腾速率、气孔导度和Fv/Fm均高于对照,而胞间CO2浓度及qN低于对照,说明生育后期自然光照度已导致光抑制现象发生;在旺长后期遮阴处理烟叶SOD活性、POD活性、超氧阴离子产生速率和过氧化氢含量与对照相比差异不显著;但成熟期适度遮阴烟叶CAT、POD活性升高,超氧阴离子产生速率、过氧化氢含量显著低于对照,表明成熟期适度遮光可抑制强光对烟叶保护酶系统的破坏,降低生物膜受损指标.通过对烤后烟叶化学成分的分析可以看出,遮阴网适度遮阴有利于降低烟叶刺激性,优化烟叶的等级.研究表明,湘南烟区生育后期适度降低大田自然光照度有利于减轻光损伤,缓解烟叶“高温逼熟”现象.

为探讨前膜后草覆盖栽培对烤烟生长及产量与质量的影响,2017年进行了前膜后草覆盖方式烤烟生长的影响试验.结果表明,前膜后草处理较不盖地膜且不盖稻草对照处理株高有增加;在茎围与节距、叶面积上影响不明显;各处理之间比较均无显著性差异.产量和产值前膜后草处理较不盖地膜且不盖稻草对照均有显著差异,但未达极显著水平;均价前膜后草处理和不盖地膜且不盖稻草对照之间无显著差异.上等烟比例及中上等烟比例前膜后草处理均大于不盖地膜且不盖稻草对照,但各处理之间上等烟比例与中上等烟比例都无明显差异.

为解决烤烟高温逼熟现象,2017年进行了不同烤烟品种喷施高温逼熟防控药剂对烟叶质量的影响试验.结果表明,在旺长期和成熟期各喷施1次高温逼熟防控药剂,不同品种、不同药剂烟叶均价比较,以喷施水杨酸处理(24.07元/kg)表现最好,其次是喷施氯化钙处理(23.49元/kg),再次是喷施亚硝铁氟化钠处理(23.22元/kg)和喷施腐胺处理(22.51元/kg),喷清水对照(22.14元/kg)最差,各处理之间无显著差异.不同品种、不同药剂上等烟比例以喷施水杨酸处理(47.74％)效果最好,其次是喷施氯化钙处理(46.28％),再次是喷施亚硝基铁氟化钠处理(43.62％)和喷施腐胺处理(38.99％),喷清水对照(32.01％)效果最差,喷施水杨酸、喷施氯化钙和喷清水对照之间有显著差异,但未达到极显著水平,其他各处理之间无显著差异.综合来看,不同品种、不同药剂比较,水杨酸表现最好.同一品种不同药剂烟叶均价比较,云烟87喷施水杨酸处理均价(25.84元/kg)最高,湘烟5号喷施亚硝基铁氰化钠处理均价(23.91元/kg)最高,K326喷施氯化钙处理均价(24.07元/kg)最高.同一品种不同药剂中上等烟比例比较,云烟87喷施清水对照中上等烟比例(98.58％)最高,湘烟5号喷施亚硝基铁氰化钠中上等烟比例(96.17％)最高,K326喷施氯化钙中上等烟比例(93.97％)最高.综合来看,同一品种不同药剂处理比较,云烟87喷施水杨酸表现最好,湘烟5号喷施亚硝基铁氰化钠表现最好,K326喷施氯化钙表现最好.

为探索废弃烟梗资源化利用途径,以烟梗废料为培养基的主要原料,以杀虫微生物白僵菌(Beauveria bassiana)为目标产物,采用固态发酵法研究了烟梗粒度和含水率对白僵菌产孢量的影响,并通过Plackett-Burman(PB)试验对废弃烟梗发酵产白僵菌的条件进行了初步优化.结果表明:烟梗适宜作为白僵菌的主要发酵培养基质,粒度以原梗较好,且当烟梗与水质量比为13.5时,白僵菌生长和孢子萌发状况最好,孢子产量较高;通过PB试验与分析,在选择的9个相关因素中,培养时间、蛋白胨和酵母粉添加量是影响白僵菌孢子产量的3个关键因素;在PB试验条件下,白僵菌孢子最大产量可达8.59×109个/g.研究证实了以烟梗废料发酵生产杀虫微生物白僵菌的可行性,为烟梗废料的综合利用开辟了新途径.

Tobacco wastewater contains large amounts of soluble sugar,nitrogen compounds,minerals and other nutrients.For resource utilization of tobacco wastewater,using L-lactic acid-producing Lactobacillus paracasei ZG19 which isolated and preserved in laboratory as fermentation strain,with tobacco wastewater as fermentation substrate and L-lactic acid yield as response value,the fermentation process was optimized using response surface methodology (RSM).By Plackett-Burman (PB) design,the most significant factors affecting the yield of L-lactic acid were screened.Based on the results of PB design,the steepest ascent experiment was designed to approach the optimal region,and by BBD-RSM optimization,the optimal fermentation process was analyzed and determined.The results showed that with conditions of inoculum 10％,fermentation temperature 39 ℃ and time 30 h,the actual yield of L-lactic acid was 15.16 g/L and the fitting degree was 98.76％ with the predicted value of 15.35 g/L.The yield of L-lactic acid increased significantly after optimization.Therefore,the optimal fermentation process was reasonable and available,which had practical application value.

In order to realize the resource utilization of waste tobacco stems and improve the application status on waste tobacco stem, using waste tobacco stem as fermentation substrate, and the production of Beauveria bassiana as response value, the fermentation process of Beauveria bassiana was optimized using the response surface methodology (RSM). Through Plackett-Burman(PB)design,the most significant factors affecting the production of Beauveria bassiana using waste tobacco stem as fermentation substrate were identified. Based on the results of the Plackett-Burman (PB) experiment, the steepest ascent experiment was used to approach the optimal region, and the mathematical model of optimization of Box-Behnken Design (BBD) response surface was established to determine the optimal conditions. The results showed that when the incubation time was 9.40 days, concentration of peptone and yeast powder was 1.21% and 1.03% respectively, the prediction spore yield of Beauveria bassiana was 1.17×1010/g, the actual spore yield was 1.13×1010/g, and the fitting degree was 96.58%. Therefore, the optimal fermentation conditions from RSM are reasonable, and have practical application value in microbial prevention and control and the comprehensive utilization of waste tobacco stems.

To effectively control the mildew of stored tobacco leaf, a Bacillus strain LL17 was isolated from tobacco rhizosphere soil. A dual culture method and Oxford cup method were used to study the antagonistic effects of strain LL17 on three kinds of mildew fungi. The bacteria cultural, morphological and biochemical features were observed, and in addition 16S rDNA sequence analysis was used to identify the strain. The results showed that strain LL17 was identified as Bacillus subtilis on the taxonomy. The inhibition zones were clearly observed in PDA by using the dual culture method and Oxford cup method, indicated that strain LL17 was strongly antagonistic towards three kinds of mildew fungi. Sterile filtrate of strain LL17 was antagonistic towards 2 kinds of Aspergillus conidiospores. The sterile filtrate could affect spore germination or induce hyphae deformation. Therefore, strain LL17 might have the potential application prospect and value in biological control of tobacco mildew during storage.

10 strains of Bacillus spp.were isolated from soil samples.Using Bacillus cereus as the indicator strain,strain LWM1 with strong antibacterial activity was isolated by Oxford cup diffusion method.Combined with morphological,physiological and biochemical tests,matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rDNA gene sequence analysis,strain LWM1 was preliminarily identified as Bacillus amyloliquefaciens.The inhibition effect of the fermented supematant fluid of strain LWM1 in PDW and NB medium on Bacillus cereus was researched.The results showed that PDW medium was more favorable for the growth of strain LWM1 and the production of inhibitive substance.Therefore,the inhibitive substance of strain LWM1 could be prepared by large-scale fermentation with PDW medium as the main substrate.

Objective To seek low-cost substrate for L-lactic acid fermentation and resource utilization of waste tobacco stems.Methods Tobacco stem extraction (TSE) were prepared from waste tobacco stem and L-lactic acid was produced by fermentation of TSE by Lactobacillus casei XJL.Besides,Plackett-Burman design was performed to evaluate some variables which were relevant to L-latic acid production of Lactobacillus casei XJL.Results Among ten selected variables,yeast powder and CaCO3 were found to significantly influence L-latic acid production via Plackett-Burman design and the following statistic analysis with the culture time at 24 h,whereas the key factors were yeast powder,fructose,sodium dihydrogen phosphate and CaCO3 at 48 h.Under Plackett-Burman design conditions,the maximum production of L-latic acid at 24 h and 48 h reached to 307.5 g/kg and 315.0 g/kg.Conclusion The fermentation time had little effect on L-lactic acid yield,while nutritional additives can improve the production of L-lactic acid.

To improve floating seedling system for tobacco, tobacco seedling culturing experiments were conducted in a floating system with in-house designed novel ventilated seedling tray. By taking common seedling tray as a control, the effects of ventilated seedling tray on the emergence rate, botanical characters and activities of protective enzymes and nitrate reductase (NR) of tobacco seedling were studied. The results showed that comparing with the control, the time of seedling culturing in the ventilated seedling tray was shortened by 5 d and emergence rate increased by 3.67%; the leaf size, root development and comprehensive growth of tobacco seedlings were significantly improved. On the 35th, 45th and 55th days after sowing, the root vigor of seedling increased by 14.26%, 20.45% and 19.31%, respectively; chlorophyll contents and the activities of catalase, peroxidase, superoxide dismutase and NR significantly increased comparing with the control. Therefore, the novel ventilated seedling tray has shown a clear promoting effect on seedling growth.

Monofactorial experiments were performed to study the effects of six operational parameters, including fermentation means, fermentation duration, loading capacity, inoculum size, temperature and initial pH, on the preparation of L-lactic acid by the fermentation of waste tobacco stems with Lactobacillus casei XJL. According to the results of monofactorial experiments, four factors (inoculum size, loading capacity, initial pH and temperature) were selected and orthogonally tested at three levels. The results showed that: 1) Initial pH, inoculum size and loading capacity significantly affected the yield of L-lactic acid. 2)The optimum operational parameters were initial pH 7.0, loading capacity 120/250 mL (120 mL tobacco stem extraction loaded in a conical flask of 250 mL), inoculum size 7.5% (the volume ratio of transferred inoculant solution to total culture solution after inoculation), static culture for 48 hours at 37 ℃; under the above conditions, the maximum yield of L-lactic acid reached 157.50 g/kg.