消除共生质粒的费氏中华根瘤菌HND29SR在大豆根圈的定殖动态

比较研究了费氏中华根瘤菌（Sinorhizobium fredii)HN01(出发菌）、发光酶基因标记菌HNO1L（参照菌）、消除HN01共生质粒的菌株HND29SR在无菌砂培条件下的大豆根圈定殖动态。供试菌单独接种时：HN01、HN01L和HND29SR的定殖动态基本一致,其早期定殖密度下降较快,播种后第16天时HN01和HN01L分别达到较高的定殖水平6.49logcfu/g鲜根和6.78logcfu/g鲜根,然后维持相对稳定的定殖水平。但HND29SR的定殖密度持续下降到播种后第16天时才开始上升,至第35天时仍维持相对稳定的定殖密度6.94logcfu/g鲜根。等量混合接种时供试菌在根圈定殖群体中各自定殖密度在测定过程中基本相等。结果表明消除HN01的共生质粒对其在大豆根圈中定殖能力无显著影响。     

费氏中华根瘤菌HN01DL在大豆根圈的定殖动态与结瘤研究

以发光酶基因luxAB为标记,在根盒缩影条件下研究了费氏中华根瘤菌HN01DL在大豆根圈的定殖动态、分布范围及其结瘤情况.结果表明,HN01DL在根盒灭菌土壤和非灭菌土壤缩影中的大豆全根系定殖动态与水平明显不同,前者在第12d时达到最高定殖密度(8.65logcfu·g^-1),而后者的早期定殖数量下降较快,且于第15d时达到最高定殖密度(6.88logcfu·g^-1).HN01DL在大豆播种5d后在大豆根部的A(0～4cm)区根段上达到最高定殖密度(7.05logcfu·g^-1),然后开始缓慢下降,至第19d时仍维持相对稳定,在第33d时又开始回升.至播种后第46d时HN01DL可散布至种子下方16cm处的根段部位.HN01DL在A区根段的定殖密度持续较高,所形成的发光根瘤总数(16.3个)及发光比例(68.8%)最高,且发光根瘤主要集中于该区段主根上.发光根瘤比例沿A-E区段逐渐下降,在E区段未检测到发光根瘤.     

土壤有益细菌在植物根际竞争定殖的影响因素

在土壤有益微生物应用于生物肥料、生物杀虫剂、植物生长刺激剂和生物处理剂的过程中,根际定殖具有重要作用。细菌在植物根际定殖是一个比较复杂的过程,影响定殖能力的因素也是复杂多样的。本文综述了参与根部竞争定殖的生物因素,包括受细菌遗传控制的某些特性如鞭毛/运动性、趋化性、多糖、位点特异重组酶/菌落阶段变异、NADH脱氢酶,植物根的分泌物和植物种类等;影响微生物根际定殖的非生物因素如土壤类型、土壤特性和土壤温度等,探讨了影响微生物根际定殖的主要研究方向。     

外源基因标记的紫云英根瘤菌在水稻根部的定殖研究

前期研究中已证实紫云英根瘤菌(Rhizobium astragalus)JS5A16菌株对水稻生长有一定的促生作用,利用gusA基因标记的JS5A16菌株(编号为JS5A16G)接种水稻种子并检测其在水稻(汕优63)生长初期的根固定殖动态及分布。结果表明菌株JS5A16G在水稻出苗后2d根固定殖密度大量增加,第4天达到最大值16d后趋于稳定。将水稻根表面灭菌后,检测菌株JS5A16G在根内的定殖情况,发现在“汕优63”出苗后2d检测不出菌株JS5A16G,第4天可检测出。根部直接染色显示,菌株JS5A16G在根部的分布并不均匀,主要是在根系的某些部位形成微菌落。同时利用luxAB发光酶基因标记紫云英根瘤菌JS5A16菌株(编号为JS5A16L)研究其在不同品种水稻根部的定殖动态。结果表明,菌株JS5A6L在不同水稻品种“汕优63”、“汕优64”和“马协l18—2”根部的定殖密度不同且可以进入不同水稻品种的根内。在整个水稻生长期内菌株JS5A16L在“汕优64”根部的定殖密度明显高于其在“汕优63”根部的定殖密度,在“马协ll8—2”的定殖密度与其在“汕优63”、“汕优64”根部相比没有显著差异。但菌株JS5A16L在不同水稻根部的定殖动态相似,数量均在水稻生长到60-75d时(即水稻的孕穗期)达到最高值。     

植物根际促生细菌定殖研究进展

植物促生菌(plant grwth-promoting bacterium,PGPB)因可有效抑制根际病原菌,促进植物生长,增加作物产量等作用,在农业生产领域展现出巨大前景。以了解植物根际促生细菌的种类,理解定殖相关的促生机制为目的,展示了根际细菌的定殖过程及影响细菌在根部定殖的因素,通过抗生素标记、免疫学方法、外源基因标记等方法进行定殖微生物的检测,以高通量测序技术在根际定殖中的广泛应用为结果,得出结论应从遗传水平上对菌株进行解读,获得植物根际土壤微生物群落多样性,功能基因等研究,这项工作对预测根际促生细菌与植物的交互作用,生物菌剂的田间应用有重要意义,应是植物根际促生细菌今后的研究方向。     

接种量对费氏中华根瘤菌HN01DL根圈定殖与结瘤的影响

在灭菌土和未灭菌土盆栽系统中 ,研究了大豆种子的表面初始接种量对费氏中华根瘤菌HN0 1DL在大豆根圈中的定殖动态与结瘤的影响。结果表明 ,与 3个接种量对应的早期根圈定殖动态和水平有明显差异 ,但随着宿主植物根系的生长其差异逐渐减小 ,整个定殖动态曲线的变化和定殖密度趋向一致 ,并且发现 3个不同的初始接种量对HN0 1DL在大豆黑农 33根系上的结瘤数量和占瘤率没有显著影响。     

丛枝霸王（Zygophyllum dumosum）根际AM真菌生态学研究

AM真菌是一类广泛分布的土壤真菌,与宿主植物形成共生结构后,对于植物生长和植被恢复有多种有益的生理学和生态学作用。1999年11月至2000年10月,通过每月分别从0－10cm和10－20cm土层采集根际土样,对以色列荒漠地区丛枝霸王（Zygophyllum dumosum Boiss）根际AM真菌进行了系统的生态学研究。AM真菌的分布和定殖与季节变化和采样浓度密切相关。菌根真菌的最高定殖率并不伴随有最大的孢子密度,最高的定殖率发生在1999年11月,而最大的孢子密度则出现在2000年9月。10－20cm土层中的菌根真菌定殖率和孢子密度明显高于0－10cm土层。土壤温度与所有菌根结构的定殖率呈正相关,土壤有机质含量与泡囊和丛枝定殖率呈正相关,而土壤总可溶性氮对泡囊和丛枝定殖有显著正效应,对孢子密度有显著负效应。结果建议,孢子密度、泡囊和丛枝定殖程度可作为检测荒漠土壤状况的生态指标。研究应用于我国特别是西部荒漠地区的植被建立和恢复,可望发挥重任作用：（1）AM真菌能与绝大多数高奶系形成共生联合体,促进根系对土壤矿质营养和水分的吸收,提高植物对干旱、高温、盐碱、根部病害等的抗性,提高逆境条件下植物的成活率,深入研究荒漠生态系统中AM真菌动态分布,以及筛选优势AM菌种和人工接种,进行菌根化育苗,为植被建立和恢复提供优质苗木;（2）菌根的丛枝定殖时间短,主要发生在幼根,泡囊定殖时间长,主要发生在老根,而AM真菌的生长发育和繁殖所需的碳水化合物来自植物根系的分泌活动,所以,通过检测不同时期菌根各种结构的定殖程度和孢子的丰富度,可以获得宿主植物根系的生长状况,进而对土壤环境作出科学的评估。     

接种丛枝菌根真菌对柑橘生长与次生代谢的影响

为了解丛枝菌根(AM)真菌对根、根系分泌物中次生代谢物和植物生长的影响,以AM真菌(Glomusepigaeum)接种柑橘(Citrusreticulata),对柑橘的酚类物质、抗氧化酶和生长情况进行研究。结果表明,温室盆栽6个月后,接种AM真菌显著提高柑橘酚酸类物质的积累,但是酚酸组分在根和根分泌物中存在差异,根中原儿茶酸和丁香酸含量为29.98和18.32μg/g,分别是未接种的4.58和2.26倍。而根系分泌物中香豆酸、苯甲酸和根皮苷含量为0.36、6.04和12.32μg/g,分别是未接种的1.71、1.94和1.25倍,而香草醛仅在未接种柑橘根中检测到。接种AM真菌的柑橘苯丙氨酸解氨酶、多酚氧化酶和过氧化物酶活性为38.36、0.51和28.62 U/(g·min),分别是未接种AM真菌的1.99、2.83和3.10倍。同时菌根定殖也显著提高柑橘的株高、茎粗和叶片数。因此,AM真菌定殖能促进植物生长,改变柑橘次生代谢产物的积累。     

巨大芽胞杆菌luxAB标记菌株的根际定殖研究

通过三亲本杂交方法成功地用发光酶基因luxAB标记巨大芽胞杆菌ATCC14581,所获得的标记菌株ATCC14581-L在不同的条件下能稳定发光.将该标记菌株制成微生物接种剂,并利用土壤微缩系统将其接种小麦进一步研究它在小麦根际的定殖动态和散布规律.结果发现,ATCC14581-L在灭菌土壤中的定殖水平高于不灭菌土壤,在垂直方向上主要的定殖在0～7cm根段间,且随深度增加而降低.ATCC14581-L在小麦种后第7d之前就已达到最高定殖水平,在初始接种量为3.40×107cfu/g根情况下,第7d时灭菌土壤处理的根际菌数为2.54×105cfu/g根,而不灭菌土壤的根际菌数为8.87×104cfu/g根;随着时间的增长,定殖数量明显降低.     

费氏中华根瘤菌HN01DL在大豆根圈能定殖动态与结瘤研究

以发光酶基因luxAB为标记,在根盒缩影条件下研究了费氏中华根瘤菌HN01DL在大豆根圈的定殖动态、分布范围及其结瘤情况．结果表明,HN01DL在根盒．灭菌土壤和非灭菌土壤缩影中的大豆全根系定殖动态与水平明显不同,前者在第12d时达到最高定殖密度(8．65log cfu·g^-1),而后者的早期定殖数量下降较快,且于第15d时达到最高定殖密度(6．88log cfu·g^-1)．HN01DL在大豆播种5d后在大豆根部的A(0～4cm)区根段上达到最高定殖密度(7．05log cfu·g^-1),然后开始缓慢下降,至第19d时仍维持相对稳定,在第33d时又开始回升．至播种后第46d时HN01DL可散布至种子下方16cm处的根段部位．HN01DL在A区根段的定殖密度持续较高,所形成的发光根瘤总数(16．3个)及发光比例(68．8％)最高,且发光根瘤主要集中于该区段主根上．发光根瘤比例沿A-E区段逐渐下降,在E区段未检测到发光根瘤．     

发光酶基因标记的荧光假单胞菌X16L2在小麦根圈的定殖动态

在土壤微宇宙系统内及田间土壤中,采用发不酶基因标记检测技术研究了荧光假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓ ｆｌｕｏｒｅｓｃｅｎｓ,简称Ｐｆ）Ｘ１６Ｌ２在小麦根圈的定殖动态,研究结果表明：在不同灭菌灰潮土微宇宙中,Ｐｆ．Ｘ１６Ｌ２在小麦播种后３６ｄ定殖密度可达最高水平（３．２＊１０＾４ｃｆｕ．ｇ＾－１根）,然后开始下降,最后保持在一个相对稳定的较低水平（约３．２＊１０＾２ｃｆｕ．ｇ＾－１根））。在田间条     

Survival of genetically modified Pseudomonas fluorescens introduced into subtropical soil microcosms

Abstract A genetically modified strain of Pseudomonas fluorescens and its parent showed grossly similar decline rates following introduction into subtropical clay and sandy soils. In unplanted clay soit at pH 6.9 and 25°C, population densities declined progressively from about 10⁸ to 10³ colony forming units (cfu) g⁻¹ dry soil over 75 days, but in unplanted sandy soil the introduced populations could not be detected after 25 days. In clay soil at pH 8.7 or 4.7, or at environmental temperature, decay rates were enhanced as compared to those at pH 6.9 and 25°C. Counts of introduced strains in clay bulk soil and in rhizosphere and rhizoplane of maize suggested that the introduced bacteria competed well with the native bacteria, and colonized the roots at about 10⁶ cfu g⁻¹ dry root at 25°C, over 20 days. However, rhizoplane colonization was lower at environmental temperature. The decay rate of both strains was slower in planted than in unplanted sandy soil. The population densities in the rhizosphere and rhizoplane in the sandy soil were significantly lower than those in the clay soil. Both introduced strains colonized the maize roots in both soils, using seeds coated with bacteria in 1% carboxymethyl cellulose. Introduced cells were localized at different sites along the roots of plants developing in clay soil, with higher densities in the original (near the seeds) and root hair zones as compared to the intermediate zones. No significant difference was observed between the extent of root colonization of the genetically modified strain and its parent.     

LuxAB标记的N2106-L在小麦根圈的定植动态

目的研究小麦PGPR（植物根际促生菌）菌株的个体生态学及其在小麦根圈的定植动态。方法采用三亲本杂交法将发光酶基因luxAB转人具有固氮能力的小麦根际促生菌Azotobacter N2106菌株中,获得标记菌株N2106-L,将标记菌株接种到灭菌和非灭菌的黄褐土、红壤和黄潮土中研究其存活状况,采用根盒试验追踪标记菌株在小麦根圈的定植动态。结果标记菌株N2106-L具有发光活性和对km、str、tet三种抗生素的抗性,且具有较好的遗传稳定性。N2106-L在灭菌土壤中的数量稍高于非灭菌土壤;在3种土壤中的数量依次为：黄褐土〉黄潮土〉红壤。N2106-L在小麦根表定植密度大于根际定植密度;在小麦根际,小麦播种10d时标记菌株在0-2cm深度根际土壤定植达到最大值（2．17±0．25）×10^6CFU／g土,20d时在2-4cm深度的根际土壤中达到最高定植水平（3．92±0．47）×10^5CFU／g土;在小麦根表,标记菌株在小麦播种10d时在所有深度的根段均达到最高定植水平,0-2cm根段定植密度为（3．60±0．60）×10^6CFU／g鲜根,12cm以下根段达到（2．78±0．56）×10^4CFU／g鲜根。结论标记菌株随着根的伸长不断向根尖方向扩散,且较为稳定地在小麦根圈定植,研究结果为小麦PGPR菌株的应用提供了可靠实验数据。     

Survival of gfp-tagged antagonistic bacteria in the rhizosphere of tomato plants and their effects on the indigenous bacterial community

The survival and colonization patterns of Pseudomonas putida PRD16 and Enterobacter cowanii PRF116 in the rhizosphere of greenhouse-grown tomato plants and the effects of their inoculation on the indigenous bacterial community were followed by selective plating, molecular fingerprinting, and confocal laser scanning microscopy (CLSM) over 3 weeks. Both strains, which showed in vitro antagonistic activity against Ralstonia solanacearum, were previously tagged with gfp. Seed and root inoculation were compared. Although plate counts decreased for both gfp-tagged antagonists, PRD16 showed a better survival in the rhizosphere of tomato roots independent of the inoculation method. Analysis of 16S rRNA gene fragments amplified from total community DNA by denaturing gradient gel electrophoresis and CLSM confirmed the decrease in the relative abundance of the inoculant strains. Pronounced differences in the Pseudomonas community patterns for plants inoculated with PRD16 compared to the control were detected 3 weeks after root inoculation, indicating a longer-lasting effect. Analysis by CLSM showed rather heterogeneous colonization patterns for both inoculant strains. In comparison with seed inoculation, root inoculation led to a much better colonization as evidenced by all three methods. The colonization patterns observed by CLSM provide important information on the sampling strategy required for monitoring inoculant strains in the rhizosphere.     

Measuring the spermosphere colonizing capacity (spermosphere competence) of bacterial inoculants

Spermosphere establishment by bacteria which were coated onto seeds was studied using soybean seeds treated with four bacterial strains at levels of log10 1 to 4 colony-forming units (cfu) per seed planted in a field soil mix, and incubated 48 h. Each strain at every inoculum level developed spermosphere population densities of log10 4 to 8 cfu/seed, demonstrating an average multiplication of log10 3 cfu/seed. An alternative method was developed to differentially rank bacteria for spermosphere colonizing capacity, based upon incorporation of bacteria into a soil and monitoring the resulting spermosphere population densities around noninoculated seeds after 4 days at 14 degrees C. Fifty-seven bacterial strains which were isolated from soybean roots or from water samples, including Pseudomonas putida, P. putida biovar B, P. fluorescens, Serratia liquefaciens, Enterobacter aerogenes, and Bacillus spp. were tested in the spermosphere colonization assay. Average spermosphere population densities for the 57 strains ranged from 0 to log10 7.0 cfu/seed. Strains of a given taxon demonstrated marked diversity with ranges from 0 to log10 6.0 cfu/seed for Bacillus spp. and from log10 1.4 to 7.0 cfu/seed for Pseudomonas putida. The relative ranking of representative strains was consistent in repeating experiments. The potential usefulness of the assay for efforts to develop competitive bacterial inoculants for crop seeds is discussed.     

Fluorescent pseudomonads in the rhizosphere of plants and their relation to root exudates

Fluorescent pseudomonads were present in chernozem soil not influenced by plant roots (10(3)-10(4) per g dry soil) in the rhizosphere soil of various plants (10(4)-10(5) per g soil) and on roots (10(3) to 10(7) per g fresh roots), depending on the species and age of the plant. Relative species representation of fluorescent pseudomonads changed on the roots and in the plant rhizosphere as compared with free soil. Pseudomonas fluorescens, representing 60-93% of the population of fluorescent pseudomonads predominated on the roots of all plants investigated. Somewhat different results were obtained in rhizosphere soil. Relatively higher numbers of P. fluorescens were detected in the rhizosphere soil of cucumber and maize, numbers in the rhizosphere soil of wheat were practically the same as in free soil and higher numbers of P. putida were found in the rhizosphere soil of barley. Almost all components contained in the root exudates of the plants studied, including beta-pyrazolylalanine from the root exudates of cucumbers were utilized as carbon and energy sources. Root exudates of wheat and maize were utilized by the strain P. putida K2 with an efficiency of 73-91%, depending on species and age of the plant.     

Nitrogen-fixing pseudomonads isolated from roots of plants grown in the canadian high arctic

Root-associated bacteria capable of reducing acetylene to ethylene (biological nitrogen fixation) were isolated from various native plants grown in the Canadian High Arctic. All the strains belonged to the genus Pseudomonas but varied in several physiological characteristics. The rates of acetylene reduction at 14 or 20 degrees C were higher than at 25 or 9 degrees C. Six strains reduced acetylene at 4 degrees C. All the strains exhibited chemotaxis to l-asparagine in semisolid agar at 4 to 25 degrees C. Eleven strains colonized roots of canola (Brassica campestris cv. Tobin) in field soil at population densities of log 4.3 to log 5.1 CFU/g of fresh root at 14 degrees C and log 4.0 to log 5.2 CFU/g of fresh root at 25 degrees C. Some of these nitrogen-fixing pseudomonad strains demonstrated a competitive advantage for root colonization over other rhizosphere bacteria at low temperatures. The combined capabilities of nitrogen fixation and root colonization by diazotrophic pseudomonads may be useful for the development of a biofertilizer inoculant for temperate and cold regions.