大豆根瘤菌突变株的细菌学鉴定(简报)

大豆根瘤菌优良菌种的选育,对提高根瘤菌的接种效果起着重要的作用。以往大都从自然界筛选的办法来获得高固氮高结瘤率的菌株,R. L. Maier等人应用诱变的手段对美国商业用大豆根瘤菌进行处理,得到的诱变菌株较野生菌株结瘤早、固氮力高,在生长室内并能提高植株     

内蒙古乌拉尔甘草根瘤菌的遗传多样性分析

从内蒙古乌拉尔甘草根瘤中共分离到54株具有根瘤菌属特征的菌株。采用ERIC-PCR分析方法,发现这些菌株存在特异的扩增条带,能够分辨出从不同年份植株上分离菌株的差异。选取有代表性的菌株进行回接并对结瘤效果好的菌株进行序列分析,发现R31与Mesorhizobium temperatum(T)AF508208、R50与Meso-rhizobium mediterraneum(T)L38825相似性都为99%,属于中慢生根瘤菌属。     

应用BOX分子标记技术筛选南苜蓿高效根瘤菌菌株

【目的】应用BOX-PCR技术对12株南苜蓿(Medicago polymorpha)接种根瘤菌菌株在土壤中的竞争结瘤能力进行研究,以占瘤率的高低配合植物生长性状最终确定与南苜蓿共生的高效根瘤菌菌株。【方法】对接种根瘤菌产生的南苜蓿根瘤进行分离培养,选择部分结瘤菌株和12株全部供试接种菌株进行BOX分子指纹图谱研究,经过对图谱的比较与分析获得了各接种菌株的占瘤率。【结果】在供试菌株中,来自于云南盈江的菌株SWF67523占瘤率最高,为93.33%,说明菌株SWF67523具有较强的竞争结瘤能力,该菌株对南苜蓿干重增幅也较高,为100%;菌株SWF67409来自于云南楚雄,其占瘤率略低于菌株SWF67523,但其对提高植物干重的贡献最大(增幅106.5%);来自于云南楚雄的菌株SWF67394占瘤率较低,但其结瘤率高,对植物生长的影响作用也较明显。【结论】将根瘤菌菌株的竞争结瘤能力纳入南苜蓿高效根瘤菌菌株的筛选中,研究获得了一株竞争结瘤能力强、显著提高植物生物量的菌株SWF67523,以及促生根瘤菌菌株SWF67409和SWF67394,为生产高效根瘤菌菌剂提供了物质基础。     

普通结瘤基因探针鉴定根瘤菌的研究

普通结瘤基因（ｎｏｄＡＢＣ）是所有根瘤菌所特有的、最为保守的基因,用苜蓿根瘤菌结瘤基因（ｎｏｄＡＢＣ）和豌豆根瘤菌的（ｎｏｄＣ）基因片段为探钉,与５２株包括常见土壤细菌、已知根瘤菌、根瘤未知分离物的总ＤＮＡ进行斑点杂交,探索用普通结瘤基因（ｎｏｄＡＢＣ）或（ｎｏｄＣ）探针鉴定根瘤菌的可能性。结果,未找到合适实验条件,使来自这两个种的结瘤基因只能与根瘤菌菌株杂交,而不与土壤细菌的菌株杂交。但在高温条件下,两种探针都专一性的和种内菌株杂交。此结果表明：在一定的实验条件下,普通结瘤基因探针用做根瘤菌的鉴定,只能     

南苜蓿高效共生根瘤菌土壤的筛选

利用云南德宏盈江县3种类型的土壤,对分离自云南楚雄、德宏等地的SWF67523、SWF67409、SWF67456等12株根瘤菌菌株进行了南苜蓿接种效果的研究。结果表明,在所有供试菌株中,菌株SWF67523、SWF67409、SWF67394表现最为优良,结瘤率较高的菌株是SWF67523、SWF67501、SWF67394和SWF67350,结瘤率均达到95％以上;而菌株SWF67523、SWF67409和SWF67394对植株株高影响最大;菌株SWF67409、SWF67523和SWF67394对提高植株干重贡献最大,其中菌株SWF67409比空白对照增产106.5%;供试菌株对含氮量的影响也很显著,接种菌株SWF67409、SWF67523和SWF67394的苜蓿植物含氮量相比其他菌株较高,影响显著。综合以上结果,发现来源于盈江南苜蓿的根瘤菌菌株SWF67523较其他菌株表现突出,同时发现根瘤菌菌株的接种效果受土壤因子的影响,在含有效钾中等的土壤平原镇Ⅱ上,接种根瘤菌的南苜蓿地上部分干重普遍高于其他两种有效钾含量为较缺的土壤,说明根瘤菌菌株与宿主和土壤环境存在密切关系,在适合的宿主和生活环境中将会发挥最大的共生固氮作用。     

紫云英根瘤菌基因文库的构建及含完整结瘤基因的重组质粒pRaZ15的分离

以紫云英根瘤菌菌株7653R为材料,制备总DNA,经EcoRⅠ限制酶部分酶解,通过10—50％蔗糖梯度离心,分离到20一30 kb的DNA片段。利用能在革兰氏阴性菌中转移和复制的广谱寄主载体——pLAFRl质粒,构建了紫云英根瘤菌基因文库。通过与苜蓿根瘤菌102l菌株中8．7kb的共同结瘤基因(作探针DNA)杂交,从基因文库中分离到紫云英根瘤菌共同结瘤基因片段。以紫云英根瘤菌不结瘤突变株7653R+1(7653R消除共生质粒)为受体、构建的7653R基因文库(E．Coli C600)为供体,通过协助转移质粒pRK2013(LE392)进行三亲交配,在含四环素的根瘤菌台成培养基(sM)上选择接合转移子。将得到的所有接台转移子混合在一起接种植物,通过植物结瘤试验,分离到含紫云英根瘤菌结瘤基因的重组质粒pRaz15。将该质粒用EcoRⅠ完全酶切,得到25kb左右的外源DNA片段,该片段携带完整的结瘤基因簇。     

格拉姆柱花草(Stylosanthes gracilis H.B.K.cv.Graham)凝集素的纯化与性质研究

为了探讨凝集素在豆科植物与根瘤菌的识别过程中的作用,用ＤＥＡＥ－３２离子交换层析和ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤分离、纯化格拉姆柱花草种子凝集素（ＳＧＬ）,其分子量约为４５ｋＤ,由两个相同的亚基组成,等电点约ｐＨ５．８,它是一种糖蛋白,含糖量约为２．６％．ＳＧＬ的热稳定性强．ＳＧＬ的血凝活性能被甘露糖所抑制．ＳＧＬ对红细胞的凝集作用可能具有种属专一性;ＳＧＬ具有强的促有丝分裂作用;荧光标记实验显示：９株能与格拉姆柱花草植株结瘤的菌株有７株能与ＳＧＬ结合,６株不能与之结瘤的菌株,只有１株能与ＳＧＬ结合,这表明不同根瘤菌菌株对ＳＧＬ的结合能力,和它们在格拉姆柱花草上结瘤能力之间可能具有一定的生物学相关性．     

VA菌根真菌、根瘤菌双接种对大豆增产效果的影响

本文论述了在自然状态下的土壤里,VA菌根真菌、根瘤菌双接种对大豆结瘤、菌根真菌侵染率、固氮酶活性及植株养分吸收状况的影响.研究结果表明,VA菌根真菌、根瘤菌双接种明显提高大豆结瘤能力与固氮酶活性,并可促进植物对养分的吸收,双接种比单接种VA菌根真菌处理提高产量140%,比单接种根瘤菌处理提高55.84%.     

山西锦鸡儿根瘤菌资源分布与生态学研究

研究了山西锦鸡儿根瘤菌资源分布、菌种形态特征、理化特性及其生态学适应性与生态效应。结果表明,山西锦鸡儿根瘤菌资源分布广、结瘤率高,根瘤大,固氮周期长,耐盐碱,用高效抗逆菌株接种,明显促进植株的生长发育,可增加干重28．9％—79．5％。     

紫花苜蓿内生和非内生根瘤菌多样性及共生差异

【背景】对根瘤菌多样性的研究有助于推进根瘤菌种质资源的利用。【目的】研究紫花苜蓿内生和非内生根瘤菌的表型和遗传多样性,比较菌株在5个苜蓿品种上的共生效应,验证根瘤菌群体共生效应由苜蓿品种决定的假设。【方法】从甘肃省白银会宁旱作区、兰州安宁灌区、武威凉州灌区3个栽培区域的陇中、清水、WL168HQ、甘农3号、甘农9号等紫花苜蓿品种中分离内生(植株种子、花、叶、茎、根表皮、根中柱和根瘤)和非内生(根际土壤和田间土壤)根瘤菌菌株,通过表型数值分类、 16SrRNA基因限制性片段长度多态性(Restrictionfragmentlengthpolymorphism,RFLP)、16S rRNA基因测序、持家基因多位点序列分型(Multilocus sequence typing,MLST),以及结瘤基因nodC和固氮基因nifH片段序列测定,研究紫花苜蓿根瘤菌的表型和遗传多样性,并采用主成分分析研究根瘤菌菌株在5个紫花苜蓿品种上的共生效应差异。【结果】共分离得到43株内生根瘤菌和10株非内生根瘤菌,叶片和花中没有分离到根瘤菌菌株。53株根瘤菌以及对照菌株R.GN5和S.12531表型特征数值分类聚为8个群,菌株表型多样性丰富。经16SrRNA-RFLP分析共形成22种RFLP分型组合,基因型Ⅰ分布最广泛(24),其次为基因型Ⅻ(5)、ⅩⅤ(5)和ⅩⅨ(3),其余16株菌各代表1种基因型,菌株遗传多样性丰富。16S rRNA基因测序和MLST分析将所有菌株划分为Rhizobiumradiobacter、R.rosettiformans和Ensifermeliloti。仅从7株E.meliloti代表菌株和对照菌株S.12531中扩增到nodC和nifH基因,说明E.meliloti菌株均能结瘤固氮。E.meliloti菌株G3L3接种甘农3号,LP3、LL1和LL2接种陇中,QL2接种清水,LL1、LL2和WLP2接种WL169HQ苜蓿均能显著促进植株的单株结瘤数、地上干重和粗蛋白含量。E.meliloti菌株接种甘农3号、甘农9号和清水苜蓿品种后所有参数值在PC1轴上分别聚在-1-1之间,在PC3轴上聚在-1.5-1.5之间;接种陇中和WL168HQ苜蓿的参数值较分散,PC1轴上分散在-1.5-4之间,PC3轴上分散在-3-4之间。【结论】紫花苜蓿内生和非内生根瘤菌菌株多样性丰富,表型和遗传多样性与其来源没有直接关系。菌株G3L3与甘农3号,LP3、LL1、LL2与陇中,QL2与清水、LL1、LL2、WLP2与WL169HQ苜蓿品种共生匹配和适应能力强。在甘农3号、甘农9号和清水紫花苜蓿品种上群体共生效应相似,在陇中和WL168HQ紫花苜蓿上共生效应差异明显。本研究内生和非内生根瘤菌菌株的群体共生效应根据苜蓿品种而定,根瘤菌菌株与苜蓿品种间的信号识别程度存在差异。     

新疆苦豆子根瘤菌的数值分类研究

苦豆子(Sophora alopecuroides)对于干旱荒漠地区的畜牧业发展有着非常重要的意义,其生长特性与根瘤菌有密切关系。我们对分离自新疆苦豆子根瘤的67株根瘤菌及36个模式菌株进行了118项表型性状的测定,包括唯一碳源利用、唯一氮源利用、对抗生素和染料的抗性、耐盐性、初始pH值生长范围、生长温度范围及石蕊牛奶反应、氧化酶、过氧化氢酶和脲酶。对测定结果用聚类分析方法进行了分析,获得数值分类树状图。结果表明：新疆苦豆子根瘤菌在碳氮源利用、抗生素敏感性以及对染料的抗性程度等方面存在着差异。新疆苦豆子根瘤菌能耐受低温,并具有较强的耐盐、碱能力,所有供试菌株均能在初始pH值为9－12的YMA培养基上生长,92.5％的菌株能耐受3.0％的NaCl,91.0％的菌株能耐受4.0％的NaCl,有18株菌甚至能耐受5.0％和6.0％的NaCl。聚类结果表明, 在84.8%的相似性水平上,67个供试菌株构成了4个新的表观群,第Ⅰ、Ⅱ、Ⅲ、Ⅳ类群分别有21、7、4、3个菌株,中心菌株分别为NWBC152、NWTKX101、NWYJS12、NWLP112。此外,数值分类结果还表明,苦豆子根瘤菌与模式菌株的相似性较低,它们所形成的4个独立群可能有新种出现。     

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.     

毛乌素沙地中间锦鸡儿根瘤菌的多样性及其抗逆性

从内蒙古毛乌素沙地优势物种中问锦鸡儿收集根瘤,分离根瘤菌15株,对其进行同工酶电泳,聚类分析证明中间锦坞儿的遗传多样性对其耐盐性、耐酸碱性、生长温度范围、产酸产碱性、过氧化氢酶活性以及唯一碳源利用能力进行比较研究,结果表明,所有中间锦鸡儿根瘤菌均分泌H^+,都具有过氧化氢酶活性,73.3%的菌株可以在含3％NaCl的YMA培养基上生长,80％的菌株可以抵抗50℃高温,除对乳糖和淀粉利用表现出差异外,中间锦鸡儿根瘤菌对其余碳源没有选择性,证明中间锦鸡儿根瘤菌具有较强的抗逆性,但仍存在菌株间差异,是对毛乌素沙地多种景观生态类型的适应作为一种新的种质资源,中间锦鸡儿根瘤菌可用于恶劣生境固氮。     

Competitiveness of a <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. Strain in Soils Containing Indigenous Rhizobia

The success of rhizobial inoculation on plant roots is often limited by several factors, including environmental conditions, the number of infective cells applied, the presence of competing indigenous (native) rhizobia, and the inoculation method. Many approaches have been taken to solve the problem of inoculant competition by naturalized populations of compatible rhizobia present in soil, but so far without a satisfactory solution. We used antibiotic resistance and molecular profiles as tools to find a reliable and accurate method for competitiveness assay between introduced Bradyrhizobium sp. strains and indigenous rhizobia strains that nodulate peanut in Argentina. The positional advantage of rhizobia soil population for nodulation was assessed using a laboratory model in which a rhizobial population is established in sterile vermiculite. We observed an increase in nodule number per plant and nodule occupancy for strains established in vermiculite. In field experiments, only 9% of total nodules were formed by bacteria inoculated by direct coating of seed, whereas 78% of nodules were formed by bacteria inoculated in the furrow at seeding. In each case, the other nodules were formed by indigenous strains or by both strains (inoculated and indigenous). These findings indicate a positional advantage of native rhizobia or in-furrow inoculated rhizobia for nodulation in peanut.     

毛乌素沙地中间锦鸡儿根瘤菌的多样性及其抗逆性

从内蒙古毛乌素沙地优势物种中间锦鸡儿收集根瘤,分离根瘤菌15株,对其进行同工酶电泳,聚类分析证明中间锦鸡儿的遗传多样性.对其耐盐性、耐酸碱性、生长温度范围、产酸产碱性、过氧化氢酶活性以及唯一碳源利用能力进行比较研究,结果表明,所有中间锦鸡儿根瘤菌均分泌H⁺,都具有过氧化氢酶活性,73.3%的菌株可以在含3%NaCl的YMA培养基上生长,80%的菌株可以抵抗50℃高温,除对乳糖和淀粉利用表现出差异外,中间锦鸡儿根瘤菌对其余碳源没有选择性,证明中间锦鸡儿根瘤菌具有较强的抗逆性,但仍存在菌株间差异,是对毛乌素沙地多种景观生态类型的适应.作为一种新的种质资源,中间锦鸡儿根瘤菌可用于恶劣生境固氮.     

Diversity and effectiveness of rhizobial strains from <Emphasis Type="Italic">Gliricidia sepium</Emphasis> native to Reunion Island,Kenya and New Caledonia

The aim of this study was to use several approaches to assess the diversity occuring in a collection of rhizobial strains that had been isolated from Gliricidia sepium (Jacq.) Walp. originating from Kenya, Reunion Island and New Caledonia. Results showed that G. sepium establishes an effective symbiosis only with fast-growing rhizobia. This suggests that this legume nodulates preferentially with this type of rhizobia, although ineffective nodules were observed on root systems of plants inoculated with slow-growing rhizobia. It was possible to distinguish several groups of strains which have the same behaviour in terms of utilization of amino acids and sources of carbon and intrinsic resistance to antibiotics and salinity. Six molecular groups were distinguished by profiles obtained using PCR/RFLP techniques. No relationship was demonstrated between phenotypic groups and molecular groups. Neither was it possible to find a relationship between the geographic origins of the strains and their distribution in the several phenotypic and/or molecular groups. In an experiment carried out under glasshouse conditions, we observed that all strains from the collection significantly improved the growth of G. sepium cultivated in a substrate totally devoid of nitrogen. The strain GsK5 induced the greatest stimulation of plant growth.     

Survival of several <Emphasis Type="Italic">Rhizobium/Bradyrhizobium</Emphasis> strains on different inoculant formulations and inoculated seeds

The effect of a variety factors on the survival of several rhizobia strains on inoculants and inoculated seeds has been evaluated. Since the rhizobia strains showed different cell-density-evolution patterns on peat-based inoculants and on inoculated seeds, several inoculant formulations with highly effective Rhizobium/Bradyrhizobium strains (for Lupinus, Hedysarum, Phaseolus and Glycine max.) were monitored under the following storage conditions: (a) the inoculants were kept refrigerated (at 4 °C), or (b) at room temperature (25 °C). The effect of water content (30–50%, w/w) in the inoculants as well as that of several seed-coating adhesives were also investigated. Alternative carriers including perlite and vermiculite were tested. For all of the strains, survival on sterile peat-based inoculants was higher than on the corresponding unsterile peat formulation; for the latter, refrigerated storage conditions are recommended to ensure high bacterial densities. The water content of the inoculants had a differential effect on strain survival depending on the sterility of the peat, such that a high water content was more detrimental when unsterilized peat was employed. The best adherent for rhizobia survival was a gum arabic/water solution. Perlite was as effective as peat in maintaining a high population of rhizobia, at least for 6 months of storage. Electronic Publication     

刺槐根瘤菌的表型多样性研究

从杨陵地区采集豆科树种刺槐的根瘤,经分离、纯化,获得40株未知菌株,并选取35株参比菌株,进行唯一碳源、氮源利用、对抗生素和染料的抗性、耐盐性、初始pH生长、生长温度范围及石蕊牛奶反应等共105项生理生化测定。结果表明：供试的刺槐根瘤菌在碳、氮源利用、抗生素敏感性、对染料的抗性程度等方面存在着差异。部分菌株具有较强的耐盐碱能力,其中42．5％的菌株能耐受3.0％的NaCl,17．5％的菌株可在初始pH12的YMA培养基上生长。从数值分类树状图可见,在86％的相似水平上未知菌株构成了3个新的类群,其中第1、2类群各有10株菌,中心菌株分别为NWYC113和NWYCl29,第3类群有7株菌,中心菌株为NWYC147。其分类地位需进一步研究和确定。     

卷荚相思根瘤菌的生物学特性

为探讨卷荚相思根瘤菌的生物学特性,从广西高峰林场卷荚相思林分采集分离到12株根瘤菌,对这些菌株进行了生理生化试验,并利用这些菌株对厚荚相思苗木进行接种结瘤试验。结果表明:(1)依据生长代时和单个菌落形成测定结果,分离获得的12个菌株可以分为9个快生菌株和3个慢生菌株。(2)大部分的供试根瘤菌菌株可以利用无机氮源作为其生长所需的氮源,但对供试用的糖源利用存在着较大的差别。(3)所有供试菌株在3一酮基乳糖反应、牛肉膏蛋白胨、淀粉水解试验中呈阴性,所有供试的12个菌株均能进行利用柠檬酸盐,在BTB试验中均产酸,在石蕊牛奶反应均产碱。(4)12个卷荚相思根瘤菌菌株接种到厚荚相思苗木后,均能使其根部结瘤,并进一步筛选出结瘤效果和固N效果比较好的供试菌株是快生型菌株(J003、J004、J005、J008)和慢生型菌株(J012)。这为从华南的酸性土壤中寻找和利用土著根瘤菌资源提供了科学依据。     

Strain selection for improvement of Bradyrhizobium japonicum competitiveness for nodulation of soybean

A Bradyrhizobium japonicum USDA 110-derived strain able to produce wider halos in soft-agar medium than its parental strain was obtained by recurrent selection. It was more chemotactic than the wild type towards mannitol and three amino acids. When cultured in minimal medium with mannitol as a single carbon-source, it had one thick subpolar flagellum as the wild type, plus several other flagella that were thinner and sinusoidal. Root adsorption and infectivity in liquid media were 50-100% higher for the selected strain, but root colonization in water-unsaturated vermiculite was similar to the wild type. A field experiment was then carried out in a soil with a naturalized population of 1.8 x 10(5) soybean-nodulating rhizobia g of soil(-1). Bradyrhizobium japonicum strains were inoculated either on the soybean seeds or in the sowing furrows. Nodule occupation was doubled when the strains were inoculated in the sowing furrows with respect to seed inoculation (significant with P<0.05). On comparing strains, nodule occupation with seed inoculation was 6% or 10% for the wild type or selected strains, respectively, without a statistically significant difference, while when inoculated in the sowing furrows, nodule occupation increased to 12% and 22%, respectively (differences significant with P<0.05).