Symbiotic Nitrogen Fixation Ability Research Articles

Independent Component Analysis Reveals the Transcriptional Regulatory Modules in Bradyrhizobium diazoefficiens USDA110.

The dynamic adaptation of bacteria to environmental changes is achieved through the coordinated expression of many genes, which constitutes a transcriptional regulatory network (TRN). Bradyrhizobium diazoefficiens USDA110 is an important model strain for the study of symbiotic nitrogen fixation (SNF), and its SNF ability largely depends on the TRN. In this study, independent component analysis was applied to 226 high-quality gene expression profiles of B. diazoefficiens USDA110 microarray datasets, from which 64 iModulons were identified. Using these iModulons and their condition-specific activity levels, we (1) provided new insights into the connection between the FixLJ-FixK2-FixK1 regulatory cascade and quorum sensing, (2) discovered the independence of the FixLJ-FixK2-FixK1 and NifA/RpoN regulatory cascades in response to oxygen, (3) identified the FixLJ-FixK2 cascade as a mediator connecting the FixK2-2 iModulon and the Phenylalanine iModulon, (4) described the differential activation of iModulons in B. diazoefficiens USDA110 under different environmental conditions, and (5) proposed a notion of active-TRN based on the changes in iModulon activity to better illustrate the relationship between gene regulation and environmental condition. In sum, this research offered an iModulon-based TRN for B. diazoefficiens USDA110, which formed a foundation for comprehensively understanding the intricate transcriptional regulation during SNF.

A Lipopolysaccharide Synthesis Gene rfaD from Mesorhizobium huakuii Is Involved in Nodule Development and Symbiotic Nitrogen Fixation.

Rhizobium lipopolysaccharide (LPS) is an important component of the cell wall of gram-negative bacteria and serves as a signal molecule on the surface of rhizobia, participating in the symbiosis during rhizobia-legume interaction. In this study, we constructed a deletion mutant of ADP-L-glycerol-D-mannoheptosyl-6-exoisomerase (rfaD) of Mesorhizobium huakuii 7653R and a functional complementary strain. The results showed that the deletion of rfaD did not affect the free-living growth rate of 7653R, but that it did affect the LPS synthesis and that it increased sensitivity to abiotic stresses. The rfaD promoter-GUS reporter assay showed that the gene was mainly expressed in the infection zone of the mature nodules. The root nodules formation of the rfaD mutant was delayed during symbiosis with the host plant of Astragalus sinicus. The symbiotic phenotype analyses showed that the nodules of A. sinicus lost symbiotic nitrogen fixation ability, when inoculated with the rfaD mutant strain. In conclusion, our results reveal that the 7653R rfaD gene plays a crucial role in the LPS synthesis involved in the symbiotic interaction between rhizobia and A. sinicus. This study also provides new insights into the molecular mechanisms by which the rhizobia regulate their own gene expression and cell wall components enabling nodulation in legumes.

ECOLOGICAL POTENTIAL OF GRAIN PEGULUM CROPS IN MODERN INTENSIVE CROP ROTATIONS

The article discusses the ecological significance of leguminous crops grown in modern intensive crop rotation. In particular, the area under crops of common leguminous crops in Ukraine and the level of their productivity have been analyzed. A comparison is made with the acreage of the most widespread grain crops. The emphasis of the article is aimed at establishing the level of nitrogen fixation of leguminous crops, which have the largest sown areas in Ukraine. The volume of accumulation by these leguminous crops of by-products in the form of their straw and stubble is also calculated. A comparison is made according to these indicators with the most widespread grain crops grown in Ukraine. The data on the content of the main nutrients in the by-products of leguminous crops - nitrogen, phosphorus, potassium are given. On the basis of these indicators, a calculation was made of the accumulation of the main nutrients in the soil, which can come with the by-products of leguminous crops with an average yield of their seeds. We also compared the obtained indicators with the input of nitrogen, phosphorus and potassium into the soil with by-products of the most common grain crops. Based on this, a conclusion was made about the most effective leguminous crops, the cultivation of which in the modern intensive crop rotation contributes most to the stabilization of the agro-ecological state of the soil. According to the State Statistics Service in Ukraine in 2019, the largest sown area among leguminous crops belonged to peas - 347.0 thousand hectares, which is 61.3% in the structure of all leguminous crops. In total, the sown area for leguminous crops in Ukraine is 566.0 thousand hectares, which is about 2% of the total sown area and this is a very low indicator. Considering the average yield in Ukraine, beans can return more by-products to the soil - 3.5 t/ha, soybeans and peas - by 8.6% less, beans - by 37.1%, and least of all - chickpeas and lentils - 1.7 - 1.8 t/ha. The content of the main macronutrients in the by-products of all leguminous crops is similar and is: nitrogen - 10.0-12.0 kg/t, phosphorus - 3.4-3.6 kg/t, potassium - 4.6-5.0 kg/t. It has been proven that an increase in the area of leguminous crops in an intensive crop rotation will have a positive effect on the agro-ecological state of the soil. In particular, growing beans allows you to get the highest mass of by-products that can be ploughed into the soil - 3.5 t/ha. Also, by-products of beans are characterized by a high content of mineral phosphorus - 3.6 kg/t, which ensures the supply of all mineral phosphorus to the soil - 12.6 kg/ha of all leguminous crops, as well as potassium - 16.5 kg/ha. Soybean by-products are characterized by a high nitrogen content - 12.0 kg/t, phosphorus - 3.6 kg/t and potassium - 5.0 kg/t. This allows, after growing soybeans, to accumulate in the soil with by-products more mineral nitrogen - 38.4 kg/ha. Also, soybeans are characterized by a high symbiotic nitrogen-fixing ability among all leguminous crops - 120 kg/ha. By-products of leguminous crops have a high content of nitrogen - 2.3-2.7 times, phosphorus - 1.5-1.6 times compared to by-products of grain crops. Also, when plowing soybean by-products into the soil, there will be 2 times more mineral nitrogen and 1.1-1.3 times more phosphorus than when plowing winter wheat by-products. Key words: egumes, by-products, nitrogen fixation, nutrients, accumulation, soil.

EFFECT OF GOAT’S-RUE RHIZOBIA ON THE FORMATION AND FUNCTIONING OF THE SOYBEAN – BRADYRHIZOBIUM JAPONICUM 634B SYMBIOSIS

Objective. Study the peculiarities of nodule formation upon the formation of the symbiotic sys-tem soybean-Bradyrhizobium japonicum 634b, as well as the symbiotic nitrogen-fixation ability and plant growth and development under the influence of goat’s-rue rhizobia. Methods. Microbiologi-cal, physiological, statistical, gas chromatography. Results. In green house experiments, using sand as a substrate for growing plants, the mixed microbial cultures combining soybean nodule bacteria B. japonicum 634b and goat’s-rue nodule bacteria R. galegae 0702 or R. galegae 0703 in the ratio of 1 : 1 differed from the monoculture bradyrhizobium by their influence on the nodulation, nitro-gen-fixation ability of soybean-rhizobial symbiosis and development of soybean plants (variety Almaz). Increased nodulation activity in the primordial leaf and budding phases, as well as a signif-icant decrease in the level of symbiosis nitrogen fixation during budding, were observed when used in binary bacterial compositions of strain R. galegae 0703. These rhizobia of goat’s-rue suppressed the development of the root system of soybeans, but had no significant effect on the formation of the aerial part of the plants throughout the observation period. R. galegae 0702 strain slightly slowed the formation of nodules by bacteria in the primordial leaf phase, which caused a decrease in the number of soybean plants that formed symbiosis with B. japonicum 634b. Goat’s-rue nodule bacte-ria R. galegae 0702 improved the formation of the root system, and stimulated the growth and de-velopment of the aerial part of the macro symbiont in the phase of two trigeminal leaves. Conclu-sion. Combined inoculation of the rhizobia of goat’s-rue with nodule bacteria B. japonicum 634b showed a multidirectional effect on the formation of symbiosis by soybean plants of variety Almaz and functioning of soybean rhizobial symbiosis. The nature of the influence of R. galegae depended on their strain affiliation.

Methods for Isolation and Characterization of Nitrogen-Fixing Legume-Nodulating Bacteria.

Symbiotic nitrogen fixation (SNF) is a characteristic feature of nodulating legumes. The wild legumes are comparatively less explored for their SNF ability; hence, it is essential to study nodulation and identify the microsymbiont diversity associated with them. This chapter aims to describe the methodology for nodule hunting; trapping, isolation, and characterization of root nodule bacteria (RNB) at phenotypic, genotypic, and symbiotic levels. The documentation of nodulating native legume species and the rhizobial diversity associated with them in various parts of world has gained attention as this symbiotic association provides fixed nitrogen, improves productivity of plants in an ecofriendly manner. Before field-based applications the symbiotic bacteria need to be assessed for their N fixing ability as well as characterized at molecular level. The phylogeny based on symbiosis-essential genes supplemented with the host-range studies helps in better understanding of the symbiotaxonomy of rhizobia. More efficient symbiotic couples need to be screened by cross-nodulation studies for their application in agricultural practices.

Genotypic differences in symbiotic nitrogen fixation ability and seed yield of climbing bean

AimsSymbiotic nitrogen fixation (SNF) contributes to improve grain yield under nitrogen (N) deficiency. Climbing beans are known to be superior to bush beans in their potential for SNF. The main objectives of this study were to: (i) quantify genotypic differences in SNF ability of climbing beans using 15N natural abundance method; (ii) identify climbing bean genotypes that combine high SNF ability with high yield potential that could serve as parents in the breeding program; and (iii) test whether δ15N in seed can be used instead of δ15N in shoot for estimating SNF ability.Methods98 Climbing bean genotypes were evaluated for SNF ability in terms of nitrogen derived from the atmosphere (%Ndfa). Field trials were conducted at two locations in Colombia.ResultsSignificant genotypic differences were observed in SNF ability. Good yielding lines with 4.6 t ha−1 fixed as much as 60% of their N (up to 92 kg of N fixed ha−1) without application of N fertilizer to soil.ConclusionsBased on evaluations from both locations, seven climbing bean lines (ENF 235, ENF 234, ENF 28, ENF 21, MAC 27, CGA 10 and PO07AT49) were identified as promising genotypes. Seed samples can be used to determine SNF ability, to select for genotypes with superior SNF ability.

Root traits and their potential links to plant ideotypes to improve drought resistance in common bean

Drought stress limits growth and yield of crops, particularly under smallholder production systems with minimal use of inputs and edaphic limitations such as nitrogen (N) deficiency. The development of genotypes adapted to these conditions through genetic improvement is an important strategy to address this limitation. The identification of morpho-physiological traits associated with drought resistance contributes to increasing the efficiency of breeding programs. A set of 36 bean genotypes belonging to the Middle American gene pool was evaluated. A greenhouse study using soil cylinders was conducted to determine root vigor traits (total root length and fine root production) under drought stress. Two field trials were conducted to determinate grain yield, symbiotic nitrogen fixation (SNF) ability and other shoot traits under drought stress. Field data on grain yield and other shoot traits measured under drought were related with the greenhouse data on root traits under drought conditions to test the relationships between shoot traits and root traits. Response of root vigor to drought stress appeared to be related with ideotypes of water use (water savers and water spenders). The water spender ideotypes presented deeper root system, while the water saver ideotypes showed a relatively shallower root system. Increase in SNF ability under drought stress was associated with greater values of mean root diameter while greater acquisition of N from soil was associated with finer root system. We identified seven common bean lines (SEA 15, NCB 280, SCR 16, SMC 141, BFS 29, BFS 67 and SER 119) that showed greater root vigor under drought stress in the greenhouse and higher values of grain yield under drought stress in the field. These lines could serve as parents for improving drought resistance in common bean.

Phenotypic evaluation and QTL analysis of yield and symbiotic nitrogen fixation in a common bean population grown with two levels of phosphorus supply

Common bean is an important staple crop in Eastern Africa and Latin America. Low soil fertility is a major limitation to agronomic productivity. Symbiotic nitrogen fixation (SNF) is an important property of legumes, leading to high protein levels and high nutritional value. Nitrogen (N) metabolism and yield traits were evaluated in the common bean population DOR 364 × BAT 477 in field experiments under moderate and low phosphorus (P) soil conditions resembling environments found on farmers’ fields. Low P availability in soil severely limits seed yield, and trait correlations with yield reveal that high biomass as well as early maturity and efficient seed filling are important for good performance in low P stress, resembling drought resistance. Investigation of SNF and soil N uptake under low P stress showed reduced seed nitrogen levels and major variation in soil-derived N. In low P conditions, no significant reduction of %N derived from the atmosphere (%Ndfa) was observed; however, %Ndfa was correlated with yield, indicating that under stress SNF becomes an important asset. Significant genetic variation was observed for yield, yield components, and SNF ability suggesting that traits can be improved by breeding. Quantitative trait loci (QTLs) for %Ndfa and seed N concentration were discovered on chromosomes Pv07 and Pv02; independent yield QTLs were identified on the same chromosomes. Two QTL hotspots that affect several traits including yield components were found on Pv02 and Pv06; the latter represents a constitutive QTL hotspot independent from the environment. QTLs may be used for marker design and molecular breeding.

Estimation of phenotypic variability in symbiotic nitrogen fixation ability of common bean under drought stress using 15N natural abundance in grain.

Common bean (Phaseolus vulgaris L.) is the most important food legume, cultivated by small farmers and is usually exposed to unfavorable conditions with minimum use of inputs. Drought and low soil fertility, especially phosphorus and nitrogen (N) deficiencies, are major limitations to bean yield in smallholder systems. Beans can derive part of their required N from the atmosphere through symbiotic nitrogen fixation (SNF). Drought stress severely limits SNF ability of plants. The main objectives of this study were to: (i) test and validate the use of 15N natural abundance in grain to quantify phenotypic differences in SNF ability for its implementation in breeding programs of common bean with bush growth habit aiming to improve SNF, and (ii) quantify phenotypic differences in SNF under drought to identify superior genotypes that could serve as parents. Field studies were conducted at CIAT-Palmira, Colombia using a set of 36 bean genotypes belonging to the Middle American gene pool for evaluation in two seasons with two levels of water supply (irrigated and drought stress). We used 15N natural abundance method to compare SNF ability estimated from shoot tissue sampled at mid-pod filling growth stage vs. grain tissue sampled at harvest. Our results showed positive and significant correlation between nitrogen derived from the atmosphere (%Ndfa) estimated using shoot tissue at mid-pod filling and %Ndfa estimated using grain tissue at harvest. Both methods showed phenotypic variability in SNF ability under both drought and irrigated conditions and a significant reduction in SNF ability was observed under drought stress. We suggest that the method of estimating Ndfa using grain tissue (Ndfa-G) could be applied in bean breeding programs to improve SNF ability. Using this method of Ndfa-G, we identified four bean lines (RCB 593, SEA 15, NCB 226 and BFS 29) that combine greater SNF ability with greater grain yield under drought stress and these could serve as potential parents to further improve SNF ability of common bean.

Evaluation of symbiotic nitrogen fixing ability of legume crops in Central Himalaya, India

Symbiotic biological nitrogen fixation is the major source of nitrogen input in traditional agriculture and plays a significant role in improving soil fertility and productivity, though it has hardly been estimated in Himalayan region. In the present study, nitrogenase activity of selected legumes was estimated in pure-legume and legume-Eleusine coracana mixed cropping systems to assess the potential of legumes in maintaining soil fertility in the mountain region of Central Himalaya. Assessment of nitrogenase activity and effect of legume crop cultivation on soil chemical properties reveals that legume crops possess great potential for nitrogen fixation and soil fertility improvement for sustainable rainfed agriculture system.

Effect of a Nitrogen-Fixing Actinorhizal Shrub on Herbaceous Vegetation in a Mixed Conifer Forest of Central Himalaya

In this study, we examined the effect of a nitrogen-fixing shrub Coriaria nepalensis Wall on herb species composition, diversity and biomass. The effect was measured in terms of species richness, diversity and biomass of herb species in three sites varying in Coriaria density viz. site 1 (low Coriaria density; 20 ha-1), site-2 (medium Coriaria density; 120 ha-1) and site-3 (high Coriaria density 190 ha-1). Species richness was minimum at Site-1 (16 species), and maximum at site-2 (27 species). G. aparine dominated site-1 and Arthraxon sp dominated site-2 and 3. The individual herb density ranged between 0.40 - 42.40 m-2, and total herb density ranged between 138- 170.4 m-2 and was maximum at site-2. Value for species richness (27) and Shannon Index (3.72) was highest for medium Coriaria density site and lowest for low Coriaria density site. Simpson Index ranged between 0.11 and 0.14 and was lowest for site-2(medium Coriaria density) indicating that at this the dominance was shared by many species. Along the gradient of Coriaria density, maximum biomass was recorded at site-3 with highest Coriaria density and lowest at site-2 with medium Coriaria density. This may be due to the symbiotic nitrogen fixing ability of Coriaria that improve the habitat quality. The facilitative effect of C. nepalensis in terms of soil amelioration and herb growth can be used to regenerate degraded forest ecosystems.

N2 fixation ability of different dry bean genotypes

Farid, M. and Navabi, A. 2015. N2 fixation ability of different dry bean genotypes. Can. J. Plant Sci. 95: 1243–1257. Common bean (Phaseolous vulgaris L.) is generally known as a weak N2 fixer compared with other legumes. The availability of genetic variation for N2 fixation potential of common bean supports the idea that symbiotic nitrogen fixation (SNF) in common bean can be improved through breeding. To assess the potential N2 fixation of selected common bean genotypes, 12 bean varieties including three Andean and nine Middle American were screened for SNF ability and related traits in controlled environments and field trials in Ontario, Canada. A non-nodulating mutant, R99, was used as the reference plant to estimate nitrogen derived from the atmosphere (Ndfa) through the natural 15N abundance method. Significant variation was found among the bean genotypes for Ndfa and its related traits. Environmental and genotype by environmental effects significantly influenced Ndfa and its related traits. The three Andean bean genotypes were superior to Middle American genotypes for nodulation ability, while the Middle American genotypes were generally stronger nitrogen fixers in optimum soil moisture conditions. In general, nitrogen fixation was found to be significantly associated with seed yield and carbon isotope discrimination, an indicator of water use efficiency.

Association of Nodule Performance Traits with Shoot Performance Traits of Common Bean Under Drought Stress

Although symbiotic nitrogen fixation (SNF) and nodule characteristics have been evaluated before, their use as phenotypic markers for drought tolerance in beans (Phaseolus vulgaris L.) and their association with aboveground traits under water deficiency have not been quantified widely. Therefore, the effect of drought stress on the association of nodule performance traits with shoot performance traits was investigated using six common bean lines with contrasting differences in their adaptation to drought and low phosphorus (P) availability in soil. Drought stress reduced leaf water potential and gas-exchange characteristics (CO2 assimilation, stomatal conductance) in all lines tested, but two drought-tolerant lines, BAT 477 and BT_34-1,-1, were superior in their ability to adjust their leaf water status under drought. These two lines were also superior in root growth, rate of CO2 assimilation, vegetative biomass production, and nitrogen-fixing ability under drought stress. A direct relation of SNF and nodule fresh weight with gas-exchange traits and biomass production existed, suggesting that relative growth of shoot vs. root depends on the provision of nitrogen by SNF and photosynthetic carbon assimilation. Overall, differences found in nodule, root, and shoot performance traits indicated that the P-efficient bean line (BT_34-1-1) was superior to the other lines in adaptation to drought. Marked association of nodule performance traits with shoot performance traits under drought stress was detected. We suggest that nodule characteristics and SNF ability should be included with above- and below-ground traits as phenotypic markers in germplasm evaluation and breeding programs aimed at improving drought tolerance in common bean.

Assessment of nifH diversity in rhizobial isolates of different origin and the role of antioxidant in respiratory protection

Rhizobia diversity is considered as one of the most useful resources for bioprospecting due to their symbiotic nitrogen-fixing ability with members of Leguminosae. The highly conserved nature of the nitrogenase reductase gene (nifH) makes it an ideal molecular tool to determine the potential for biological nitrogen fixation in any environment. In the present investigation, 250 rhizobial strains were isolated from legumes belonging to different geographical locations of Chhattisgarh, India. Genetic diversity of the nitrogenfixing bacterial community was analyzed using the nifH gene-specific primer. The polymorphism was found among the nitrogen-fixing population of different sources and origin but not in same source of rhizobia. Further, the symbiotic plasmid DNA was characterized on the basis of size and copy number of plasmids. The plasmid number varying from one to three in different rhizobial isolates had a size greater than 23 kb, while in some rhizobial isolates plasmids were absent. In addition, to examine the role of ascorbate in respiratory protection, the clear black spot margin of ascorbate was observed in the endodermis region of the nodule whereas scarcely dispersed in the infected region. Therefore, our findings demonstrated that knowing the rhizobial nifH gene diversity along with copy number of the plasmid is important for strain identification, deciding its fertility, productivity standards, and potential of biological nitrogen fixation across the geographical region.

Identification of a Novel Gene for Biosynthesis of a Bacteroid-Specific Electron Carrier Menaquinone

Ubiquinone (UQ) has been considered as an electron mediator in electron transfer that generates ATP in Rhizobium under both free-living and symbiosis conditions. When mutated, the dmtH gene has a symbiotic phenotype of forming ineffective nodules on Astragalus sinicus. The gene was isolated from a Mesorhizobium huakuii 7653R transposon-inserted mutant library. The DNA sequence and conserved protein domain analyses revealed that dmtH encodes demethylmenaquinone (DMK) methyltransferase, which catalyzes the terminal step of menaquinone (MK) biosynthesis. Comparative analysis indicated that dmtH homologs were present in only a few Rhizobia. Real-time quantitative PCR showed dmtH is a bacteroid-specific gene. The highest expression was seen at 25 days after inoculation of strain 7653R. Gene disruption and complementation tests demonstrated that the dmtH gene was essential for bacteroid development and symbiotic nitrogen fixation ability. MK and UQ were extracted from the wild type strain 7653R and mutant strain HK116. MK-7 was accumulated under microaerobic condition and UQ-10 was accumulated under aerobic condition in M. huakuii 7653R. The predicted function of DmtH protein was confirmed by the measurement of methyltransferase activity in vitro. These results revealed that MK-7 was used as an electron carrier instead of UQ in M. huakuii 7653R bacteroids.

Generation of Bradyrhizobium japonicum Mutants with Increased N 2 O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N(2)O) reductase (N(2)OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N(2)O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction into B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N(2)O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N(2)OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N(2)OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.

Selection of chickpea (Cicer arietinum) for yield and symbiotic nitrogen fixation ability under salt stress

L'objectif de cette recherche est de selectionner des genotypes de pois chiche pour la capacite de fixation symbiotique de l'azote (SNF) sous des contraintes salines. Deux cents lignees marocaines ont ete evaluees pour l'activite de la nitrogenase, la matiere seche, la masse des nodules, la couleur de la plante et l'azote total sous 0 mM, 25 mM et 50 mM NaCl en serre sur du sable depourvu d'azote. L'inoculum comprend un melange de 4 souches locales de Rhizobium tolerantes a la salinite. Sept lignees tolerantes a la salinite pour la SNF ont ete selectionnees. Les lignees selectionnees ont ete evaluees en hiver 1998 sur un sol salin et un autre non salin en station experimentale. Les resultats ont confirme la superiorite des lignees tolerantes au sel par rapport aux temoins pour le rendement et les caracteres associes a SNF en sol salin. Le rendement a ete reduit de 26 a 38 % en sol salin selon les genotypes. Sur la base des donnees des deux sols, les lignees MCA103, MCA131 et MCA250 ont ete selectionnees pour leur tolerance au stress salin pour la fixation symbiotique et le rendement. En 1999 ces 3 lignees ont ete evaluees pour la production a la ferme dans 2 regions. Les resultats ont montre leur superiorite par rapport aux temoins Douyet et Rizky. MCA250 est la lignee la plus performante dans les 2 sites.

Variabilité génotypique de l'aptitude à la fixation symbiotique de l'azote chezAcacia cyanophyllaLindl.

Genotypic variability of the symbiotic nitrogen fixation ability on Acacia cyanophylla Lindl. The phenotypic and functional characteristics of four rhizobium strains isolated from nodules of Acacia cyanophylla Lindl., syn. Acacia saligna (Labill.) H. Wendl., and the inoculated host-plant genetic variability were evaluated. The evaluation used permitted the ranking of the tested rhizobium strains and seedlots according to their performance. The growth pattern of these strains indicated that A. cyanophylla was able to nodulate and fix N2with slow- and fast-growing rhizobium strains. The acetylene reduction activity test reflected a variable effectiveness of the strains depending on their site of isolation. Strains RFH383 and RFH483 were more effective than RFH183 and RFH283. The high antibiotic resistance acquisition by RFH183 and RFH283 strains seemed to be inversely related to their effectiveness. Acacia cyanophylla seedlings from five seedlots displayed a high variability regarding their growth, nodulation, and ability to fix nitrogen. The ability to fix nitrogen was greater in the seedlings from KL seedlot than those from EN, KR, and TZ seedlots. The qualitative assessment of the seedlots using correspondence factorial analysis showed that the seedlings from KL seedlot preformed better than those from KR and TZ. The seedlings from TN and EN seedlots displayed an intermediate behaviour pattern. Seed origin seemed to affect the growth, nodulation, and nitrogen fixation of inoculated A. cyanophylla. The behaviour of this species and that of its associated rhizobium strains appeared to be very sensitive to variations in site characteristics. Results suggested that the association between plants derived from KL seedlot and RFH383 strain may represent the best partnership allowing the improvement of symbiotic nitrogen fixation.Key words: Acacia cyanophylla, rhizobium, nodulation, N2fixation, genotypic variability, correspondence factorial analysis.

Duplication of DNA regions carrying repetitive sequence RS? in Bradyrhizobium japonicum 110

L. D. Kuykendall, M. E. Barnett and J. N. Mathis. 1997. RSα is a repeated DNA sequence found within the nitrogen-fixation gene cluster of Bradyrhizobium japonicum, a symbiotic nitrogen-fixing bacterium that nodulates soybean. Bradyrhizobium japonicum strain 110spc4 contains 12 repeats, each located on a separate XhoI DNA restriction fragment between 1.2 and 14 kb in length. Although Fix+ and Fix− derivatives of B. japonicum USDA 110 were first reported more than two decades ago, genotypic differentiation, on the basis of RSα hybridization pattern, was reported only recently. Bradyrhizobium japonicum strain USDA 110 had only single copies of the RSα-hybridizing bands, but a particular Fix− derivative, MSDJGl, carried doublets of two distinct XhoI fragments that carry RSα3 and RSα4. In this study, RSα hybridization patterns were analysed further in both Fix+ and Fix− derivatives of strain 110 to test for duplication of these particular genomic regions. It was concluded that the duplication, or not, of genetic regions carrying RSα3 and RSα4 in strain USDA 110 derivatives is unrelated to symbiotic nitrogen-fixation ability. Like Fix− MSDJGl, Fix+ strain 110 derivatives I-110 and MN-110 had duplications of the XhoI DNA restriction fragments carrying RSα3 and RSα4, but Fix− strain 110 derivative L2–110 lacked these duplications. Thus, it is now clear that Fix− derivatives MSDJG1 and L2–110 arose via distinct genetic mechanisms. Interestingly, Fix+ derivatives of strain 110 from the laboratories of Elkan and Hennecke differed in RSα hybridization profile.

Analysis of the Symbiotic Performance of Bradyrhizobium japonicum USDA 110 and Its Derivative I-110 and Discovery of a New Mannitol-Utilizing, Nitrogen-Fixing USDA 110 Derivative

Previously, Bradyrhizobium japonicum USDA 110 was shown to contain colony morphology variants which differed in nitrogen-fixing ability. Mannitol-utilizing derivatives L1-110 and L2-110 have been shown to be devoid of symbiotic nitrogen fixation ability, and non-mannitol-utilizing derivatives I-110 and S-110 have been shown to be efficient at nitrogen fixation. The objectives of this study were to determine the effect of media carbon sources on the symbiotic N(2)-fixing ability of strain USDA 110 and to compare the effectiveness of strain USDA 110 and derivative I-110. Based on acetylene reduction activity and the nitrogen content of 41-day-old soybean plants, neither derivative I-110 nor cultures of USDA 110 grown in media favoring non-mannitol-using derivatives had symbiotic nitrogen fixation that was statistically superior to that of cultures of USDA 110 grown in media favoring mannitol-using derivatives. In another experiment 200 individual nodules formed by strain USDA 110 grown in yeast extract gluconate were screened for colony morphology of occupying variant(s) and acetylene reduction activity. Nodules occupied by mannitol-using derivatives (large colony type on 0.1% yeast extract-0.05% K(2)HPO(4)-0.08% MgSO(4) . 7H(2)O-0.02% NaCl-0.001% FeCl(3) . 6H(2)O [pH 6.7] with 1% mannitol [YEM] plates) had a mean acetylene reduction activity equal to that of nodules occupied by non-mannitol-using derivatives (small colony type on YEM plates). A total of 20 large colonial derivatives and 10 small colonial derivatives (I-110-like) were isolated and purified by repeated culture in YEM and YEG (same as YEM except 1% gluconate instead of 1% mannitol) media, respectively, followed by dilution in solutions containing 0.05% Tween 40. After 25 days of growth, soybean plants inoculated with the large colony isolates had mean whole-plant acetylene reduction activity, whole-plant dry weight, and whole-plant nitrogen contents equal to or better than those of plants inoculated with either the small colony isolates (I-110-like) or the I-110 (non-mannitol-using) derivative. Hence, the existence of a mannitol-utilizing derivative that fixes nitrogen in a culture of strain USDA 110 obtained from the U.S. Department of Agriculture, Beltsville, Md., was established. This new USDA 110 derivative was designated as MN-110 because it was a mannitol-utilizing nitrogen-fixing USDA 110 derivative. This derivative was morphologically indistinguishable from the non-nitrogen-fixing derivative L2-110 found in cultures obtained earlier from the U.S. Department of Agriculture, Beltsville. DNA-DNA homology and restriction enzyme analyses indicated that MN-110 is genetically related to other USDA 110 derivatives that have been characterized previously.