A rhizobia strain isolated from root nodule of gymnosperm Podocarpus macrophyllus

Symbiotic nitrogen fixation of rhizobia and leguminous plants is considered as the most important biologic nitrogen fixation system on earth. Symbiotic nodulation of gymnosperm Podocarpus macro-phyllus and rhizobia has never been reported. In this study, 11 endophytic bacteria strains were isolated from root nodules of P. macrophyllus and its variation P. macrophyllus var. maki. The plant infection tests on these strains indicated that the isolated strains could be nodulated on P. macrophyllus plants, and weak nitrogenase activity of nodules was found in acetylene reduction method. According to the physiological and biochemical characteristics of the 11 strains, GXLO 02 was selected as the representative strain. 16S rDNA full-length sequence analysis of GXLO 02 confirmed that the representative strain GXLO 02 belongs to Rhizobium sp.     

罗汉松根瘤内生细菌的分离和特性

罗汉松Ｐｏｄｏｃａｒｐｕｓｍａｃｒｏｐｈｙｌｌｕｓ(Ｔｈｕｎｂ .)Ｄ .Ｄｏｎ是裸子植物亚门罗汉松科一种 ,在我国主产长江以南各省 ,为用材及园林绿化的优良树种 ,种子和根可入药。关于罗汉松根际微生物的研究 ,梁秀棠[1] 、花晓梅[2 ] 曾报道其根与菌根真菌共生形成外生或内生菌根。国外有学者发现 ,几种其它的罗汉松科植物Ｐ .ｎｕｂｉｇｅｎｕｓ等具有结瘤固氮现象[3～ 6] ,但对其与细菌共生形成根瘤以及根瘤内生细菌的分离鉴定至今国内外尚未见报道。作者在广西的南宁、桂林、扶绥及广东的佛山等地进行野外调查 ,证明罗汉松在自然界…     

Effects of Engineered Sinorhizobium meliloti on Cytokinin Synthesis and Tolerance of Alfalfa to Extreme Drought Stress

Cytokinin is required for the initiation of leguminous nitrogen fixation nodules elicited by rhizobia and the delay of the leaf senescence induced by drought stress. A few free-living rhizobia have been found to produce cytokinin. However, the effects of engineered rhizobia capable of synthesizing cytokinin on host tolerance to abiotic stresses have not yet been described. In this study, two engineered Sinorhizobium strains overproducing cytokinin were constructed. The tolerance of inoculated alfalfa plants to severe drought stress was assessed. The engineered strains, which expressed the Agrobacterium ipt gene under the control of different promoters, synthesized more zeatins than the control strain under free-living conditions, but their own growth was not affected. After a 4-week inoculation period, the effects of engineered strains on alfalfa growth and nitrogen fixation were similar to those of the control strain under nondrought conditions. After being subjected to severe drought stress, most of the alfalfa plants inoculated with engineered strains survived, and the nitrogenase activity in their root nodules showed no apparent change. A small elevation in zeatin concentration was observed in the leaves of these plants. The expression of antioxidant enzymes increased, and the level of reactive oxygen species decreased correspondingly. Although the ipt gene was transcribed in the bacteroids of engineered strains, the level of cytokinin in alfalfa nodules was identical to that of the control. These findings suggest that engineered Sinorhizobium strains synthesizing more cytokinin could improve the tolerance of alfalfa to severe drought stress without affecting alfalfa nodulation or nitrogen fixation.     

Physiological Characteristics of Cowpea Rhizobia: Evaluation of Symbiotic Efficiency in Vigna unguiculata

One fast-growing and three slow-growing strains of Rhizobium (isolated from cowpeas) were evaluated for symbiotic performance on Vigna unguiculata (L.) Walp. cultivar California no. 5 blackeyes. Plants inoculated with slow-growing strains 176A22, 176A30, and 176A32 developed a maximum acetylene reduction activity of 24.6, 27.0, and 32 μmol of ethylene formed per plant per h, respectively, versus 6.4 μmol per plant per h in plants inoculated with the fast-growing strain 176A28. When inoculated with approximately equal proportions of rhizobia, the fast-growing strain 176A28 produced 95% of the nodules when challenged with the slow-growing strain 176A22, but formed only 6% of the nodules when challenged with the slow-growing strain 176A30. Consequently, there was no relation between the growth rate in vitro and the capability of rhizobia to compete for nodule-forming sites. Plants inoculated with strain 176A28 and subjected to drought during the vegetative growth period recovered to the same level of nitrogen fixation and nodulation as those that received adequate irrigation. On the other hand, plants inoculated with strains 176A22, 176A30, and 176A32 failed to achieve the same levels of nodulation and nitrogen fixation under drought as compared with irrigated conditions.     

Competition between rhizobia under different environmental conditions affects the nodulation of a legume

Mutualistic symbiosis and nitrogen fixation of legume rhizobia play a key role in ecological environments. Although many different rhizobial species can form nodules with a specific legume, there is often a dominant microsymbiont, which has the highest nodule occupancy rates, and they are often known as the “most favorable rhizobia”. Shifts in the most favorable rhizobia for a legume in different geographical regions or soil types are not well understood. Therefore, in order to explore the shift model, an experiment was designed using successive inoculations of rhizobia on one legume. The plants were grown in either sterile vermiculite or a sandy soil. Results showed that, depending on the environment, a legume could select its preferential rhizobial partner in order to establish symbiosis. For perennial legumes, nodulation is a continuous and sequential process. In this study, when the most favorable rhizobial strain was available to infect the plant first, it was dominant in the nodules, regardless of the existence of other rhizobial strains in the rhizosphere. Other rhizobial strains had an opportunity to establish symbiosis with the plant when the most favorable rhizobial strain was not present in the rhizosphere. Nodule occupancy rates of the most favorable rhizobial strain depended on the competitiveness of other rhizobial strains in the rhizosphere and the environmental adaptability of the favorable rhizobial strain (in this case, to mild vermiculite or hostile sandy soil). To produce high nodulation and efficient nitrogen fixation, the most favorable rhizobial strain should be selected and inoculated into the rhizosphere of legume plants under optimum environmental conditions.     

兰坪铅锌尾矿区豆科根瘤菌遗传多样性ARDRA分析

通过16S rDNA扩增产物限制性片段长度多态性分析(ARDRA),对兰坪铅锌尾矿区豆科植物根瘤菌的遗传多样性进行了研究。采用限制性内切酶Hae Ⅲ、Hind Ⅲ、Hinf Ⅰ和Taq Ⅰ对16S rDNA扩增产物进行了酶切分型,根据ARDRA酶切图谱的不同,进行树状聚类。结果表明:49株根瘤菌在40%的相似水平上按氮含量不同及铅锌含量的采集地不同分别聚为OTU1、OTU2和OTU33个群,说明根瘤菌的遗传多样性及分布与土壤中的氮含量和铅锌含量有关。代表菌株的16S rDNA测序结果分析表明,它们在系统发育树上属于Rhizobium sp.、Sinorhizobium sp.和Bradyrhizobium sp.3个系统发育分支,进一步说明兰坪铅锌尾矿区豆科植物根瘤菌多样性较丰富。     

Effectiveness of nitrogen fixation in rhizobia

Biological nitrogen fixation in rhizobia occurs primarily in root or stem nodules and is induced by the bacteria present in legume plants. This symbiotic process has fascinated researchers for over a century, and the positive effects of legumes on soils and their food and feed value have been recognized for thousands of years. Symbiotic nitrogen fixation uses solar energy to reduce the inert N₂ gas to ammonia at normal temperature and pressure, and is thus today, especially, important for sustainable food production. Increased productivity through improved effectiveness of the process is seen as a major research and development goal. The interaction between rhizobia and their legume hosts has thus been dissected at agronomic, plant physiological, microbiological and molecular levels to produce ample information about processes involved, but identification of major bottlenecks regarding efficiency of nitrogen fixation has proven to be complex. We review processes and results that contributed to the current understanding of this fascinating system, with focus on effectiveness of nitrogen fixation in rhizobia.     

Assessing the suitability of antibiotic resistance markers and the indirect ELISA technique for studying the competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions in South Africa

Background  

Symbiotic N2 fixation in legumes is constrained by many factors, including the paucity of suitable soil rhizobia To maximise growth of legume species therefore often requires the application of effective rhizobia as inoculants. But where native strains out-compete introduced rhizobia for nodule formation, it is important that the competitiveness of selected strains is tested in the field and glasshouse prior to their recommendation as commercial inoculants. However the methodology for strain identification inside nodules has often proved difficult and thus limited this field of research. In this study, the suitability of the antibiotic resistance technique (both intrinsic low-resistance fingerprinting and high-resistance marking) and the serological indirect ELISA method were assessed for their ability to detect selected Cyclopia rhizobia under glasshouse and field conditions. The four rhizobial strains that were used, namely PPRICI3, UCT40a, UCT44b and UCT61a, were isolated from wild Cyclopia species growing in the Western Cape fynbos of South Africa.     

MALDI mass spectrometry‐assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula–Sinorhizobium meliloti symbiosis

Symbiotic associations between leguminous plants and nitrogen‐fixing rhizobia culminate in the formation of specialized organs called root nodules, in which the rhizobia fix atmospheric nitrogen and transfer it to the plant. Efficient biological nitrogen fixation depends on metabolites produced by and exchanged between both partners. The Medicago truncatula–Sinorhizobium meliloti association is an excellent model for dissecting this nitrogen‐fixing symbiosis because of the availability of genetic information for both symbiotic partners. Here, we employed a powerful imaging technique – matrix‐assisted laser desorption/ionization (MALDI)/mass spectrometric imaging (MSI) – to study metabolite distribution in roots and root nodules of M. truncatula during nitrogen fixation. The combination of an efficient, novel MALDI matrix [1,8–bis(dimethyl‐amino) naphthalene, DMAN] with a conventional matrix 2,5–dihydroxybenzoic acid (DHB) allowed detection of a large array of organic acids, amino acids, sugars, lipids, flavonoids and their conjugates with improved coverage. Ion density maps of representative metabolites are presented and correlated with the nitrogen fixation process. We demonstrate differences in metabolite distribution between roots and nodules, and also between fixing and non‐fixing nodules produced by plant and bacterial mutants. Our study highlights the benefits of using MSI for detecting differences in metabolite distributions in plant biology.     

Effect of dinitroaniline herbicides on rhizobia, nodulation and N2(C2H2) fixation of four groundnut cultivars

Field, greenhouse and laboratory investigations were conducted to determine the effect of four dinitroaniline herbicides on rhizobia, nodulation and nitrogen fixation of four groundnut cultivars. Benefin, dinitramine and nitralin used at recommended levels decreased nodule dry weight, nitrogenase activity and total nitrogen of groundnut tops and pod yield in three cultivars Kadiri 71-1, Kadiri-2, ICGS-11 and not for a fourth cultivar, Kadiri-3 of groundnut (Arachis hypogaea L.), but fluchloralin used at the recommended level increased the nodulation rate, nitrogenase activity and total nitrogen of groundnut tops and pod yield compared to untreated plants. Studies were conducted in vitro to determine the relative toxicity of the herbicides on four Rhizobium strains isolated from the nodules of four cultivars of groundnut. It was found that various strains of rhizobia differ in their sensitivity to different rates of the herbicides tested. Carbon dioxide exchange rate (CER) of all the cultivars which received herbicide treatment was measured at different time intervals to determine the relationship between photosynthesis and inhibition of nodulation. The lack of adverse effect on the CER of four cultivars when treated at recommended concentrations indicated that nitrogen fixation was affected in cultivars Kadiri 71-1, Kadiri-2 and ICGS-11 due to inhibition of nodulation.     

Increased Effectiveness of Competitive Rhizobium Strains upon Inoculation of Cajanus cajan

A field study was conducted in lysimeters containing ¹⁵N-enriched soil to determine the effects of four competitive rhizobium strains upon yield parameters of pigeon peas (Cajanus cajan). The greatest differences observed were in seed yields; strain P132 effected the highest seed yield (121 ± 20 g per plant), and the control strain (indigenous rhizobia) effected the lowest yield (43.9 ± 8 g per plant). With the exception of seeds and pods, the dry matter weights were not different. Although there appeared to be no effect by inoculum strains on the fractional content of N derived from biological nitrogen fixation when the total plant biomass was considered, strains P132 and 401 partitioned more of the N derived from fixation into seeds and leaves than did the other strains. Because the seeds comprised the major portion of plant N, more total N and more N derived from biological nitrogen fixation (about half of total N) were found in plants inoculated with P132, whereas the smallest amount was found in the uninoculated controls. P132 was also the best competitor with respect to indigenous rhizobia and acounted for all of the nodules found on the plants in which it was inoculated.     

Improvement of biological nitrogen fixation in Egyptian winter legumes through better management of Rhizobium

The diversity of rhizobia nodulating common bean ( Phaseolus vulgaris), berseem clover (Trifolium alexanderinum) and lentil (Lens culinaris) was assessed using several characterization techniques, including nitrogen fixation efficiency, intrinsic antibiotic-resistance patterns (IAR), plasmid profiles, serological markers and rep-PCR fingerprinting. Wide diversity among indigenous rhizobial populations of the isolates from lentil, bean and clover was found. Strikingly, a large percentage of the indigenous rhizobial population was extremely poor at fixing nitrogen. This emphasizes the need to increase the balance of highly efficient strains within the rhizobial population. Use of high-quality inocula strains that survive and compete with other less-desired and less-efficient N2-fixing rhizobia represents the best approach to increase biological nitrogen fixation of the target legume. In field-grown lentils, the inoculant strains were not able to outcompete the indigenous rhizobia and the native lentil rhizobia occupied 76–88% of the total nodules formed on inoculated plants. Nitrogen fixation by lentils, estimated using the ¹⁵N isotope dilution technique, ranged between 127 to 139 kg ha-1 in both inoculated and un-inoculated plants. With berseem clover, the inoculant strains were highly competitive against indigenous rhizobia and occupied 52–79% of all nodules. Inoculation with selected inocula improved N2 fixation by clover from 162 to 205 kg ha-1 in the three cuts as compared with 118 kg ha-1 in the un-inoculated treatment. The results also indicated the potential for improvement of N2 fixation by beans through the application of efficient N2-fixing rhizobia.     

Isolation and symbiotic characterization of transposon Tn5-induced arginine auxotrophs of Sinorhizobium meliloti

Seventeen arginine auxotrophic mutants of Sinorhizobium meliloti Rmd201 were isolated by random transposon Tn5 mutagenesis using Tn5 delivery vector pGS9. Based on intermediate feeding studies, these mutants were designated as argA/argB/argC/argD/argE (ornithine auxotrophs), argF/argI, argG and argH mutants. The ornithine auxotrophs induced ineffective nodules whereas all other arginine auxotrophs induced fully effective nodules on alfalfa plants. In comparison to the parental strain induced nodule, only a few nodule cells infected with rhizobia were seen in the nitrogen fixation zone of the nodule induced by the ornithine auxotroph. TEM studies showed that the bacteroids in the nitrogen fixation zone of ornithine auxotroph induced nodule were mostly spherical or oval unlike the elongated bacteroids in the nitrogen fixation zone of the parental strain induced nodule. These results indicate that ornithine or an intermediate of ornithine biosynthesis, or a chemical factor derived from one of these compounds is required for the normal development of nitrogen fixation zone and transformation of rhizobial bacteria into bacteroids during symbiosis of S. meliloti with alfalfa plants.     

我国蚕豆根瘤菌的数值分类及其16SrDNA PCR-RFLP研究

对分离自我国11个省24个地区49株蚕豆根瘤菌及11株参比菌株进行了唯一碳源、氮源、抗生素、耐逆性和酶活性等138个表型性状测定,并用MINTS软件进行聚类分析。结果表明,全部供试菌株在59％的相似水平上聚在一起,在80％的相似水平上可分为6个群。其中群4与参比菌株聚在一起,而其他5个群均由未知菌组成。进一步对36株菌进行了16S rDNA PCR—RFLP分析,在85％相似水平上供试菌可分为4个群和1个独立的分支,其聚群结果与数值分类结果有较好的一致性。表型及遗传型分析结果表明,我国蚕豆根瘤菌具有极大的多样性。     

Nodulation studies on legumes exotic to Australia: Hedysarum coronarium

Abstract Symbiotic experiments in glasshouse, controlled environment cabinet, and field were conducted with four lines of sulla ( Hedysarum coronarium ) and 15 strains of Rhizobium spp. This plant is highly Rhizobium -specific and appropriate strains are most unlikely to occur naturally in Australia. Under several sets of experimental conditions, H. coronarium nodulated abundantly and effectively with homologous rhizobia introduced from Spain and Italy. The optimum temperature for nitrogen fixation was relatively low (approx. 21°C) but significant interactions between line of host, strain of rhizobia, and growth temperature were frequent. The rhizobia were persistent in soil.     

我国蚕豆根瘤菌的数值分类及其16S rDNA PCR-RFLP研究*

对分离自我国11个省24个地区49株蚕豆根瘤菌及11株参比菌株进行了唯一碳源、氮源、抗生素、耐逆性和酶活性等138个表型性状测定,并用M INTS软件进行聚类分析。结果表明,全部供试菌株在59%的相似水平上聚在一起,在80%的相似水平上可分为6个群。其中群4与参比菌株聚在一起,而其他5个群均由未知菌组成。进一步对36株菌进行了16S rDNA PCR-RFLP分析,在85%相似水平上供试菌可分为4个群和1个独立的分支,其聚群结果与数值分类结果有较好的一致性。表型及遗传型分析结果表明,我国蚕豆根瘤菌具有极大的多样性。     

Nitrogen fixation in pure culture by rhizobia isolated from stem nodules of tropical Aeschynomene species

Abstract Asymbiotic nitrogenase activity was investigated in rhizobia strains isolated from stem and root nodules of severa Aeschynomene species. All isolated from stem-nodulating species were able to develop nitrogenase activity ex planta in the presence or in the absence of combined nitrogen, whereas root isolates from Aeschynomene species related to the cowpea group of plants showed little or no activity. Nitrogenase activity in soft-agar and in liquid cultures displayed by strains ORS310 and ORS322, isolated from stem nodules of A. indica and A. afraspera respectively, was of the same order of magnitude as that found for Azorhizobium caulinodans ORS571 and ten times higher than for Bradyrhizobium strain CB756. Furthermore, like A. caulinodans ORS571, strains ORS310 and ORS322 were able to use atmospheric nitrogen as sole nitrogen source for growth.     

Inoculation Response of Legumes in Relation to the Number and Effectiveness of Indigenous Rhizobium Populations

The response of legumes to inoculation with rhizobia can be affected by many factors. Little work has been undertaken to examine how indigenous populations or rhizobia affect this response. We conducted a series of inoculation trials in four Hawaiian soils with six legume species (Glycine max, Vigna unguiculata, Phaseolus lunatus, Leucaena leucocephala, Arachis hypogaea, and Phaseolus vulgaris) and characterized the native rhizobial populations for each species in terms of the number and effectiveness of the population for a particular host. Inoculated plants had, on average, 76% of the nodules formed by the inoculum strain, which effectively eliminated competition from native strains as a variable between soils. Rhizobia populations ranged from less than 6 × 10⁰/g of soil to 1 × 10⁴/g of soil. The concentration of nitrogen in shoots of inoculated plants was not higher than that in uninoculated controls when the most probable number MPN counts of rhizobia were at or above 2 × 10¹/g of soil unless the native population was completely ineffective. Tests of random isolates from nodules of uninoculated plants revealed that within most soil populations there was a wide range of effectiveness for N₂ fixation. All populations had isolates that were ineffective in fixing N₂. The inoculum strains generally did not fix more N₂ than the average isolate from the soil population in single-isolate tests. Even when the inoculum strain proved to be a better symbiont than the soil rhizobia, there was no response to inoculation. Enhanced N₂ fixation after inoculation was related to increased nodule dry weights. Although inoculation generally increased nodule number when there were less than 1 × 10² rhizobia per g of soil, there was no corresponding increase in nodule dry weight when native populations were effective. Most species compensated for reduced nodulation in soils with few rhizobia by increasing the size of nodules and therefore maintaining a nodule dry weight similar to that of inoculated plants with more nodules. Even when competition by native soil strains was overcome with a selected inoculum strain, it was not always possible to enhance N₂ fixation when soil populations were above a threshold number and had some effective strains.     

Characterization of high temperature-tolerant rhizobia isolated from Prosopis juliflora grown in alkaline soil

A method was developed for the fast screening and selection of high-temperature tolerant rhizobial strains from root nodules of Prosopis juliflora growing in alkaline soils. The high-temperature tolerant rhizobia were selected from 2,500 Rhizobium isolates with similar growth patterns on yeast mannitol agar plates after 72 h incubation at 30 and 45 degrees C, followed by a second screening at 47.5 degrees C. Seventeen high-temperature tolerant rhizobial strains having distinguishable protein band patterns were finally selected for further screening by subjecting them to temperature stress up to 60 degrees C in yeast mannitol broth for 6 h. The high-temperature tolerant strains were NBRI12, NBRI329, NBRI330, NBRI332, and NBRI133. Using this procedure, a large number of rhizobia from root nodules of P. juliflora were screened for high-temperature tolerance. The assimilation of several carbon sources, tolerance to high pH and salt stress, and ability to nodulate P. juliflora growing in a glasshouse and nursery of the strains were studied. All five isolates had higher plant dry weight in the range of 29.9 to 88.6% in comparison with uninoculated nursery-grown plants. It was demonstrated that it is possible to screen in nature for superior rhizobia exemplified by the isolation of temperature-tolerant strains, which established effective symbiosis with nursery-grown P. juliflora. These findings indicate a correlation between strain performance under in vitro stress in pure culture and strain behavior under symbiotic conditions. Pure culture evaluation may be a useful tool in search for Rhizobium strains better suited for soil environments where high temperature, pH, and salt stress constitutes a limitation for symbiotic biological nitrogen fixation.     

Taxonomic and symbiotic diversity of bacteria isolated from nodules of Acacia tortilis subsp. raddiana in arid soils of Tunisia

A collection of rhizobia isolated from Acacia tortilis subsp. raddiana nodules from various arid soils in Tunisia was analyzed for their diversity at both taxonomic and symbiotic levels. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at 40 °C. Genetic characterization emphasized that most of the strains (42/50) belong to the genus Ensifer, particularly the species Ensifer meliloti, Ensifer garamanticus, and Ensifer numidicus. Symbiotic properties of isolates showed diversity in their capacity to nodulate their host plant and to fix atmospheric nitrogen. The most effective isolates were closely related to E. garamanticus. Nodulation tests showed that 3 strains belonging to Mesorhizobium genus failed to renodulate their host plant, which is surprising for symbiotic rhizobia. Furthermore, our results support the presence of non-nodulating endophytic bacteria belonging to the Acinetobacter genus in legume nodules.