Manipulation of rhizobia microflora for improving legume productivity and soil fertility: A critical assessment

Inputs of biologically fixed nitrogen derived from the symbiotic relationship between legumes and their root-nodule bacteria into terrestrial ecosystems amount to at least 70 million metric tons per year. It is obvious that this enormous quantity will need to be augmented as the world's population increases and as the natural resources that supply fertilizer nitrogen diminish. This objective will be achieved through the development of superior legume varieties, improvement in agronomic practice, and increased efficiency of the nitrogen fixation process itself by better management of the symbiotic relationship between plant and bacteria. This paper considers ways and means by which populations of root-nodule bacteria, established and introduced, can be manipulated ecologically, agronomically, edaphically and genetically to improve legume productivity and, as a consequence, soil fertility.     

Characterization of rhizobia nodulating Lotus subbiflorus from Uruguayan soils

Generation times, acid production, carbon utilization, immunological properties, plasmid content, protein profile and symbiotic properties of 15 isolates of rhizobia nodulating Lotus subbiflorus were studied. Based on specific growth rates, carbon source utilization and acid production, 13 out of the 15 isolates could be assigned to the slow-growing group of rhizobia (bradyrhizobia). Using antisera against whole cells of three isolates, we separated the 15 isolates into three serogroups. Only the slow-growing isolate Ls4 and the fast-growers Ls5 and Ls552 lacked cross-reactivity with any of the sera tested. Electrophoretic mobilities of whole cell protein from seven out of the eight isolates included in the serogroup represented by strain Ls31 were identical. Similarly, isolates Ls1B3 and Ls1B4, both in serogroup Ls1B3, had the same pattern of cell proteins. In contrast, isolates Ls3 and Ls7, belonging to serogroup Ls7, differed in protein profile. Plant growth experiments carried out under bacteriologically controlled conditions revealed that all of the isolates effectively nodulated L. subbiflorus and L. pedunculatus, but were unable to form effective nodules on L. tenuis and L. corniculatus. All isolates showed similar effectiveness in symbiosis with L. subbiflorus, except isolate Ls7, which gave significantly higher plant dry weight.Abbreviations ELISA enzyme linked immuno-sorbent assay - kDa kiloDalton - MM mineral medium - PBS phosphate-buffered saline - RE relative efficiency - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - YEM deyeast extract mannitol     

H2-Recycling system in mungbean Rhizobium in relation to N2-fixation

Thirty isolates of mungbean Rhizobium were tested for the presence of H₂-recycling system. All the isolates were preliminary screened for detecting H₂-recycling system in free culture using triphenyltetrazolium chloride reduction as screening procedure. The isolates which reduced the dye rapidly at early stages of growth were found to recycle hydrogen both in vivo as well as in vitro. Nitrogen fixing efficiency of hydrogenase positive, hydrogenase negative isolates and Hup^– mutants was compared by green house experiments. There was 13–56% increase in dry matter and 21–46% increase in total nitrogen of the plants inoculated with H₂-recycling isolates over the plants inoculated with non-recycling isolates. There was reduction in dry matter and total nitrogen content of the plants inoculated with Hup^– mutants as compared to plants inoculated with wild type strain. The per cent decrease due to inoculation with Hup^– mutants over wild type strain was 19–22 and 20–26 of dry weight and total nitrogen in plants, respectively.Abbreviations TTC triphenyltetrazolium chloride     

Evaluation of strain competitiveness in Rhizobium leguminosarum bv phaseoli using a nod + fix− natural mutant

Competition from native soil rhizobia is likely to be an important factor limiting Phaseolus vulgaris L. inoculant response in Latin America. We used UMR 1116, a nod ⁺ fix^– natural mutant of Rhizobium leguminosarum bv phaseoli strain CC511, as a reference strain to study competition for nodulation sites in this species. When P. vulgaris cv Carioca was planted in soils containing different proportions of UMR 1116 and the effective and competitive strain UMR 1899, UMR 1116 occupied more than 50% of the nodules at all inoculant ratios tested, though increasing the proportion of UMR 1899 in the inoculant did enhance the number and percentage of effective nodules and plant dry weight. Sixty two strains of bean rhizobia were tested in competition with UMR 1116. An inoculant ratio of 1:1 was used, with all strains applied to the soil rather than to seeds. Strains varied in the number and percentage of effective nodules produced in competition with UMR 1116, and in plant dry weight, and there was a strong correlation between variation in each of these traits and plant N accumulation. Seven of the strains (UMR 1073, 1084, 1102, 1125, 1165, 1378 and 1384) were identified as both superior in competitive ability and active in N₂ fixation. Site of placement of the inoculant and ambient temperature influenced strain response.Journal paper 16736, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

Mutants of Rhizobium capable of fixing N2 in the free-living condition

Six mutant strains of Rhizobium were isolated after UV treatment which could exhibit nitrogenase activity in Burk's N-free medium without any supplement. The activity ranged between 99.5 and 113 nmol/mg cell dry weight and hour. Two of the parent strains belonged to soybean, and one each to mungbean and Sesbania sp. Both the parent and mutant strains exhibited nitrogenase activity in CS 7 medium. One of the mutants retained its capacity to produce nodules on soybean roots.List of Abbreviations C.D. Critical difference - EMS ethylmethane sulphonate - NTG N-methyl-N-nitro, N-nitrosoguanidine     

Can mushrooms fix atmospheric nitrogen?

It is generally reported that fungi likePleurotus spp. can fix nitrogen (N₂). The way they do it is still not clear. The present study hypothesized that only associations of fungi and diazotrophs can fix N₂. This was testedin vitro. Pleurotus ostreatus was inoculated with a bradyrhizobial strain nodulating soybean andP. ostreatus with no inoculation was maintained as a control. At maximum mycelial colonization by the bradyrhizobial strain and biofilm formation, the cultures were subjected to acetylene reduction assay (ARA). Another set of the cultures was evaluated for growth and nitrogen accumulation. Nitrogenase activity was present in the biofilm, but not when the fungus or the bradyrhizobial strain was alone. A significant reduction in mycelial dry weight and a significant increase in nitrogen concentration were observed in the inoculated cultures compared to the controls. The mycelial weight reduction could be attributed to C transfer from the fungus to the bradyrhizobial strain, because of high C cost of biological N₂ fixation. This needs further investigations using¹⁴C isotopic tracers. It is clear from the present study that mushrooms alone cannot fix atmospheric N₂. But when they are in association with diazotrophs, nitrogenase activity is detected because of the diazotrophic N₂ fixation. It is not the fungus that fixes N₂ as reported earlier. Effective N₂ fixing systems, such as the present one, may be used to increase protein content of mushrooms. Our study has implications for future identification of as yet unidentified N₂ systems occurring in the environment.     

Characterization of rhizobia isolated from Albizia spp. in comparison with microsymbionts of Acacia spp. and Leucaena leucocephala grown in China

This is the first systematic study of rhizobia associated with Albizia trees. The analyses of PCR-RFLP and sequencing of 16S rRNA genes, SDS-PAGE of whole-cell proteins and clustering of phenotypic characters grouped the 31 rhizobial strains isolated from Albizia into eight putative species within the genera Bradyrhizobium, Mesorhizobium and Rhizobium. Among these eight rhizobial species, five were unique to Albizia and the remaining three were shared with Acacia and Leucaena, two legume trees coexisting with Albizia in China. These results indicated that Albizia species nodulate with a wide range of rhizobial species and had preference of microsymbionts different from Acacia and Leucaena. The definition of four novel groups, Mesorhizobium sp., Rhizobium sp. I, Rhizobium sp. II and "R. giardinii", indicates that further studies with enlarged rhizobial population are necessary to better understand the diversity and to clarify the taxonomic relationships of Albizia-associated rhizobia.     

New techniques for studying competition by Rhizobia and for assessing nitrogen fixation in the field

One of the key factors limiting the proper assessment and use of rhizobial strains in the field is the lack of suitable methodology to screen the success of individual isolates in competing for nodule occupancy with different cultivars of legumes and in different soil and agronomic conditions. The use of marker genes enables individual rhizobial strains to be identified by a simple colour assay, thus enabling a dramatic increase in throughput of strain screening. One such marker system for rhizobial ecology, the GUS system, is already in use to facilitate rapid screening of rhizobial isolates. Other markers, which will allow the competitive behaviour of several strains to be studied at once, are under development.Likewise, breeding of the host legume for a high efficiency of nitrogen fixation is hampered by the difficulty in assessing this property. The method which currently gives the highest throughput of analysis, and has been successfully used in soybean breeding programs, is the ureide technique. However, it remains somewhat laborious for use in routine breeding programs. In this paper we discuss the potential use of reporter genes to provide information on the relative levels of ureides and other nitrogenous compounds in plants growing in the field. This would greatly increase the rate at which this trait could be scored, and would thus enable routine assays for increased symbiotic nitrogen fixation for breeding or management purposes in legume crops such as soybean (Glycine max) and common bean (Phaseolus vulgaris).     

Influence of rhizobia,azotobacter and blue-green algae on N content and yield of rice

Summary Inoculation with root-nodule bacteria had favourable influence on N-uptake and yield of wheat. Since waterlogged root region of rice permits higher nitrogenase activity a pot culture experiment was conducted using same nine strains of rhizobia,Azotobacter chroococcum and bluegreen algae as inoculants.R. leguminosarum in combination with 50 kg N ha⁻¹;R. japonicum and a strain of rhizobium isolated from moong bean increased the yield of paddy cv. Pusa-33. On the other hand an adverse effect of bacterial inoculation and of applied N was observed in case of Azotobacter, and rhizobia isolated from green gram, cicer, soyabean and clover. The importance of plant type, growth conditions and application of inorganic N in determining the success of plant-rhizobial associations is emphasised.     

Iron uptake and metabolism in the rhizobia/legume symbioses

Iron-containing proteins figure prominently in the nitrogen-fixing symbioses between bacteria of the genera Azorhizovium, Bradyrhizobium and Rhizobium and their respective plant hosts. Although iron is abundant in the soil, the acquisition of iron is problematic due to its low solubility at biological pH under aerobic conditions. The study of iron acquisition as it pertains to these economically important symbioses is directed at answering three questions: 1) how do rhizobial cells acquire iron as free-living microorganisms where they must compete for this nutrient with other soil inhabitants 2) how do the plant hosts acquire enough iron for the symbiosis and 3) how do rhizobia acquire iron as symbionts? Production and/or utilization of ferric-specific ligands (siderophores) has now been documented in the laboratory for a number of rhizobial species, but there is limited information on whether production and/or untilization occurs either in the soil or in planta. Studies with rhizobial mutants which can no longer produce and/or utilize siderophores should address whether siderophores contribute to functional symbioses. In addition, the ability to produce and/or utilize siderophores may affect the outcome of both interstrain and interspecific competition in the rhizosphere and in bulk soil. Some progress has been made at documenting the effects of iron deficiency on nodule development. Studies are also underway to determine whether, in addition to its central structural role, iron may also play a regulatory role in the symbioses. This review is an attempt to give an overview of the field, and hopefully will stimulate further research on the iron nutrition of these symbioses which account for such a significant proportion of the world's biologically fixed nitrogen.     

Response of soybean-Rhizobium symbioses to mineral nitrogen

Summary The effect of mineral nitrogen on establishment and activity of symbioses between soybean and several strains ofRhizobium japonicum and on the establishment of nodules ofR. japonicum isolated from nodules of field crops is studied. All strains were highly susceptible to the effects of 200 ppm NO₃–N on the establishment of symbiosis; 50 ppm NO₃–N had little effect. Response of symbioses establishhed in the absence of mineral N to short term exposure to nitrate or ammonium varied significantly between strains. Nodule isolates from soybean crops growing in nitrifying soil were no less susceptible to the inhibitory effects of mineral N on nodule formation than a laboratory culture of the commercial inoculant strain.     

Mimosine produced by the tree-legume Leucaena provides growth advantages to some Rhizobium strains that utilize it as a source of carbon and nitrogen

Growth of most Rhizobium strains is inhibited by mimosine, a toxin found in large quantities in the seeds, foliage and roots of plants of the genera Leucaena and Mimosa. Some Leucaena-nodulating strains of Rhizobium can degrade mimosine (Mid⁺) and are less inhibited by mimosine in the growth medium than the mimosine-nondegrading (Mid^-) strains. Ten Mid⁺ strains were identified that did not degrade 3-hydroxy-4-pyridone (HP), a toxic intermediate of mimosine degradation. However, mimosine was completely degraded by these strains and HP was not accumulated in the cells when these strains were grown in a medium containing mimosine as the sole source of carbon and nitrogen. The mimosine-degrading ability of rhizobia is not essential for nodulation of Leucaena species, but it provides growth advantages to Rhizobium strains that can utilize mimosine, and it suppresses the growth of other strains that are sensitive to this toxin.     

Morphology and general characteristics of phages of chickpea rhizobia

Six rhizobiophages designated as RC1, RC2, RC3, RC4, RC5 and RC6, infective against six strains of chickpea Rhizobium were isolated from field soils. Seasonal incidence, morphology, host range and inactivation pattern of the phages to heat and UV-light were studied. Four investigated phages were differentiated into two morphological types; one with hexagonal head and a long flexible tail (RC1 and RC5), the other with hexagonal head and a very short tail (RC2 and RC3). Electron microscopic examination of phage RC1 infected cells revealed that phage multiplication occurred at one pole of the cell. Phage RC3 appeared to be more thermal sensitive than others and exhibited one component inactivation while relatively resistant phages (RC1 and RC2) revealed two component inactivation. The six phages could be grouped into two classes on the basis of UV sensitivity; relatively resistant (RC1, RC2 and RC5) and sensitive (RC3, RC4 and RC6).     

Release of transgenic bacterial inoculants - rhizobia as a case study

The current debate on the release of genetically modified organisms to the environment must be informed by scientific data obtained from field studies. Many of the microorganisms that have potential applications outside the laboratory, especially in agriculture and horticulture, could be improved by genetic modification. Rhizobia, the bacteria that form N₂-fixing symbioses with leguminous plants, have a long history of safe use as seed inoculants, their biology is relatively well known, and they represent a relevant model system. There have been several field releases of genetically modified (GM) rhizobia in the USA and Europe, which provide information on various aspects of their ecology and efficacy. This review summarises the rationale for each release, details of the methods used for monitoring, and the results. Novel properties of rhizobia did not always have the predicted effects. Most studies revealed that rhizobial numbers dropped rapidly after application to soil or seeds but then numbers stabilised for months or years. The monitoring of survival and spread of rhizobia was greatly improved by the presence of novel marker genes. Tagging of rhizobia with marker genes provided more accurate information compared to the use of conventional strains, illustrating an important application of genetic modification, for tracking bacteria in the environment.     

Nodulation and nitrogen fixation by fast- and slow-growing rhizobia strains of soybean on several temperate and tropical legumes

Three slow-growingBradyrhizobium japonicum (G₃, USDA-110 and KUL-150) of diverse origins and two fast-growing strains ofRhizobium fredii (USDA-192 and USDA-193) were tested with a cropped soybean (Glycine max L. Merrill) cultivar, two cowpeas (Vigna unguiculata), one mung-bean (Phaseolus radiata), one winged-bean (Psophocarpus tetragonolobus) and one field bean (Phaseolus vulgaris) varieties.TheR. fredii strains nodulated and fixed Nitrogen as effectively as the strains ofB. japonicum in a modern european soybean cultivar, namely Fiskeby V. The other western bred soybeans tested were not nodulated by theseR. fredii strains. All of the soybean rhizobia produced nodules in both cowpeas and in mung-bean; theR. fredii strains showed effective N₂-fixation in the cowpeas, particularly USDA-193, yielding shoot dry weights greater than those from theB. japonicum. The symbiotic performance of theR. fredii strains with soybean and other legumes indicated that they should be placed in an intermediate group between the slow-growingB. japonicum and cowpearhizobium sp.The hydrogen uptake activites suggested a possible host effect on the expression of such genes in one out of theB. japonicum strains tested. Furthermore, the slow-growing rhizobia showed significantly higher nitrate-reduction than theR. fredii in the nodules.     

Phosphate solubilization activity of rhizobia native to Iranian soils

Agricultural soils in Iran are predominantly calcareous with very low plant available phosphorus (P) content. In addition to their beneficial N₂-fixing activity with legumes, rhizobia can improve plant P nutrition by mobilizing inorganic and organic P. Isolates from different cross-inoculation groups of rhizobia, obtained from Iranian soils were tested for their ability to dissolve inorganic and organic phosphate. From a total of 446 rhizobial isolates tested for P solubilization by the formation of visible dissolution halos on agar plates, 198 (44%) and 341(76%) of the isolates, solubilized Ca₃(PO₄)₂ (TCP) and inositol hexaphosphate (IHP), respectively. In the liquid Sperber TCP medium, phosphate-solubilizing bacteria (Bacillus sp. and Pseudomonas fluorescens) used as positive controls released an average of 268.6 mg L⁻¹ of P after 360 h incubation. This amount was significantly (P < 0.05) higher than those observed with all rhizobia tested. The group of Rhizobium leguminosarum bv. viciae mobilized in liquid TCP Sperber medium significantly (P < 0.05) more P (197.1 mg L⁻¹ in 360 h) than other rhizobia tested,. This group also showed the highest dissolution halo on the TCP solid Sperber medium. The release of soluble P was significantly correlated with a drop in the pH of the culture filtrates indicating the importance of acid production in the mobilization process. None of the 70 bradyrhizobial isolates tested was able to solubilize TCP. These results indicate that many rhizobia isolated from soils in Iran are able to mobilize P from organic and inorganic sources and this beneficial effect should be tested with crops grown in Iran.     

Methods for enhancing symbiotic nitrogen fixation

Biological nitrogen fixation of leguminous crops is becoming increasingly important in attempts to develop sustainable agricultural production. However, these crops are quite variable in their effectiveness in fixing nitrogen. By the use of the ¹⁵N isotope dilution method some species have been found to fix large proportions of their nitrogen, while others like common bean have been considered rather inefficient. Methods for increasing N₂ fixation are therefore of great importance in any legume work. Attempts to enhance nitrogen fixation of grain legumes has been mainly the domain of microbiologists who have selected rhizobial strains with superior effectiveness or competitive ability. Few projects have focused on the plant symbiont with the objective of improving N₂ fixation as done in the FAO/IAEA Co-ordinated Research Programme which is being reported in this volume. The objective of the present paper is to discuss some possibilities available for scientists interested in enhancing symbiotic nitrogen fixation in grain legumes. Examples will be presented on work performed using agronomic methods, as well as work on the plant and microbial symbionts. There are several methods available to scientists working on enhancement of N₂ fixation. No one approach is better than the others; rather work on the legume/Rhizobium symbiosis combining experience from various disciplines in inter-disciplinary research programmes should be pursued.     

Cyanide assimilation in Rhizobium ORS 571: influence of the nitrogenase catalyzed hydrogen production on the efficiency of growth

When cyanide is gradually added to a nitrogenfixing culture, Rhizobium ORS 571 is capable of assimilating large amounts of cyanide using its nitrogenase. Under these conditions the molar growth yield on succinate (Y _succ) increases from 27 at the start of cyanide addition to 38 at the end. The respiratory chain of cells grown at a concentration of 7 mM cyanide is still very sensitive to cyanide. The increase in growth yield is explained by a decrease in hydrogen production by nitrogenase as soon as cyanide is assimilated. This is confirmed by calculating the influence of hydrogen production on Y _succ. Hydrogen production by nitrogenase has a greater influence on growth yields than the presence or absence of hydrogenase activity. At the end of cyanide addition when all cell nitrogen is synthesized from cyanide and no nitrogen fixation occurs, nitrogenase will be in a very oxidized state.     

Calcium requirement in nitrogen fixation in the cyanobacterium Synechococcus RF-1

Under diurnal 16/8-h light-dark cycles, ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) at 1 mM completely blocked the appearance of rhythmic N₂-fixing activity in Synechococcus RF-1. Ca²⁺ at 2 mM, when supplied either together with or several hours after the EGTA application, restored the nitrogenase activity, whereas, when Ca²⁺ was supplied several hours later, the peak of nitrogenase activity was shifted from the dark to the light period in which the activity is normally suppressed. Sr²⁺ also reversed the inhibition by EGTA, but only partially. When O₂ in the gas phase above the culture was below 1%, the inhibition of nitrogenase activity by EGTA was reduced to less than 20% of the control value without EGTA. Thus Ca²⁺ appears to be required by the cell to protect its nitrogenase from inactivation by O₂. In media without EGTA, a close correlation between nitrogenase activity and concentrations of Ca²⁺ was also observed.Abbreviation EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid     

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.