苹果根际自毒物质降解菌的筛选鉴定及降解特性研究

【目的】从苹果根际土壤筛选苹果根系自毒物质降解细菌,并探究分离菌株对根皮苷、邻苯二甲酸、对羟基苯甲酸以及焦性没食子酸的降解能力。【方法】分别采用邻苯二甲酸、焦性没食子酸为唯一碳源富集并筛选其降解菌株。通过对分离菌株的生理生化特征测定及16S r RNA基因序列分析,采用MEGA 5构建系统发育树,进行菌株鉴定。采用紫外分光光度法和高效液相色谱法测定分离菌株对4种自毒物质的降解能力。【结果】共分离5株有降解能力的细菌,编号为BL1、BL2、BL3、BJ1和BJ2,经鉴定BL1为钩虫贪铜菌Cupriavidus necator,BL2为生脂固氮螺菌Azospirillum lipoferum,BL3为氧化烃微杆菌Microbacterium hydrocarbonoxydans,BJ1为Paenibacillus phyllosphaerae,BJ2为Ochrobactrum cytisi。BL1、BL2、BL3菌株对邻苯二甲酸、对羟基苯甲酸、根皮苷、焦性没食子酸的降解率均高于50%。其中BL2菌株的降解效果最好,分别达到66%、72%、84%和84%。【结论】首次发现钩虫贪铜菌、生脂固氮螺菌和氧化烃微杆菌对4种自毒物质均具有很好的降解能力,对缓解自毒物质引起的连作障碍具有潜在的应用价值。     

微小杆菌(Exiguobacterium sp.)对肉桂酸降解行为

【目的】为有效缓解自毒物质肉桂酸对西瓜等作物生长的危害,从宁夏中卫硒砂瓜连作土壤中分离筛选得到一株高效降解肉桂酸的菌株,研究其基本降解特性。【方法】分离筛选得到一株能有效利用肉桂酸生长的菌株,采用16S r RNA基因序列分析进行菌株鉴定,运用高效液相色谱法和西瓜幼苗生长毒性实验检测降解特性。【结果】从多年西瓜连作土壤中筛选得到一株高效降解肉桂酸的细菌R30,鉴定为Exiguobacterium sp.,其96 h内对肉桂酸的降解率可达99%以上,最适降解温度和p H分别为30°C、p H 7.0。除肉桂酸外,该菌也能够高效降解香豆酸、阿魏酸、苯甲酸等其他酚酸类物质,表现出一定的底物广谱性;检测96 h降解液对西瓜种子萌发直至幼苗生长阶段的影响表明,该菌株可有效缓解肉桂酸对西瓜幼苗的生长抑制作用。【结论】菌株R30在肉桂酸、香豆酸、阿魏酸、苯甲酸等酚酸类物质导致的农作物连作障碍治理领域具有潜在的开发应用价值。     

缓解花生连作障碍的根际促生菌分离及功能鉴定

【目的】长期连作障碍严重降低花生生产的产量及品质,根际促生菌可有效降解土壤中自毒化感物质、抑制植物病原菌生长及促进植物生长,从而有效缓解连作障碍问题。筛选优化具有缓解花生连作障碍能力的多功能根际益生微生物,验证其益生作用能力,为根际促生菌株在连作障碍中的应用提供理论依据及技术支持。【方法】采集连作12年地块花生根际土壤,利用以酚酸为唯一碳源的筛选培养基获得具有酚酸自毒化感物质降解及利用能力的根际促生菌,通过16S rRNA基因测序进行系统发育分析,确定根际促生菌菌株的分类地位,并验证其对植物病原菌生长抑制能力及解磷、解钾、产植物激素吲哚乙酸能力。【结果】从连作12年的花生发病土壤中获得7株可高效降解酚酸类自毒物质且降解底物多样的根际微生物菌株,经16S rRNA测序比对分别为克雷伯氏菌B02 (Klebsiella sp. B02)、克雷伯氏菌B07(Klebsiella sp. B07)、克雷伯氏菌B15 (Klebsiella sp. B15)、芽孢杆菌B28 (Bacillus sp. B28)、不动杆菌P09(Acinetobacter sp. P09)、布鲁氏杆菌VA05 (...     

节杆菌(Arthrobacter sp.)降解阿魏酸的效能

【背景】蓄积在土壤中的阿魏酸类化感自毒物质对农作物生长产生危害,利用有益微生物分解该类物质是一项有效的治理措施。【目的】从自然界土壤分离获得能高效降解阿魏酸的菌株,并评估典型环境因子对降解效能的影响,以期为该菌在阿魏酸类自毒物质降解领域中的应用提供理论依据。【方法】采用一次性投加高浓度化合物的驯化方法分离筛选得到能有效降解阿魏酸的菌株,采用16S rRNA基因序列分析进行菌株鉴定,采用高效液相色谱法跟踪不同条件下该菌株对阿魏酸的降解情况。【结果】分离筛选获得的菌株为节杆菌属细菌(Arthrobacter sp.),命名为J6;在≤1.20%盐度(即12g/LNaCl)、pH5.0-9.0、20-40℃、有/无葡萄糖和有/无酵母粉的条件下,菌株J6均能高效降解阿魏酸。【结论】菌株J6在阿魏酸类自毒物质的生物修复领域具有较高的应用潜能。     

三七连作障碍研究进展

三七是中国名贵中药材之一,但其在种植环节中存在严重的连作障碍现象。连作障碍产生的原因主要有根际土壤微生物群落变化、土传病虫害增加、化感物质积累及其自毒作用、土壤理化性状恶变等。以前三七连作障碍研究多集中于单一因素对连作障碍的影响及三七病虫害防治,疏于综合分析各种因素间的内在联系和相互影响。本文总结了作物连作障碍形成机制的最新研究进展,综合分析了各因素间可能存在的关系,首次提出了各因素对三七连作障碍影响指数的评估方法,建议了未来研究方向,旨在为揭示三七连作障碍形成机制提供参考,并为连作障碍消减提供理论依据。     

芦笋连作障碍中的自毒物质研究进展

芦笋的自毒作用是导致连作障碍的主要原因之一。关于芦笋连作障碍中自毒物质的研究, 包括芦笋自毒作用的解读、自毒物质来源、自毒物质种类、及芦笋品种间化感自毒作用差异性存在的研究, 对于这些方面的综述旨在为芦笋化感自毒作用的研究提供参考资料。并总结了缓解自毒作用的途径, 包括轮作、生物炭、降解菌及其他途径, 以期为解决芦笋连作障碍中的自毒作用提供新思路。     

烟草化感自毒物质降解复合菌剂的优化及应用效果评价

烟草(Nicotianatabacum)长期连作导致土壤中烟草自身分泌的化感自毒物质的积累,加剧了烟草青枯病(tobacco bacterial wilt, TBW)的发生,严重威胁烟草的生产并造成了巨大的经济损失。【目的】获得对长期连作烟田青枯病具有显著防治效果的复合菌剂。【方法】利用筛选到的烟草化感自毒物质降解菌株构建复合菌剂,通过正交试验和单因素试验对菌种配比和助剂进行优化,对其在温室盆栽和连作烟田生产的应用效果进行评价。【结果】正交试验结果表明,复合菌剂中芽孢杆菌(Bacillus sp.) NO1、布鲁氏菌(Brucella sp.) NO8、芽孢杆菌(Bacillus sp.) NO9和NO10的最佳菌种配比为1:3:4:2;单因素试验结果表明最佳的载体为白炭黑;最佳润湿剂和分散剂分别为六偏磷酸钠和丁基萘磺酸钠,其最佳比例为1:1,最佳用量各为2%;稳定剂甘油的最佳浓度为1.0%。盆栽试验结果显示,复合菌剂对6种化感自毒物质的降解率和对青枯病的抑制率均达到78%以上。大田试验结果显示,100倍稀释的复合菌剂对连作15年烟田中的6种化感自毒物质具有显著的降解效果,并且能明显增...     

花生植株和土壤水浸液自毒作用研究及土壤中自毒物质检测

通过盆栽试验及生物测试研究花生植株、土壤水浸液对其种子萌发和幼苗生长的自毒作用,利用单因子方差分析结合多重比较分析各处理组生物活性差异;采用液相色谱法(HPLC)对正茬、连作2、4 a和6 a的花生根际土壤中酚酸类物质进行定性和定量分析。结果表明:花生植株不同部位及根际土壤水浸液对花生种子萌发和幼苗生长均具有不同程度的抑制作用,且抑制作用具有浓度梯度效应,浓度越高抑制作用越强,16 g/10 mL水浸液的抑制作用最大。花生生长受自毒物质的影响,此影响作用强度因自毒物质的来源部位不同而存在差异。种子萌发以茎水浸液的自毒作用最为明显。但针对幼苗生长的不同测定指标其部位差异不一致,其中以茎水浸液对幼苗株高和叶面积的自毒作用最为明显,根际土壤水浸液对幼苗主根长、单株鲜重和单株干重的自毒作用最为明显。在根际土壤水浸液中鉴定到4种酚酸物质,分别为对羟基苯甲酸、香草酸、香豆酸和香豆素,其中香草酸和香豆素含量较高且变化规律性明显,在土壤中的含量随连作年限的增加而上升,连作6 a后土壤中的含量达到0.289μg/g和0.025μg/g干土。     

根皮苷降解真菌的筛选、鉴定及降解特性研究

筛选高效降解根皮苷的功能菌以期为生物降解根皮苷、缓解苹果连作障碍奠定基础。采用以根皮苷为唯一碳源选择性培养基筛选菌种,对筛选的菌种进行生理生化和分子生物学鉴定,并研究了不同培养条件下的降解特性。结果显示,从环渤海苹果主产区的12个县市地区的苹果根际土壤中筛选出一株能够高效降解根皮苷的菌株AMCC300110,经形态学和分子生物学方法初步鉴定该菌为土曲霉(Aspergillus terreus),该菌在根皮苷初始浓度为2 mmol/L、p H5.0,接种量2%条件下,40℃、160 r/min摇床培养96 h,降解率可达88.96%。降解特性研究结果表明,不同培养条件下摇床培养12 h,该菌分别在p H为5.0,培养温度40℃,初始根皮苷浓度为6 mmol/L时有最大的降解率,同时该菌还具有广谱降解酚酸的能力。通过选择性培养基筛选得到一株土曲霉,该菌具有高效降解根皮苷的能力,在生物降解酚酸类自毒物质,缓解苹果连作障碍方面有一定的应用潜力。     

一株纤维素降解细菌的筛选、鉴定及产酶条件分析

目的筛选高活性的纤维素降解细菌,并进行初步鉴定和产纤维素酶条件分析。方法采集吉首旗帜山松树林的土壤样品,通过富集培养和刚果红平板染色法筛选分离纤维素降解细菌;通过形态观察、生理生化特性检测和基于16S rRNA基因序列的系统发育分析对分离的菌株进行初步鉴定。利用单因素实验对产纤维素酶条件进行优化。结果分离获得1株高活性纤维素降解细菌JDM11,初步鉴定其为Bacillus velezensis;菌株JMD11产纤维素酶最佳培养温度、最适初始pH和培养时间分别为28℃、7.0～7.5和32h,在该条件下其滤纸酶(FPase)和羧甲基纤维素酶(CMCase)活力分别为260.32U/ml和651.75U/ml。结论菌株JDM11是1株高活性纤维素降解的Bacillus velezensis。     

小花老鼠簕可培养细菌多样性及其生物学活性研究

小花老鼠簕(Acanthus ebracteatus)是一种生长在红树生态系统的珍稀真红树植物,具有较高的药用价值。为研究小花老鼠簕内生及根际可培养细菌多样性,挖掘其潜在新物种及具有特殊生物学活性的菌株,该文利用7种不同培养基,通过传统稀释涂布法对小花老鼠簕各植物组织及根际土壤可培养细菌进行分离,基于16S rRNA基因序列解析其内生及根际细菌群落结构和多样性特征,应用植物病原菌平板对峙实验和平铺捕食活性测试分析其可培养细菌的抗菌活性。结果表明：(1)基于16S rRNA基因序列分析,发现从小花老鼠簕的根、茎、叶、花及根际土壤中分离得到144株可培养细菌,这些细菌隶属于18目26科37属66种,芽孢杆菌属(Bacillus)和链霉菌属(Streptomyces)为优势菌属,分别占细菌种数的15.1%和13.6%;(2)拮抗多种植物病原菌试验结果显示,获得29株具有拮抗植物病原菌活性的细菌,10株具有广谱抑菌活性,其中链霉菌属菌株拮抗作用最强且菌株Y129为潜在新物种。(3)捕食活性测试结果显示,有5株细菌对金黄色葡萄球菌(Staphylococcus aureus)、耐甲氧西林金黄色葡...     

Manipulation of ginsenoside heterogeneity of <Emphasis Type="Italic">Panax notoginseng</Emphasis> cells in flask and bioreactor cultivations with addition of phenobarbital

Structure-similar ginsenosides have different or even totally opposite biological activities, and manipulation of ginsenoside heterogeneity is interesting and significant to biotechnological application. In this work, addition of 1 mM phenobarbital to cell cultures of Panax notoginseng at a relatively high inoculation size of 7.6 g dry cell weight (DW)/L enhanced the production of protopanaxatriol-type (Rg₁ + Re) ginsenosides in both shake flask and airlift bioreactor (ALR, 1 L working volume). The content of Rg₁ + Re in the ALR was increased from 42.5 ± 4.0 mg per gram DW in untreated cell cultures (control) to 56.4 ± 4.6 mg per gram DW with addition of 1.0 mM phenobarbital. The maximum productivity of Rg₁ + Re in the ALR reached 5.66 ± 0.38 mg L⁻¹ d⁻¹, which was almost 3.3-fold that of control. The maximum ratio of the detectable ginsenosides protopanaxatriol:protopanaxadiol (Rb₁) was 7.6, which was about twofold that of control. The response of protopanaxadiol 6-hydroxylase (P6H) activity to phenobarbital addition coincided with the above-mentioned change of ginsenoside heterogeneity (distribution). Phenobarbital addition is considered as a useful strategy for manipulating the ginsenoside heterogeneity in bioreactor with enhanced biosynthesis of protopanaxatriol by P. notoginseng cells.     

Conversion of Major Ginsenoside Rb1 to Ginsenoside F2 by Caulobacter leidyia

Ginsenoside Rb₁ is the most predominant ginsenoside in Panax species (ginseng) and the hydrolysis of this ginsenoside produces pharmaceutically active compounds. Caulobacter leidyia GP45, one of the isolates having strong β-glucosidase-producing activity, converted ginsenoside Rb₁ to the active metabolites by 91%. The structures of the resultant metabolites were identified by NMR. Ginsenoside Rb₁ had been consecutively converted to ginsenoside Rd (1), F₂ (2) and compound K (3) via the hydrolyses of 20-C β-(1→6)-glucoside, 3-C β-(1→2)-glucoside, and 3-C β-glucose of ginsenoside Rb₁.     

Efficient elicitation of ginsenoside biosynthesis in cell cultures ofPanax notoginseng by using self-chemically-synthesized jasmonates

A series of fluorine and hydroxyl containing jasmonate derivatives, which were chemically synthesized in our institute, were investigated for their effects on the biosynthesis and heterogeneity of ginsenosides in suspension cultures ofPanax notoginseng cells. Compared to the control (without addition of elicitors), 100 μM of each of the jasmonate was added on day 4 to the suspension cultures ofP. notoginseng cells. It was observed that, jasmonates greatly enhanced the ginsenoside content and the ratio of Rb group to Rg group (i.e. (Rb₁+Rd)/(Rg₁+Re)) in theP. notoginseng cells. Some of the synthetic jasmonates, such as pentafluoropropyl jasmonate (PFPJA), 2-hydroxyethyl jasmonate (HEJA) and 2-hydroxyethoxyethyl jasmonate (HEEJA), could promote the ginsenoside content to 2.55±0.11, 3.65±0.13 and 2.94±0.06 mg/100 mg DW, respectively, compared to that of 0.64±0.06 mg/100 mg DW for the control and 2.17±0.04 mg/100 mg DW by the commercially available methyl jasmonate (MJA); and they could change the respective Rb: Rg ratio to 1.60±0.04, 1.87±0.01 and 1.56±0.05, compared to that of 0.47±0.01 for the control and 1.42±0.06 by MJA. The results suggest that suitable esterification of MJA with fluorine or hydroxyl group could increase the elicitation activity to induce plant secondary metabolism. The information obtained from this study is useful for hyper-production of heterogeneous products by plant cell cultures.     

Effects of rhizosphere remediation and bioaugmentation on carbofuran removal from soil

Rhizosphere soil contains important sources of nutrients for microorganisms resulting in high number of microorganisms capable of degrading various types of chemicals in the soil. Thus, this study investigated a carbofuran dissipation in rhizosphere soils of 6 weeds namely, umbrella sedge (Cyperus iria L.), fuzzy flatsedge (C. pilosus V.), small flower umbrella plant (C. difformis L.), tall-fringe-rush hoorah grass (Fimbristylis miliacea V.), cover fern (Marsilea crenata P.), and water primrose (Jussiaea linifolia V.). Rhizosphere soil of fuzzy flatsedge showed the shortest half-life (t_1/2) of carbofuran (15 days) among other soils. So, it was selected to be used in the bioaugmentation experiment using carbofuran degrader namely Burkholderia cepacia, PCL3, as inoculum in order to examine whether they would improve carbofuran degradation in soil. The results showed that the addition of PCL3 into rhizosphere soil did not improve carbofuran degradation suggesting that microorganisms in rhizosphere soil might be capable enough to remove carbofuran from soil. The number of carbofuran degraders in the rhizosphere soils was greater than in bulk soil 10–100 times which might be responsible to a rapid degradation of carbofuran in rhizosphere soils without the addition of PCL3. The ability of PCL3 to degrade carbofuran was evident in bulk soil (t_1/2 of 12 days) and autoclaved soils (t_1/2 13–14 days) when compared to soils without an inoculation (t_1/2 of 58 days) indicated that the addition of a degrader was useful in improving carbofuran degradation in soil.     

Ginsenoside Rd production from the major ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">β</Emphasis>-glucosidase from <Emphasis Type="Italic">Thermus caldophilus</Emphasis>

Under optimum conditions (pH 5, 75°C, and 0.2 U purified enzyme ml⁻¹), 4 mg ginsenoside Rd was produced from 5 mg reagent-grade ginsenoside Rb₁ in 5 ml after 30 min by β-glucosidase from Thermus caldophilus GK24. Using a ginseng root extract containing 1 mg ginsenoside Rb₁ ml⁻¹ and 3.2 mg additional ginsenosides ml⁻¹, 1.23 mg ginsenoside Rd ml⁻¹ was produced after 18 h; the concentrations of ginsenosides Rb₁, Rb₂, and Rc used for ginsenoside Rd production were 0.77, 0.17, and 0.19 mg ml⁻¹, respectively.     

Effects of antibiotics in soil on the population dynamics of transposon Tn5 carrying Pseudomonas fluorescens

The effects of kanamycin and streptomycin added to soil on the survival of transposon Tn5 modified Pseudomonas fluorescens strain R2f were investigated. Kanamycin in high (180 g g^-1 dry soil) or low (18 g g^-1) concentration or streptomycin in low concentration in Ede loamy sand soil had no noticeable effect on inoculant population dynamics in soil and wheat rhizosphere, whereas streptomycin in high concentration had a consistent significant stimulatory effect, in particular in the wheat rhizosphere. Streptomycin exerted its effect by selecting P. fluorescens with Tn5 insertion whilst suppressing the unmodified sensitive parent strain, as evidenced by comparing the behaviour of these two strains in separate and mixed inoculation studies.Soil textural type influenced the effect of streptomycin on the Tn5 carrying inoculant; the effect was consistently detected in rhizosphere and rhizoplane samples of wheat grown in Ede loamy sand after 7 and 14 days incubation, whereas it was only apparent after 7 days in rhizoplane or rhizosphere (and bulk soil) samples of wheat grown in two silt loam soils. Modification of soil pH by the addition of CaCO₃ or bentonite clay resulted in an enhancement of the selective effect of streptomycin by CaCO₃ and its abolishment by bentonite clay.The addition to soil of malic acid or wheat root exudate, but not of glucose, enhanced the streptomycin selective effect on the Tn5-modified P. fluorescens strain. Neither the streptomycin producer Streptomyces griseus nor two non-inhibiting mutants obtained following UV irradiation affected the dynamics of P. fluorescens (chr::Tn5) in soil and wheat rhizosphere.The effect of streptomycin in soil on inoculant Tn5 carrying bacteria depends on conditions such as soil type, the presence of (wheat) root exudates and the type of available substrate.     

阿尔泰银莲花根际土壤微生物多样性研究

为了解野生和栽培阿尔泰银莲花根际土壤微生物多样性的差异,该研究采用Illumina MiSeq高通量测序技术对野生和栽培阿尔泰银莲花根际土壤微生物的群落组成和多样性进行探究。结果表明:(1)野生阿尔泰银莲花根际土壤的真菌多样性显著高于栽培阿尔泰银莲花(P<0.05),而细菌多样性差异不显著(P>0.05); NMDS分析结果显示,野生和栽培阿尔泰银莲花根际土壤真菌群落结构差异更显著。(2)细菌9 566个可操作分类单元(OTUs)涉及39门127纲315目500科886属,真菌2 670个OTUs涉及15门57纲138目293科597属。在门水平上,细菌群落中的变形菌门、酸杆菌门、放线菌门及真菌群落中的担子菌门、子囊菌门、被孢霉门均为野生和栽培阿尔泰银莲花根际土壤优势菌门,但其相对丰度在不同生长方式下存在差异。(3)环境因子关联分析(RDA)结果显示,土壤有机质是影响土壤细菌群落的主要因子(P<0.05),土壤pH、碱解氮和有效磷是影响真菌群落的主要因子(P<0.05)。综上认为,野生和栽培下的阿尔泰银莲花根际土壤微生物群落组成和多样性存在显著差异,这种差异可能与不同生长条件下的土壤理化性质存在密切的联系,该研究结果对阿尔泰银莲花科学种植以及土壤改良具有一定意义。     

薇甘菊化感物质对土壤氮素矿化的影响及其化感利己作用

【目的】研究入侵植物薇甘菊提取物对土壤氮素矿化的影响及化感利己作用,为薇甘菊通过释放化感物质促进自身生长并排斥本地植物生长提供实验证据,为揭示薇甘菊的入侵机制提供理论依据。【方法】采用经典的化感生测实验、化感物质添加实验以及盆栽控制实验,对比研究薇甘菊及其本地伴生植物水提液和粗提物的化感生测效应、水提液和粗提物添加对土壤氮素的影响以及水提液添加对薇甘菊生长的影响。【结果】与本地伴生种相比,薇甘菊水提液的化感抑制作用较强且促进了土壤硝态氮的生成;而薇甘菊粗提物的化感作用较弱且抑制了土壤铵态氮和硝态氮的生成。水提液盆栽控制实验结果表明,与本地伴生种火炭母相比,薇甘菊水提液具有显著的化感利己作用,这与薇甘菊水提液处理后薇甘菊生长土壤中的蛋白酶活性增强和有效氮含量增加相一致。【结论】薇甘菊可通过水溶性化感物质促进有效氮的生成来实现化感利己作用。     

培养条件对三七愈伤组织生长和皂苷积累的影响

以MS为基础培养基,改变激素配比、氮源和光照等因素,以分光光度法和HPLC法分析三七愈伤组织培养过程中皂苷含量的变化。结果表明:培养条件对三七愈伤组织中皂苷积累有一定影响,激素配比对愈伤组织中皂苷含量的影响最大,在0.5 mg·L-12,4-D+1.0 mg·L-16-BA组合下,培养物中总皂苷含量最多,达到4.72%±0.29%;在总氮量为60 mmol·L-1条件下,45 mmol·L-1KNO3+7.5 mmol·L-1NH4NO3(NO3-/NH4+=7∶1)时,愈伤组织皂苷含量最多,达到4.71%±0.17%;分别在1 000 lx和500 lx光强下每天光照12 h的愈伤组织,皂苷含量均低于黑暗培养的愈伤组织,三者皂苷含量分别为1.94%±0.31%、2.38%±0.12%和3.57%±0.27%,光照引起愈伤组织表面变绿及细胞分化,可能是抑制愈伤组织中皂苷合成与积累的主要原因;HPLC检测发现,三七愈伤组织和根中均含有Rg1、Re、Rb1及Rd四种皂苷,但栽培三七根含有R1皂苷,而三七愈伤组织中未检测到R1,其原因需要进一步研究。该研究结果为未来愈伤组织培养成为部分代替人工栽培生产三七天然产物的潜在途径提供了研究基础。