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.     

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。     

Examining growth and survival of cowpea rhizobia in Jamaican peat

We have examined the survival of four cowpea rhizobia strains in Jamaican peat to determine its suitability as inoculant carrier. All strains survived well since more than 10⁷ cells of rhizobia per gram of peat were recovered from the inoculant after storage for 6 months at 30C. Survival of cowpea rhizobia was better when inoculants were stored at 4 than 30C. The native strains JRC29 and JRW3 (isolated in Jamaica) survived much better than the introduced strains MI-50A and IRC291 (isolated in West Africa). Survival of cowpea rhizobia was not significantly increased when peat was mixed with 1% sucrose. Our results suggest that Jamaican peat may be used as a carrier for inoculant production.     

The resistance of spores of resistant strains of Botrytis cinerea to quintozene, tecnazene and dicloran

Spores formed by strains of Botrytis cinerea resistant to quintozene, tecnazene, or dicloran did not always produce resistant colonies when grown on agar in the presence of these fungicides. Only about one half of spores produced by fungicide-vapour-resistant strains in the absence of fungicides gave resistant colonies whereas all spores produced by the same strains in the presence of fungicides formed resistant colonies on agar. Some spores produced by resistant strains that had developed on agar containing the fungicides were not viable, but those that were always gave resistant colonies on agar.     

Alteration of lipopolysaccharide and protein profiles in SDS-PAGE of rhizobia by osmotic and heat stress

The effects of osmotic and heat stress on lipopolysaccharides and proteins of rhizobia isolated from the root nodules of leguminous trees grown in semi-arid soils of the Sudan, and of agricultural legumes grown in salt-affected soils of Egypt, were determined by SDS-PAGE. The rhizobia were of three types: (1) sensitive strains, unable to grow in 3% (w/v) NaCl in yeast mannitol medium; (2) tolerant strains which could grow in 3% (w/v) NaCl; and (3) halophytic strains which grew with 3 to 10% (w/v) NaCl. The sensitive strains changed their gel pattern or the amount of lipopolysaccharide they synthesized when grown in 1% (w/v) NaCl. The tolerant and halophytic strains often modified their lipopolysaccharides in 3% NaCl, which was evident by a shift in the banding patterns towards longer chain length. Similar effects were observed in cells incubated with sucrose and, to a lesser extent, in cells incubated at growth temperatures near the recorded maximum temperature for growth. The stress-induced changes in lipopolysaccharides were not associated with specific banding patterns of the lipopolysaccharides. During incubation in medium containing elevated concentrations of NaCl or sucrose, the protein patterns of the rhizobia were also changed. A protein with relative mobility of 65 kDa appeared during temperature stress. The maximum growth temperature of the Sudanese rhizobia were up to 44.2°C.H.H. Zahran and M. Karsisto were and L.A. Räsänen and K. Lindström are with the Department of Applied Chemistry and Microbiology, University of Helsinki, POB 27, SF-00014 University of Helsinki, Finland. H.H. Zahran is now with the Department of Botany, Faculty of Science, Beni-Suef, Egypt. M. Karsisto is now with the Finnish Forest Research Institute, PL 18, SF-01301 Vantaa, Finland.     

Transfer of plasmids RP4 and R68.45 and chromosomal mobilization in cowpea rhizobia

R-plasmids RP4 and its derivatives R68.45 were transferred from Escherichia coli to two cowpea rhizobia strains. The frequency of RP4 transfer in cowpea rhizobia strains JRC23-SM20 and IRC256-HA409 was 1,000-fold higher than transfer frequency of R68.45. The transconjugants were further used to transfer R-plasmids within (isogenic) and between (non-isogenic) cowpea rhizobia strains. The plasmid transfer frequency was higher in isogenic than non-isogenic strains. The ability of R-plasmids to mobilize chromosomal genes in cowpea rhizobia was also examined. R-plasmids mediated the chromosomal transfer; however, mobilization of chromosomal markers Sm^R and Met⁺ by RP4 in isogenic strains was more efficient than by R68.45. Chromosomal mobilization has not previously been reported in cowpea rhizobia.Abbreviations Ap ampicillin - Km kanamycin - Tc tetracycline - Rif rifampicin - TYS tryptone yeast-extract sodium chloride - YEMA yeast-extract mannitol agar - YEMB yeast-extract mannitol broth Part of the work was presented in 6th International Symposium on Nitrogen Fixation at Oregon State University, Corvallis, August 4–10, 1985     

The Effects of Antimicrobial Substances on Rhizobium spp. and their Use in Selective Media

S ummary . Fortyseven strains of rhizobia were tested for sensitivity to 6 antibiotics (chloramphenicol, erythromycin, penicillin, streptomycin, sulphafurazole and tetra-cycline) using Oxoid Multodisks on yeast extract-mannitol agar (YMA); penicillin was the least inhibitory. Growth of selected strains on YMA was also compared with that on YMA containing one or more antimicrobial substances. Penicillin (1 i.u./ml of YMA) only slightly inhibited growth of some strains that were sensitive to it by the Multodisk test but higher concentrations were too inhibitory to be used in a selective medium. YMA containing pentachloronitrobenzene (5 p/m, suspended in a 5 p/m solution of Triton X-100), brilliant green (0.5 p/m) and sodium azide (0.5 p/m) did not inhibit 15 of 18 strains of rhizobia but was more inhibitory to the general microflora of 4 soils than was YMA. This medium (ABPA) was very inhibitory to several Rhizobium strains when supplemented with 1 i.u./ml of penicillin.     

Polymerase chain reaction detection and speciation of Campylobacter upsaliensis and C. helveticus in human faeces and comparison with culture techniques

A polymerase chain reaction (PCR) assay based on the 16S rRNA gene and an improved DNA extraction procedure were developed for the direct detection and differentiation of Campylobacter upsaliensis and C. helveticus in seeded human faeces. The PCR assay was compared with culture detection by a membrane filter (MF) technique and on selective agar (SA) containing 8 mg l⁻¹ cefoperazone. Both MF culture and the PCR assay detected 10⁵ colony-forming units (cfu) g⁻¹ faeces. Selective agar culture of some strains could detect as few as 10³ cfu g⁻¹ faeces. However, some strains were susceptible to cefoperazone and either failed to grow or were detected only with reduced sensitivity in the presence of the antibiotic. Detection by MF and SA both required 48–96 h incubation in a microaerobic atmosphere and did not specifically identify the isolate. By contrast, the PCR assay could be completed within 8 h and accurately identified the two phenotypically similar species, C. upsaliensis and C. helveticus.     

Preservation of Rhizobium viability and symbiotic infectivity by suspension in water

Three Rhizobium japonicum strains and two slow-growing cowpea-type Rhizobium strains were found to remain viable and able to rapidly modulate their respective hosts after being stored in purified water at ambient temperatures for periods of 1 year and longer. Three fast-growing Rhizobium species did not remain viable under the same water storage conditions. After dilution of slow-growing Rhizobium strains with water to 10(3) to 10(5) cells ml-1, the bacteria multiplied until the viable cell count reached levels of between 10(6) and 10(7) cells ml-1. The viable cell count subsequently remained fairly constant. When the rhizobia were diluted to 10(7) cells ml-1, they did not multiply, but full viability was maintained. If the rhizobia were washed and suspended at 10(9) cells ml-1, viability slowly declined to 10(7) cells ml-1 during 9 months of storage. Scanning electron microscopy showed that no major morphological changes took place during storage. Preservation of slow-growing rhizobia in water suspensions could provide a simple and inexpensive alternative to current methods for the preservation of rhizobia for legume inoculation.     

Phenotypic and genotypic characterization of rhizobia from diverse geographical origin that nodulate Pachyrhizus species

Legumes from the genus Pachyrhizus, commonly known as yam bean, are cultivated in several countries from the American continent and constitute an alternative source for sustainable starch, oil and protein production. The endosymbionts of these legumes have been poorly studied although it is known that this legume is nodulated by fast and slow growing rhizobia. In this study we have analyzed a collection of strains isolated in several countries using different phenotypic and molecular methods. The results obtained by SDS-PAGE analysis, LPS profiling and TP-RAPD fingerprinting showed the high diversity of the strains analyzed, although all of them presented slow growth in yeast mannitol agar (YMA) medium. These results were confirmed using 16S-23S internal transcribed spacer (ITS) region and complete sequencing of the 16S rRNA gene, showing that most strains analyzed belong to different species of genus Bradyrhizobium. Three strains were closely related to B. elkanii and the rest of the strains were related to the phylogenetic group constituted by B. japonicum, B. liaoningense, B. yuanmingense and B. betae. These results support that the study of rhizobia nodulating unexplored legumes in different geographical locations will allow the discovery of new species able to establish legume symbioses.     

Preservation of Rhizobium viability and symbiotic infectivity by suspension in water.

Three Rhizobium japonicum strains and two slow-growing cowpea-type Rhizobium strains were found to remain viable and able to rapidly modulate their respective hosts after being stored in purified water at ambient temperatures for periods of 1 year and longer. Three fast-growing Rhizobium species did not remain viable under the same water storage conditions. After dilution of slow-growing Rhizobium strains with water to 10(3) to 10(5) cells ml-1, the bacteria multiplied until the viable cell count reached levels of between 10(6) and 10(7) cells ml-1. The viable cell count subsequently remained fairly constant. When the rhizobia were diluted to 10(7) cells ml-1, they did not multiply, but full viability was maintained. If the rhizobia were washed and suspended at 10(9) cells ml-1, viability slowly declined to 10(7) cells ml-1 during 9 months of storage. Scanning electron microscopy showed that no major morphological changes took place during storage. Preservation of slow-growing rhizobia in water suspensions could provide a simple and inexpensive alternative to current methods for the preservation of rhizobia for legume inoculation.     

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。     

沙冬青根瘤菌的抗逆性

沙冬青(Ammopiptanthus mongolicus)是西北荒漠地区珍稀常绿阔叶灌木,综合沙冬青分布区内气候、土壤和水分等因子,选取宁夏中卫县沙坡头、内蒙古磴口县、内蒙古阿拉善左旗、内蒙古乌拉特后旗作为样区,于2004年4月中下旬沙冬青的花期调查和收集根瘤,并结合地理环境对分离得到的根瘤菌进行抗逆性初步分析。调查发现根瘤的生成及其在宿主植物上的着生部位、大小、形状、颜色等因生境条件的不同而有所差异,水分是其主要的影响因子;分离得到根瘤菌17株,对其耐盐性、耐酸碱性、生长温度范围和抗生素抗性进行了研究。结果表明,64.7%的菌株可以在含3%NaCl的YMA培养基上生长;94.1%的菌株可以在pH 5~11的范围内生长;全部菌株在60 ℃处理10 min后仍能生长;不同菌株对不同抗生素表现出不同的抗性,ZW₄和WH₄¹对各抗生素表现出较强的抗性。分离自磴口县的根瘤菌普遍表现出较强的抗酸碱和抗高温的能力,这是对其环境的适应。本研究证明沙冬青根瘤菌具有较强的抗逆性,但菌株间存在差异,这可能是对沙冬青分布区多种景观生态类型的适应。同时测定了几种伴生植物中间锦鸡儿(Caragana intermedia)、猫头刺(Oxytropis aciphylla)和柠条(Caragana corshinskii)根瘤菌的抗逆性,与沙冬青根瘤菌表现出一定的相似性,具较强的抗逆性,说明生态环境对豆科植物-根瘤菌共生体有重要的影响。沙冬青的高抗性可能与沙冬青根瘤菌的高抗性有关。沙冬青与其根瘤菌的共生关系有利于其植物群落的发育和多样性的维系。     

超慢生大豆根瘤菌DNA G+C含量和DNA同源性测定

用热变性温度法和液相复性速率法分别测定了超慢生大豆根瘤菌(ESG,extra-slow-growing soybean rhizobia)DNA G+C mol%及与其它根瘤菌间的DNA同源性.结果表明,ESG的DNA G+C mol含量在59.2—63.5%之间,且不同地区不同血清型的ESG代表菌株DNA同源率在70%以上,说明它们是遗传型一致的类群.ESG与在大豆上结瘤的快生大豆根瘤菌(Rhizobium fredii USDA205)同源率为14.8%,与慢生大豆根瘤菌(Bradyrhizobiumjaponicum)三个DNA同源组的同源率分别为20.5%,30.0%,19.4%.测定结果还表明,ESG与其它根瘤菌遗传学的亲缘关系也很远.     

Some technological properties of Penicillium roqueforti strains isolated from a home-made blue cheese

Nine strains of Penicillium roqueforti isolated from a traditional Spanish blue cheese (Valdeón cheese) along with two commercial strains were investigated for their ability to grow at different concentrations of salt and at different temperatures as well as for their proteolytic and lipolytic activities. Low concentrations of salt (1-3%) were stimulating for all the strains, with 1% salt being the concentration with the highest stimulating effect in nearly all. The rate of growth at 10°C was 2-3 times lower than at 25°C, the optimum temperature for the species. None of the strains, including the commercial cultures, showed proteolytic activity on casein agar, while all of them were lipolytic on tributyrin agar.     

Nodulation of Trifolium repens at low pH by single and paired strains of Rhizobium leguminosarum biovar trifolii

Detailed individual nodulation profiles were obtained for five strains of Rhizobium leguminosarum biovar trifolii inoculated onto roots of Trifolium repens seedlings growing on an agar medium of pH 4.5. The time of appearance and the location of every nodule were noted for a period of 10 days after inoculation. Using these nodulation frequency profiles, pairings of strains were identified and six mixed-strain inoculation (1:1 ratio) experiments were subsequently performed at pH 4.5. Results from the mixed-inoculum experiments showed that the performance of a Rhizobium strain in single culture could not be reliably used to predict the outcome of a paired-inoculation study and that some seedlings were exclusively nodulated by rhizobia that performed poorly at low pH in single-culture inoculations. Received: 26 November 1996 / Accepted: 18 April 1997     

哈茨木霉与绿色木霉对杉木种子萌发和幼苗生长的影响

以杉木种子为材料,研究不同浓度(0.003、0.03、0.3、3、30、300 mg·L^-1)哈茨木霉和绿色木霉溶液对杉木种子萌发和幼苗生长的影响.结果表明: 各浓度木霉溶液处理对杉木种子萌发和幼苗生长均有一定的促进作用,其促进效果随着处理浓度的增加均呈先升后降的趋势.与对照相比,0.03 mg·L^-1哈茨木霉和绿色木霉处理对提高杉木种子的发芽率、发芽势、发芽指数、活力指数、根长、苗高和鲜质量效果最佳,分别提高了57.6%、125.0%、51.0%、209.2%、114.3%、16.1%、24.6%和42.7%、76.7%、43.9%、185.4%、113.8%、8.6%、22.6%;0.3 mg·L^-1哈茨木霉和绿色木霉显著提高杉木幼苗超氧化物歧化酶和过氧化物酶活性,分别增加了157.6%、179.9%和127.5%、116.2%,而丙二醛含量降低了86.1%和72.4%. 0.03～0.3 mg·L^-1浓度的哈茨木霉和绿色木霉不仅能显著促进杉木种子的萌发和幼苗生长,而且能够提高其抗氧化酶活性,增强杉木幼苗的抗逆性.     

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。     

Chemical composition of extracellular polysaccharides of cowpea rhizobia

The extracellular polysaccharides (EPS) of six strains of cowpea rhizobia were examined. The strains (MI50A, M6-7B, IRC253) produced polysaccharides containing glucose, galactose and mannose in a molar ratio of 2:1.1:1, 1:1.3:3.1 and 1:1.3:3.5 respectively. Two strains (513-B and Ez-Aesch) produced polysaccharides containing galactose and mannose in a molar ratio of 2:3. Mannose was the only sugar detected in the EPS of strain IRC291. Pyruvate, acetate, glucuronic acid and galacturonic acid were not detected in any strain.Abbreviations EPS Extracellular polysaccharide - YEMA yeast-extract mannitol agar - YEMB yeast extract mannitol broth     

Effect of high salts concentrations on the growth of rhizobia and responses to added osmotica

Twenty-eight reference strains, 79 rhizobia isolated from Trigonella foenum graecum (fenugreek), 26 strains isolated from root nodules of Ceratonia siliqua (carob tree), 30 strains isolated from nodules of Adenocarpus decorticans and five isolated from Cytisus arboreus root nodules, were screened for their tolerance to increased concentrations of NaCl. Nine isolates of fenugreek were able to grow on medium containing 10% NaCl and one strain still grew at 14%. The effect of a range of salts at 2·5 and 5% (w/v) on the growth of rhizobia was assessed and it was shown that this effect depended on the ion form and the strains used. In general, NaCl appeared to be a good indicator of the tolerance of the strains to salts. The concentrations of the salts used were high and even at 5%, growth was not completely arrested in the less tolerant strains. Some substances, such as glutamate, proline, glycine betaine and CaCl₂, were tested as osmotica. The effect of the two amino acids and betaine was confirmed with all the strains used whereas the alleviating effect of CaCl₂ was not observed in all strains. This salt had different effects on two isolates of fenugreek. These results revealed a great diversity in salt tolerance, correlated with different responses to other stress conditions, which may be due to diversity in microbial ecology.