玉溪雪茄烟发酵霉变病原菌鉴定及生防菌筛选

为明确云南玉溪雪茄烟发酵过程中的霉变病原菌，采用组织分离法从霉变烟叶上分离病原菌，并依据柯赫氏法则，结合形态学和ITS基因鉴定病原物种类，以病原菌为指示菌筛选生防菌，经室内发酵验证防霉变效果及对香气成分的影响。柯赫氏试验结果表明，原分离病菌XJ-a与显症烟叶再分离病菌XJ-b在形态、显微、病状上高度相似，两株病原菌ITS序列与多株Aspergillus flavus菌聚为一支，序列一致性大于99%，综合形态学和分子生物学特征，确定雪茄烟叶发酵霉变病原菌为黄曲霉（A. flavus）。筛选获得3株抑菌活性较强的生防菌，其中菌株CC-3对黄曲霉的抑制带宽度达9.04 mm，鉴定为蜡样芽孢杆菌（Bacillus cereus）。在发酵中添加CC-3可显著降低烟叶霉变率，在发酵15 d和30d霉变率分别下降64.44%和52.66%，并且香气物质总量提高28.96%。本研究确定黄曲霉是玉溪雪茄烟发酵的主要致霉菌，添加生防菌CC-3进行发酵可降低黄曲霉的侵染同时提高香气成分含量。     

具有拮抗胶孢炭疽菌活性酵母菌的分离和筛选

从芒果果皮、叶片和芒果园土壤中分离筛选得到具有拮抗胶孢炭疽菌（Colletotrichum gloeosporioides）活性的酵母菌,研究其对芒果中胶孢炭疽菌的抑制作用。采用平板对峙法和活体实验进行筛选,采用形态学及r DNA-ITS序列分析等方法进行鉴定。通过平板对峙和活体实验筛选发现,菌株T18对芒果炭疽病病原菌胶孢炭疽菌的拮抗作用明显。抑菌实验表明,当T18菌悬液浓度为1×108cfu/m L时,对胶孢炭疽菌的抑菌半径为14.33mm。活体实验中,经T18处理的芒果果实炭疽病病斑直径仅为1.58mm,具有较强抑菌效果。通过菌落和菌体形态、生理生化、ITS序列分析对菌株T18进行鉴定,最终确定为尼泊尔德巴利酵母（Debaryomyces nepalensis）。      

不同甜酒曲中可培养真菌的多样性分析

用可培养的方法对6种甜酒曲中的真菌进行分离筛选,共分离得到25株酵母菌和13株霉菌,利用26S rDNA序列分析对酵母菌进行鉴定,结果表明,其分属于酿酒酵母（Saccharomyces cerevisiae）、扣囊复膜酵母（Saccharomycopsis fibuligera）、东方伊萨酵母（Issatchenkia orientalis）和克鲁维毕赤酵母（Pichia kluyveri）。利用ITS rDNA序列分析对霉菌菌株进行鉴定,结果表明,13株霉菌分别为米根霉（Rhizopus oryzae）、小孢根霉（Rhizopus microsporus）、小孢根霉华变种（Rhizopus microsporus var. chinesis）、小孢根霉须状变种（Rhizopus microsporus var. rhizopodiformis）、印度毛霉（Mucor indicus）和卷枝毛霉（Mucor circinelloides）。     

烘烤期烟叶霉烂病的病原鉴定

摘 要:福建省政和县发现红花大金元烟草在烘烤变黄期发生霉烂。霉变先从叶柄开始发生,逐渐向主脉和叶片扩展,引起烟叶大面积霉烂。为了明确病因和发病条件,进行了病原菌分离培养、病原菌形态特征观察、生长温度试验、致病性测定及病害发生条件观察。研究结果表明病原菌为米根霉（Rhizopus oryzae）,烟叶变黄期烤房温度达36～40℃,相对湿度75%～85%的条件下有利病菌的生长和侵染。首次确认烘烤期烟叶霉烂是由病原菌引起的一种新的侵染性病害,该病害命名为烟叶霉烂病（tobacco leaf mildew rot）。      

黄酒麦曲中主要霉菌的分子鉴定及分类

采用土豆琼脂培养基、麦汁琼脂培养基和察氏培养基分离麦曲中的霉菌。共得到18株霉菌。所得5株主要霉菌（数量级在10^5以上）经氯化苄法提取基因组DNA后,应用真菌ITS rDNA通用引物ITS1和ITS4扩增整个ITS序列（包括ITS1和ITS2）,经测序和序列比对,这5株菌分别为伞枝犁头霉（Absidia corymbifera）、微小毛霉（Rhizomucor Pusillus）、米曲霉（Aspergillus orgyze）、烟曲霉（Aspergillus fumigatus）、米根霉（Rhizopus oryzcte）。     

桃褐腐病菌拮抗酵母的筛选、鉴定及其抑菌活性初探

以北京平谷桃果实表皮上分离得到的45株酵母菌为出发菌株,采用平板对峙法,筛选对桃褐腐病菌（Monilinia fructicola）具有较高拮抗作用的生防酵母菌。结果表明,共筛选出具有较好抑制效果的酵母菌6株,其中菌株MLL-9-1拮抗活性最强,对桃褐腐病菌的抑菌率达77.91%;活体筛选试验结果表明,该菌株对桃褐腐病的抑制率达50.30%。生防相关性状检测结果显示,该菌株能够产生蛋白酶,透明圈直径达30.5 mm,说明该菌主要通过分泌蛋白酶达到抑制病原菌的效果。经形态学、生理生化试验及26S rDNA序列分析,鉴定该菌为葡萄汁有孢汉逊酵母（Hanseniaspora uvarum）。     

烟草镰刀菌根腐病拮抗细菌的筛选鉴定及促生防病效果

为挖掘对烟草镰刀菌根腐病具有较强拮抗作用的生防菌株,采用稀释涂布平板法从健康烟株根际土壤中分离纯化对烟草镰刀菌根腐病（Fusarium root rot）主要病原具有一定拮抗作用的细菌,进行形态学、生理生化特征和16S rDNA序列分析鉴定,并测定促生防病效果。经平板对峙测定,筛选出2株对烟草镰刀菌根腐病病原具有较好抑制作用的菌株YX53和YX72,对茄病镰刀菌（F.solani）的抑制效率分别为83.66%和73.54%,对尖孢镰刀菌（F.oxysporum）的抑制效率分别为57.57%和55.89%;2菌株无菌发酵滤液对病原孢子萌发抑制率为84.82%～100%,其挥发性化合物可抑制病原菌丝生长或产孢,且对烟草疫霉菌（Phytophthora nicotianae）、根串珠霉菌（Thielaviopsis basicola）等也有抑制效果。鉴定结果表明,YZ53和YX72分别为蜡样芽孢杆菌Bacillus cereus和枯草芽孢杆菌B. subtilis。经2菌株处理的烟苗胚根增长率分别达到了67.86%和161.61%,盆栽烟苗根长、最大叶面积、鲜质量等均有所增加;YX53盆栽防...     

海南雪茄烟叶霉变微生物鉴定及拮抗菌株筛选

为明确引起海南雪茄烟叶霉变的微生物种类并筛选其拮抗菌株防治烟叶霉变,分别从海南霉变雪茄烟叶表面分离、纯化得到霉变微生物和拮抗微生物,采用形态学和分子生物学相结合的方法鉴定微生物种类。鉴定和筛选结果表明,引起海南雪茄烟叶霉变的微生物主要为篮状菌属、帚霉属和曲霉属,得到3株霉变菌株分别为Talaromyces funiculosus、Scopulariopsis brevicaulis和Aspergillus occultus。筛选到2株拮抗效果良好的拮抗菌株,分别为枯草芽孢杆菌（Bacillus subtilis）和耐盐芽孢杆菌（Bacillus halotolerans）。引起海南雪茄烟叶霉变的微生物主要为篮状菌属、帚霉属和曲霉属菌株,筛选到的2株芽孢杆菌对致霉菌有良好的拮抗作用,为雪茄烟叶霉变的防治提供了参考。     

烟草疫霉菌拮抗细菌的筛选鉴定及发酵条件优化

为了筛选对烟草疫霉菌具有拮抗作用的生防细菌菌株,本研究从烟草黑胫病病圃地采集健康烟株的根际土样,采用对峙培养法筛选得到8株对烟草黑胫病具有拮抗作用的细菌菌株,其中编号为LB-9的菌株对烟草疫霉菌的抑制作用最强且效果稳定,抑菌率为60.6%,且抑菌谱广,对其进行16S rRNA基因序列分析后将该菌株鉴定为多粘芽孢杆菌。采用单因素优化试验初步探究了多粘芽孢杆菌LB-9的最适发酵条件,明确了LB-9的最适发酵培养基、最适发酵温度、最适发酵时间和最适发酵初始pH值,对其生防制剂的开发应用具有重要意义。     

一株绿色素产生菌的筛选及鉴定

目的:从土壤中筛选绿色素产生菌株并对该菌株进行鉴定。方法:采用平板法分离产绿色素菌株,利用形态、生理生化及分子生物学的方法对菌株进行菌种鉴定。结果:从四川理工学院校园土壤中分离得到一株产绿色素的放线菌t9,在高氏一号培养基上,气生菌丝为灰白色,基内菌丝为绿色。该菌株基因序列与鼠灰链霉菌Streptomyces_murinus_NBRC₁2799（T）相似度为99%,以Neighbor-Joining（N-J）法构建的系统发育树。结论:菌株t9的16S r DNA序列分析结果结合形态学特征及生理生化鉴定,确定该菌株为鼠灰链霉菌属（Streptomyces_murinu）,并将其命名为Streptomyces_murinus t9。      

Sprouting and Solid-State Bioprocessing by Rhizopus oligosporus Increase the In Vitro Antibacterial Activity of Aqueous Soybean Extracts Against Helicobacter pylori

Helicobacter pylori infection has been implicated as a major cause of gastric inflammation, peptic ulcer disease, and gastric cancer. While antibiotics have been the mainstay of current therapies for gastrointestinal disease linked to H. pylori infection, negative side-effects and antibiotic resistance issues have strengthened the need for alternative therapeutic strategies. In the search for new antimicrobial agents, much recent research has focused on the potential of dietary phenolic compounds. In this study, soybean extracts enriched for phenolic content via sprouting or solid-state bioprocessing by the dietary fungus Rhizopus oligosporus were investigated for in vitro antibacterial activity against H. pylori. Helicobacter pylori growth inhibition by soybean extracts was increased most effectively by 2 d sprouting or 2 d R. oligosporus bioprocessing. Anti-H. pylori activity was not associated with antioxidant activity, but was linked to extracts when activity of the phenolic-polymerizing enzymes guaiacol peroxidase (in sprouted soybean extracts) and laccase (in R. oligosporus-bioprocessed soybean extracts) were the highest. This suggests the potential involvement of polymeric phenolics in the anti-H. pylori activity of soybean extracts and possible mechanisms for such action are discussed.     

湖北省烟草靶斑病病原鉴定及其菌丝融合群遗传分化研究

2020—2021年湖北烟区爆发新型烟草叶部病害,本研究对病原菌进行分离培养、形态观察、镜检菌丝和科赫法则验证,并提取30个菌株的DNA使用通用引物ITS1/ITS4进行rDNA-ITS序列的PCR扩增,及MEGA-X软件进行基因序列比对及系统发育树构建,同时以Rs1F2/Rs2R1为引物,进行30个菌株的rDNA-ITS基因的PCR专化性鉴定。结果表明,该病害病原菌为立枯丝核菌（Rhizoctonia solani Kühn）,30个菌株序列都隶属于R. solani AG-3融合群且均具有AG-3融合群所特有的504 bp片段。因此,确定该病害为烟草靶斑病。这是首次从湖北省烟草产区分离鉴定到该病菌。     

烟草弯孢菌叶斑病病原菌鉴定及生物学特性研究

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。     

短小芽孢杆菌与化学杀细菌剂协同防治烟草青枯病研究

为探究短小芽孢杆菌与化学杀菌剂联合防控烟草青枯病的可行性,分别采用改良抑菌圈法和平板菌落计数法测定7种杀菌剂和短小芽孢杆菌AR03对青枯雷尔氏菌的毒力及杀菌剂与AR03的生物相容性,同时采用Horsfall法确定杀菌剂和AR03的复配比例。室内毒力试验结果表明,7种杀菌剂和AR03对青枯雷尔氏菌的生长均有较好的抑制作用。7种杀菌剂的毒力大小依次为三氯异氰尿酸、氯尿·硫酸铜、噻菌铜、溴菌·壬菌铜、甲霜·福美双、噻唑锌和中生菌素,EC₅₀值介于101.02~212.70 mg/L之间。浓度为1.0×10⁵~1.0×10⁹ cfu/mL的AR03对青枯雷尔氏菌的抑菌率介于26.13%~73.54%之间,呈现浓度依赖性。生物相容性分析发现7种供试药剂与AR03的生物相容性差异较大,短小芽孢杆菌AR03与噻菌铜、噻唑锌、甲霜·福美双生物相容性较好,尤其是噻菌铜表现最好,测试浓度100 mg/L时,菌落数大于1×10⁷cfu/mL。综合杀菌剂对青枯病菌的毒力及其与AR03生物相容性,噻菌铜表现最优。噻菌铜（EC₅₀=175.21 mg/L）与AR03（EC₅₀=6.84×10⁶ cfu/mL）复配剂在体积比为5∶5时,对青枯雷尔氏菌的抑制效果显著,增效作用明显,增效比率值I_R值为1.482。室内盆栽试验结果表明,菌药复配剂的防效（68.77%）明显优于单剂噻菌铜和生防菌AR03的防效,且混配剂中噻菌铜使用量只有单剂的1/2,大幅降低了化学药剂的使用量。     

一种烟草细菌性叶枯病病原鉴定及防治药剂筛选

为明确在贵州省六盘水市烟区发生的一种烟草细菌性叶枯病的病原并筛选出防治药剂,本研究通过组织分离法分离病原菌,结合形态学鉴定、柯赫氏法则和分子生物学等方法鉴定病原菌,并初步研究该菌的生理生化特性,采用抑菌圈法测定15种杀菌剂对该病原菌的毒力。结果表明,该病害病原菌与成团泛菌（Pantoea agglomerans）同源性达100%,为革兰氏阴性菌,可利用D-阿拉伯糖、丙三醇和硝酸盐等碳氮源;室内防治药剂筛选试验表明,1.6%噻霉酮EW、3%中生菌素WP和80%乙蒜素EC对该菌有较好的抑菌效果,EC₅₀值分别为0.004、0.0135和0.1176 mg/mL。试验表明,该烟草细菌性叶枯病致病菌为成团泛菌（Pantoea agglomerans）,1.6%噻霉酮EW可作为防治该病害的有效药剂。     

寡雄腐霉与氟噻唑吡乙酮协同防治烟草黑胫病研究

为了探究寡雄腐霉(Pythium oligandrum,PO)与氟噻唑吡乙酮(Oxathiapiprolin,OX)联合防控烟草黑胫病的可行性,采用菌丝生长速率法和平板对峙法分别测定了PO和OX的生物相容性,及OX、PO和OX+PO对黑胫病菌的毒力,并通过盆栽试验分别测定了OX、PO和OX+PO对烟草黑胫病菌的防效,以及对烟草幼苗的促生效应。生物相容性试验结果表明,不同浓度OX处理下,PO的生长速率与空白对照无显著差异。室内毒力结果显示,8.0×10^-3 mg/L OX对烟草黑胫病菌的抑制率为91%,EC₅₀为2.19×10^-3 mg/L,而OX(1.0×10^-3 mg/L)与PO联用的抑制率达96.31%。盆栽试验结果表明,OX+PO处理对烟草黑胫病的防效最高(84.57%),比OX(77.76%)或PO(76.54%)单独处理防效分别提高8.76%和10.49%。OX和PO联用对烟草促生效果显著,其株高、茎粗、叶长、叶宽分别比对照处理提高了57.10%、20.21%、33.62%、35.08%。...     

Recent advances on the use of adsorbent materials for detoxification of Fusarium mycotoxins

The extensive use of adsorbents in the livestock industry has led to the introduction of a wide range of new products on the market, most of them claiming high in vitro mycotoxin adsorption capacity. However, adsorbents that may appear effective in vitro do not necessarily retain their efficacy when tested in vivo. Studies performed in our laboratory during the past few years aiming to evaluate the efficacy of various adsorbent materials in binding Fusarium mycotoxins are reported. Adsorption experiments were performed in in vitro screening tests for Fusarium mycotoxins at different pHs; by in vivo tests using the increase of the sphinganine to sphingosine ratio in rat urine and tissues as a biomarker of fumonisin exposure; and by a dynamic, computer-controlled, gastrointestinal model simulating the gastrointestinal tract of healthy pigs. Most of the commercially available mycotoxin-binders failed in sequestering in vitro Fusarium mycotoxins. Only for a small number of adsorbent materials was the ability to bind more than one mycotoxin demonstrated. Cholestyramine was proven to be an effective binder for fumonisins and zearalenone in vitro, which was confirmed for zearalenone in experiments using a dynamic gastrointestinal model and for fumonisins in in vivo experiments. No adsorbent materials, with the exception of activated carbon, showed relevant ability in binding deoxynivalenol and nivalenol. The in vitro efficacy of activated carbon toward fumonisins was not confirmed in vivo by the biomarker assay. The dynamic gastrointestinal model was a reliable tool to study the effectiveness of adsorbent materials in reducing the bioaccessibility of Fusarium mycotoxins, as an alternative to the more difficult and time-consuming studies with domestic livestock.     

吉林省和黑龙江省烟草赤星病病原鉴定

为明确吉林省和黑龙江省烟草赤星病病原菌的种类,对吉林省15个烟区和黑龙江省7个烟区的烟草赤星病病原菌进行了分离鉴定,共分离获得312株致病菌株。通过形态学和分子生物学鉴定,共鉴定出4种链格孢属真菌,其中链格孢（Alternaria alternata）230株,细极链格孢（Alternaria tenuissima）78株,鸭梨链格孢（Alternaria yaliinficiens）2株,长柄链格孢（Alternaria longipes）2株。其中吉林省烟区鉴定出了链格孢、细极链格孢和鸭梨链格孢3种,黑龙江省烟区鉴定出了长柄链格孢、链格孢和细极链格孢3种。两个省区的优势种均为链格孢。此外,本试验利用5种基因序列片段（ITS、GDP、ACT、EF、RPB2）进行烟草赤星病菌的分子生物学鉴定,其中GDP基因仅能鉴定出链格孢和细极链格孢2种,RPB2基因能鉴定出4种链格孢,而ITS、EF、ACT基因序列比较保守,不适合区分这些链格孢种。本研究明确了吉林省和黑龙江省烟区引起烟草赤星病的病原菌种类,为下一步的定向防控奠定了基础。     

Characterization of the Effect of Sprouting or Solid-State Bioprocessing by Dietary Fungus on the Antibacterial Activity of Soybean Extracts Against Listeria monocytogenes

Listeria monocytogenes is one of the most severe food-borne bacterial infections causing Listeriosis. As L. monocytogenes can survive harsh adverse conditions - such as low pH, high NaCl, and refrigeration temperatures - as well as resist current antimicrobial measures such as the use of disinfectants and antibiotics, there is a need for alternative anti-Listeria strategies. In the search for new antimicrobial agents, much recent research has focused on the potential of dietary phenolic compounds. In this study, soybean extracts enriched for phenolic content via dark-germination sprouting or solid-state bioprocessing by the dietary fungus Rhizopus oligosporus or Lentinus edodes were investigated for in vitro antibacterial activity against L. monocytogenes.L. monocytogenes growth was inhibited most effectively by R. oligosporus bioprocessed soybean extracts, which showed anti-Listeria activity at total phenolic concentrations as low as 10 µg 100 µL^-1. In both sprouted soybean extract and L. edodes-bioprocessed soybean extract the anti-Listeria activity was not observed until at least 200 µg total phenolic content 100 µL^-1 was used. Anti-Listeria activity by soybean extract was associated with phenolic mobilization but not with antioxidant activity. Further, R. oligosporus bioprocessed soybean extracts were shown to inhibit the growth of L. monocytogenes in fish and meat systems at refrigeration temperatures. The potential involvement of mobilization of antimicrobial versus non-antimicrobial phenolics during sprouting and solid-state bioprocessing was hypothesized and discussed.     

Interactive inhibition of meat spoilage and pathogenic bacteria by lysozyme,nisin and EDTA in the presence of nitrite and sodium chloride at 24 degrees C

To develop a nisin- and lysozyme-based antimicrobial treatment for use with processed ham and bologna, in vitro experiments were conducted to determine whether inhibition enhancing interactions occur between the antimicrobials lysozyme, chrisin (a commercial nisin preparation), EDTA, NaCl and NaNO₂. Inhibitory interactions were observed between a number of agents when used against specific pathogenic and spoilage bacteria. The observed interactions included lysozyme with EDTA (Enterococcus faecalis and Weissella viridescens), chrisin with EDTA (all Gram-positive organisms), EDTA with NaCl (Escherichia coli, Salmonella enterica serovar Typhimurium, Serratia grimesii), EDTA with nitrite (E. coli, Lactobacillus curvatus, Leuconostoc mesenteroides, Listeria monocytogenes, S. Typhimurium), chrisin with nitrite (Lc. mesenteroides, L. monocytogenes), and NaCl with nitrite (S. Typhimurium, Shewanella putrefaciens). Previous reports have described interactions between nisin with EDTA that resulted in enhanced antimicrobial effect against Gram-negative bacteria, or lysozyme with nisin against Gram-positive bacteria. These interactions were not observed in these experiments. We observed that unlike previous studies, these experiments were conducted on growing cells in nutrient broth, rather than under conditions of nutrient limitation. We propose that screening of antimicrobials for use in food systems in nutrient-deficient systems is inappropriate and that new protocols should be developed.