高秆野生稻内生固氮细菌多样性

高秆野生稻(Oryza alta)是一种重要的种质资源, 其组织内也蕴藏着非常宝贵的功能微生物资源。本实验采用无氮培养基, 从高秆野生稻中分离到43株内生固氮菌, 结合乙炔还原法测定其固氮酶活性。经固氮酶基因(nifH)的PCR扩增检测, 43株内生固氮菌代表菌株均能扩增出固氮酶基因片段。利用IS-PCR DNA指纹图谱和SDS-PAGE全细胞蛋白电泳图谱将获得的菌株聚类为6个类群(I、II、III、IV、V、VI)。对各个类群的代表菌株(ZF3, ZF8, ZF13, ZF15, ZF24, ZF43)进行16S rRNA基因序列测定, 结果表明, 类群I属于土生拉乌尔菌(Raoultella terrigena), 类群II属于类肺炎克雷伯氏菌类肺炎亚种(Klebsiella quasipnmoniae subsp. quasipneumoniae), 类群III属于越南伯克氏菌(Burkholderia vietnamiensis), 类群IV的菌株代表肠秆菌属的一个类群(Enterobacter sp.), 类群V的菌株属于门多萨假单胞菌(Pseudomonas mendocina), 类群VI归属于固氮植物菌(Phytobacter diazotrophicus)。Biolog聚类结果与IS-PCR指纹图谱类型及SDS-PAGE全细胞蛋白聚类结果一致。Biolog板测定结果显示, 来自不同类群的代表菌株对碳源的利用差异显著, 说明野生稻内多样的内生固氮菌从环境中获取碳源和氮素的适应能力较强。     

攀枝花地区烤烟可培养内生固氮菌的多样性

【目的】认识烤烟(Flue-cured tobaccos)内生固氮菌多样性,挖掘内生固氮菌资源,丰富内生固氮菌基因库。【方法】运用纯培养法、重复因子扩增(BOX-PCR)分析技术、16S r RNA基因测序和系统发育分析对内生固氮菌多样性和系统发育进行研究,并测定分离菌株的固氮酶活性、溶磷溶钾特性、吲哚乙酸(IAA)含量等指标。【结果】通过Ashby培养基共分离得到62株固氮菌。基于BOX-PCR图谱选取16株代表菌株进行16S r RNA基因序列测定。16S r RNA基因序列系统发育分析显示,62株菌株分属于芽孢杆菌属(Bacillus)、泛菌属(Pantoea)、短小杆菌属(Curtobacterium)等3个属,其中芽孢杆菌属(Bacillus)为优势菌属。62株菌株中有20株菌株(占总分离菌株的32.3%)具有固氮酶活性,8株菌株(占总分离菌株的12.9%)能产IAA,有4株(占总分离菌株的6.5%)表现溶磷活性,有3株(占总分离菌株的4.8%)表现溶钾活性。【结论】攀枝花烤烟有较为丰富的内生固氮菌,具有潜在应用价值。     

北京地区水稻根际联合固氮菌的分离鉴定及其固氮酶活性的研究

从北京地区16个水稻品种的根际分离筛选出两株固氮酶活性较高的菌株D-12和D-25。对这两个菌株进行了分类鉴定和固氮酶活性的测定。根据形态特征和理化特性的测定,菌株D-12属于肺炎克雷伯氏菌(Klebsiella pneumoniae),菌株D-25类似于克雷伯氏菌但又有区别,因此暂放在胁杆菌科(Enterobacteriaceae)。两个菌株均在厌氧条件下固氮酶活性最高。在Hill's无氮蔗糖培养基中30℃条件下,其固氮酶活性的高峰出现在第16至第18小时。     

一株人参内生1-氨基环丙烷-1-1羧酸(ACC)脱氨酶活性细菌的筛选、鉴定及其对宿主生长的影响

【目的】从人参内生细菌中获得具有1-氨基环丙烷1-羧酸(ACC)脱氨酶活性的菌株,并进行促生效果的验证。【方法】结合初筛和复筛的方法筛选具有ACC脱氨酶活性的人参内生菌株;采用Ashby培养基和固氮酶基因验证其固氮潜能;菌碟法及钼锑抗比色法测定其解磷能力;CAS方法检测产生铁载体能力;通过室内及田间试验测定菌株对人参生长的促进作用。通过形态学、生理生化测定及16S rRNA序列分析明确菌株的分类地位。【结果】从120株人参内生菌中获得了一株具有较高ACC脱氨酶活性的菌株JJ8-3,其酶活性为α-酮丁酸6.7μmol/(mg·h);且具有解磷特性、固氮潜能和产生铁载体能力;能明显促进人参种子及根部的生长;经鉴定菌株JJ8-3为荧光假单胞菌(Pseudomonas fluorescens)。【结论】获得了一株具有ACC脱氨酶活性的人参内生细菌,将为其在促进植物生长中的应用和研究奠定基础。     

类芦和狗牙根内生固氮菌初步研究

为探索水土保持禾草的固氮能力,利用乙炔还原法(ARA)对类芦(Neyraudia reynaudiana)和狗牙根(Cynodon dactylon)植株内生固氮菌的固氮酶活性进行研究.结果表明:在类芦茎部和狗牙根叶片中存在高固氮酶活性的内生固氮菌群;这些菌群在无氮培养基上经划线分离纯化得到40个菌株,其中26株有微弱固氮酶活性,14株检测不到固氮酶活性;从类芦茎部和狗牙根叶片中筛选到的固氮菌群在兼性厌氧的环境中有较高的固氮酶活性,且在石蜡油密封条件下,两者的固氮酶活性均最高;在不改变自然界微生物之间的协同关系情况下,混合菌群均具有较高的固氮酶活性,而人为组配混合得到的菌群固氮酶活性较低. reynaudiana)和狗牙根(Cynodondxtylon)植株内生固氮菌的固氮酶活性进行研究.结果表明:在类芦茎部和狗牙根叶片中存在高固氮酶活性的内生固氮菌群;这些菌群在无氮培养基上经划线分离纯化得到40个菌株,其中26株有微弱固氮酶活性,14株检测不到固氮酶活性;从类芦茎部和狗牙根叶片中筛选到的固氮菌群在兼性厌氧的环境中有较高的固氮酶活性,且在石蜡油密封条件下,两者的固氮酶活性均最高;在不改变自然界微生物之间的协同关系 况下,混合菌群均具有较高的固氮酶     

地锦草内生细菌多样性、拮抗及促生特性测定

【背景】植物内生细菌既能抑制病原菌对植物的侵染,也具有促生作用。分离具有拮抗和促生活性的内生细菌可为开发微生物菌肥提供理论依据。【目的】筛选内生细菌中的优势拮抗、促生菌种资源。【方法】以地锦草为材料,采用4种分离培养基分离该植物内生细菌,通过形态特征以及16S r RNA基因序列分析,鉴定内生细菌的分类归属。采用平板对峙法,测定内生细菌对棉花立枯丝核菌(Rhizoctonia solani)、小麦赤霉病菌(Fusarium graminearum)、玉米小斑病菌(Bipolaris maydis)的拮抗活性。通过固氮、解磷、产吲哚乙酸(Indole Acetic Acid,IAA)、产铁载体能力等指标初步检测地锦草内生细菌的促生活性。【结果】共分离到133株内生细菌,分属于4门5纲8目13科25属,其中变形菌门(Proteobacteria)为优势门(52.63%),优势属为芽孢杆菌属(Bacillus),占15.79%。发现有8株菌相似性小于98.65%,可能为潜在新物种。拮抗活性结果表明,22株菌有不同程度的抑菌作用,其中菌株DHL56、DHN17、DHP3、DHP8对这3种病原菌都有抑制作用,均为芽孢杆菌属。菌株DHP8抑制作用最强,对棉花立枯丝核菌、小麦赤霉病菌抑制率分别为73.80%、71.25%,对玉米小斑病菌抑制率为61.70%。促生潜力结果表明,76株菌具有固氮能力;19株菌具有解磷能力;37株菌能产吲哚乙酸,菌株DHL55产吲哚乙酸的量达到105.67mg/L;7株菌能合成铁载体。其中有9株菌同时有固氮、解磷、产吲哚乙酸能力;菌株DHP8具有固氮、解磷、合成铁载体能力。DHP8不仅具有一定促生潜力,还对棉花立枯丝核菌、小麦赤霉病菌、玉米小斑病菌有明显的拮抗作用,需进一步研究。【结论】地锦草内生细菌种类丰富,获得多株具有优良拮抗和促生活性的菌株,为进一步开发微生物农药及菌肥资源提供新的菌株材料。     

四绺孢球腔菌科真菌地理分布及其生态学功能

根据ITS、LSU、rpb2、tef1和tub2多基因系统发育分析,将云南禾本科植物格孢腔菌目的7株内生真菌鉴定归属于格孢腔菌目Pleosporales四绺孢球腔菌科Tetraplosphaeriaceae的四绺孢属Tetraploa和假四绺孢属Pseudotetraploa以及该目下的一个未定属genera incertae sedis。羧甲基纤维素钠培养基和愈创木酚培养基筛选发现四绺孢球腔菌科的2个菌株具有较强的纤维素酶和漆酶活性,而这个未定属的菌株仅具有较弱的纤维素酶活性、无漆酶活性,表明格孢腔菌目的2个内生真菌类群的纤维素和木质素降解能力不同。多重对应分析发现四绺孢球腔菌科真菌的属与寄主、分离来源和地理位置有关联,其中四绺孢属和假四绺孢属可在活的健康植物作为内生真菌存活,并在植物凋落物和土壤中分离得到,推测四绺孢属和假四绺孢属两属为内生和腐生双生态位真菌。因此,进一步深入探究四绺孢球腔菌科内生真菌参与的禾本科植物凋落物的分解将深化我们对禾本科植物内生真菌多样性和生态学功能的认识。     

藤县药用野生稻内生固氮菌分离鉴定及系统发育分析

从广西梧州市藤县药用野生稻中筛选内生固氮菌,为微生物肥料生产收集菌种资源。采用3种无氮培养基经平板划线法筛选内生固氮菌;通过乙炔还原法测定其固氮酶活性;利用IS-PCR(Insertion sequence-based PCR)指纹图谱和全细胞蛋白电泳图谱对获得的内生固氮菌进行聚类分析;根据16S r RNA基因序列分析确定其进化关系;使用分光光度计比色法检测其产生长素能力、产铁载体能力、溶磷能力和ACC脱氨酶活性。结果显示,从野生稻中筛选获得34株内生固氮菌分为5个类群,类群I和II各有8株,类群III、IV、V分别有2株、5株和11株,其固氮酶活性介于5.0和1 036.7 nmol/(m L·h)之间。类群I代表菌株HU33与无乳固氮螺菌(Azospirillum amazonense ATCC 35119T)相似性为97.13%;类群II代表菌株HU31与变栖克雷伯氏菌(Klebsiella variicola DSM 15968T)相似性为99.71%;类群III代表菌株HU17与蒙氏假单胞菌(Pseudomonas monteilii ATCC 700476T)相似性为99.86%;类群IV代表菌株HU13与黄黄色杆菌(Xanthobacter flavus ATCC 35867T)相似性为100%;类群V代表菌株CM05与越南伯克霍尔德菌(Burkholderia vietnamiensis LMG 10929T)相似性为99.86%。药用野生稻内生固氮菌资源具有遗传多样性;有些菌株有较强的产生长素、产铁载体、ACC脱氨酶活性和溶磷能力,在农业生产上具有一定的应用潜力;类群I可能是一个新类群。     

海洋固氮菌和解磷菌的分离鉴定及发酵条件优化

【目的】从西沙喜盐草根际沉积物中分离纯化得到具有高效固氮能力及解磷能力的菌株。优化其发酵培养条件,研究其制备海洋微生物菌剂的可能性。【方法】从形态学特征、生理生化、16S rDNA及功能基因水平进行鉴定,通过乙炔还原法、钼锑抗显色法检测菌株的固氮酶活性和解磷能力,单因素法和响应面法优化其发酵培养条件,溶血试验和急性毒性实验鉴定菌株的安全性。【结果】结果表明,菌株AZ16属于星箭头菌(Sagittula stellate),革兰氏阴性菌,选择性固氮培养基中菌落呈黄圆形黏稠状,固氮酶活性达34.63 nmol C2H2/(mL·h),最适生长条件为:盐度25‰、pH 7.5、温度33°C、接种量5.0%;菌株XT37为海洋芽孢杆菌(Bacillus sp.),革兰氏阳性菌,选择性固氮培养基中菌落呈深黄色圆形褶皱,植酸酶活性达239.49μg/L,最适合生长条件为:盐度25‰、pH 6.7、温度28°C、接种量5.0%。溶血实验和急性毒性实验证明两株菌属实际无毒级别。【结论】两株菌具有高效的固氮解磷功能,以及抗高盐、强碱等环境的能力,安全无毒,因此有潜力应用于多功能混合微生物菌剂的研制。     

糖蜜草(Melinis minutiflora Beauv.)内生固氮菌分离鉴定

糖蜜草（Melinis minutiflora Beauv．）是热带地区的一种优良牧草。采用选择性培养基在厌氧和好氧两种培养条件下,从糖蜜草根、茎中都可分离得到具有较强固氮酶活性的菌株。通过SDS-PAGE全细胞蛋白电泳技术快速聚类分析表明,来源于糖蜜草中的菌株为同一类群。16S rDNA序列分析和总DNA的G＋c％含量进一步确定糖蜜草中所分离的菌株属于固氮螺菌属（Azospirillum）,与产脂固氮螺菌（Azospirillum lipoferum）亲缘关系较近。BIOLOG板测定结果显示,糖蜜草菌株TMCY243对多种碳源具有很强的适应性,与产脂固氮螺菌（A．lipoferum）的模式菌株DSM 1691存在着较大的差异。以上结果表明,糖蜜草内生固氮菌为固氮螺菌属的一个新类群。     

陕西耀州产黄芩生长发育规律的研究

通过观测陕西耀州产1年生和2年生黄芩(Scutellaria baicalensis Georgi)的物候期及2年生植株生长的动态变化,对黄芩的生长发育规律进行了研究。结果表明,1年生黄芩的各物候期均晚于2年生黄芩,其生育天数和生长天数均较短;2年生黄芩的地上部分和根系均有2个较快的生长时期,分别出现在生殖生长期前后;地上部分的干质量和鲜质量都有2个生长高峰,均出现在7月20日和9月20日,而根系的干质量和鲜质量则持续增长;2年生黄芩的根冠比在10月20日达最大值(0.50);2年生植株的株高、根直径及根系的干质量和鲜质量等均优于1年生植株。依据实验结果提出了黄芩优质高产的栽培技术。     

2种内生细菌对文冠果幼苗矿质元素吸收的影响

为探究前期筛选的具有溶磷、产铁载体、产IAA能力的内生细菌对文冠果(Xanthoceras sorbifolia Bunge)幼苗矿质元素的影响,选取一株假单胞菌属菌株XSB5和一株约克氏菌属菌株GG10作为试验菌株。用菌液浸泡经过层积催芽的文冠果种子,待植株长出第一片真叶后,用等量的菌液和清水处理文冠果幼苗,4个月后分析文冠果植株的生长状况。结果表明:用XSB5菌液处理后,文冠果植株地上和地下生物量均显著增加(P<0.05);GG10菌液处理后,文冠果地上和地下生物量均有所增加,地下生物量增加达显著水平(P<0.05);两种菌液处理后文冠果植株N、P、Mg积累量较对照显著增加(P<0.05),Fe积累量因处理时间不同而有差异;4种矿质元素的转运系数因不同菌液、不同处理时间而有差异;栽培基质中有效态N、P含量较对照显著增加(XSB5-30处理除外),有效态Mg、Fe含量与对照无显著差异。说明接种这2种内生细菌能影响栽培基质中矿质元素含量的变化,提高文冠果植株对矿质元素的吸收,改变矿质元素向上的运输能力,从而促进文冠果植株的生长。该研究可为文冠果促生菌肥的筛选提供一定的理论基础。     

柠条根瘤内生细菌的抗逆性及遗传多样性

采用16S rDNA PCR-RFLP和序列分析方法对分离自柠条根瘤的40株内生细菌的遗传多样性及系统发育进行分析,并对菌株的耐盐性、耐酸碱性和生长温度范围进行测定.结果表明:40株供试菌株共产生9种遗传图谱类型;对各类型代表菌株进行16S rDNA序列测定,结合形态特征和生理生化检测结果,表明供试菌株分别归属于芽孢杆菌属(Bacillus)、Inguilinus属、申氏杆菌属(Shinella)和不动杆菌属(Acinetobacter),遗传多样性较为丰富;57.5％的菌株可耐受4％的NaCl,75％的菌株可在pH 11.0的条件下生长,85％的菌株经60℃热激处理后仍能继续生长,显示柠条根瘤内生细菌具有较强的抗逆性,菌株LWEN 07和LWEN 15抗逆能力最为显著.     

Xylem Cavitation in Nodes and Internodes of Whole Chorisia insignis H.B. et K. Plants Subjected to Water Stress: Relations Between Xylem Conduit Size and Cavitation

Measurements of cavitation occurring in xylem conduits of differentstem parts in whole Chorisia insignis H.B. et. K. plants subjectedto water stress are reported. Pre-stressed plants were shownto undergo cavitation over 10 times greater than watered ones.The most vulnerable parts of plants were one-year-old twigswhere cavitation reached a peak of over 50 acoustic emissions(AE) min^–1 while in two-year-old twigs AE min^–1were about one half this value. Stem zones were found wherecavitation was typically very low even during water stress:these were one-year-old nodes and junctions where branches meet.Measurements of the inside diameters of xylem conduits and distributionof conduit ends in stem parts where AE were detected, showedthat nodes have a significantly larger percentage of narrowxylem conduits than internodes. Similar ‘constricted zones’were found injunctions with respect to two-year-old twigs. Hereabout 50 per cent of the xylem conduits were as narrow as 20to 50 µm in diameter. The distribution of xylem conduitends show about 3 per cent of them ending in the nodes and 1per cent in the internodes of one-year-old twigs. About 11.6per cent of xylem conduits end in the junctions and about ahalf in two-year-old internodes. Our data would give furtherexperimental evidence to the functional concept of ‘plantsegmentation’ into zones (internodes) more efficient inwater conduction, i.e. with wider xylem conduits but more vulnerableto cavitation and others (nodes and junctions) with oppositecharacteristics. Chorisia insignis, acoustic emissions, water stress, nodes, internodes, xylem conduit size, vessel ends     

Diversity and host preference of fungi co-inhabiting Cenococcum mycorrhizae

Diverse fungal assemblages colonize the fine feeder roots of woody plants, including mycorrhizal fungi, fungal root endophytes and soil saprotrophs. The fungi co-inhabiting Cenococcum geophilum ectomycorrhizae (ECM) of Abies balsamea, Betula papyrifera and Picea glauca were studied at two boreal forest sites in Eastern Canada by direct PCR of ITS rDNA. 50 non-Cenococcum fungal sequence types were detected, including several potentially mycorrhizal species as well as fungal root endophytes. Non-melanized ascomycetes dominated, in contrast to the dark septate endophytes (DSE) reported in most culture dependent studies. The results demonstrate significant differences in root associated fungal assemblages among the host species studied. Fungal diversity was also host dependent, with P. glauca roots supporting a more diverse community than A. balsamea. Differences in root associated fungal communities may well influence ecological interactions among host plant species.     

Sowing and transplantation of Adonis ramosa (Ranunculaceae) for the establishment of a new population in a seminatural broad-leaved deciduous forest floor

Adonis ramosa (Ranunculaceae) is a plant whose risk of extinction is increasing in Japan. In this study, we show that there is a significant possibility of establishing an A. ramosa population by sowing seeds or transplanting plants in a seminatural broad-leaved deciduous forest floor. Immediately after seed collection in early June, seeds were sown either in pots that were watered periodically or in a deciduous forest floor where the soil surface was prepared. In the following spring, 30–40% of the seeds germinated. Of the one-year-old plants buried at a depth of 2 cm and two-year-old plants buried at a depth of 2 or 8 cm in the fall, 96% sprouted leaves in the following spring. However, 79% of the one-year-old plants buried at a depth of 8 cm sprouted leaves. In spring, the number of leaves, the widths of individual plants, and the heights of the two-year-old plants were greater than those of the one-year-old plants. The dry weights of one- and two-year-old plants in fall after one spring growing season increased by 1.6–2.3 times and by 2.3–5.1 times, respectively, compared to the dry weight at the time of transplanting. Also, the dry weights of the plants transplanted at a depth of 8 cm were higher than those of plants transplanted at a depth of 2 cm for both one- and two-year-old plants. The results suggest that the growth conditions at a depth of 8 cm are more favorable than those at a depth of 2 cm.     

醉马草内生菌的分离、鉴定及杀虫研究

摘要：【目的】探明醉马草内生菌的种类,筛选对农作物虫害有毒杀作用的菌株。【方法】采用研磨法从健康醉马草植物的根、茎、叶和种子中进行菌种分离;通过对其形态、培养特征、生理生化及其他生物学特性的研究;16S rDNA及ITS序列的系统发育学分析进行鉴定;用玻片浸渍法和喷雾法筛选产杀虫活性物质菌株。【结果】获得细菌89株,分别属于枯草芽孢杆菌属（Bacillus）、链霉菌属(Streptomyces)、棒状杆菌属（Corynebacterium）、叶杆菌属（Phyllobacterium）、鞘脂单胞菌属（Sph     

黄芩根的解剖学研究

用常规石蜡切片方法对黄芩(Scutellaria baicalensis Georgi)一、二年生主根的发育及结构进行解剖学研究。结果表明,黄芩根的发育可分为原分生组织、初生分生组织、初生结构和次生生长等4个阶段。原分生组织由3群原始细胞组成,其细胞具有典型分生组织的细胞学特征;初生分生组织包括根冠原、表皮原、皮层原和中柱原,其中根冠原和表皮原具有相同的起源。初生结构由表皮、皮层和中柱组成。初生木质部为二原型。次生生长主要是由维管形成层和木栓形成层的活动完成,木栓形成层起源于中柱鞘细胞。二年生黄芩主根的主要结构与一年生的基本相同,其不同之处为:二年生主根的周皮增厚,次生木质部中木纤维成群分布,出现木间木栓;维管射线为多列且明显;在近周皮的韧皮部内出现包围石细胞的木栓环组织。     

Endophytic Occupation of Root Nodules and Roots of Melilotus dentatus by Agrobacterium tumefaciens

Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129^T and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules.     

黄芩根的解剖学研究

用常规石蜡切片方法对黄芩(Scutellaria baicalensis Georgi)一、二年生主根的发育及结构进行解剖学研究.结果表明,黄芩根的发育可分为原分生组织、初生分生组织、初生结构和次生生长等4个阶段.原分生组织由3 群原始细胞组成,其细胞具有典型分生组织的细胞学特征;初生分生组织包括根冠原、表皮原、皮层原和中柱原,其中根冠原和表皮原具有相同的起源.初生结构由表皮、皮层和中柱组成.初生木质部为二原型.次生生长主要是由维管形成层和木栓形成层的活动完成,木栓形成层起源于中柱鞘细胞.二年生黄芩主根的主要结构与一年生的基本相同,其不同之处为:二年生主根的周皮增厚,次生木质部中木纤维成群分布,出现木间木栓;维管射线为多列且明显;在近周皮的韧皮部内出现包围石细胞的木栓环组织.