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The importance of atmospheric nitrogen-fixing bacteria The study aimed to isolate bacteria from the roots of plants From four areas of the city of Mosul the winter agriculture, season 2020-2021 and using biochemical tests and phenotypic and agricultural characteristics to diagnose bacterial isolates and study sensitivity and resistance of bacterial isolates to ten antibiotics. if the results showed that the highest percentage of resistance was to the two antibiotics, Trimethoprim, and streptomycin, at 100%, while The least resistance was to the two antivirals Gentamycin and Rifampicin by 20%. In addition to Studying The sensitivity and resistance of bacterial isolates to four heavy Metals, namely Mercury chloride (HgCl), Cobalt chloride (CoCl), Cadmium Chloride (CdCl), and nickel chloride (NiCl). The resistance was high with Cadmium chloride salt (CdCl) and the lowest salt Mercury Chloride (HgCl). PCR techniques were used to diagnose samples based on 16S rRNA sequence analysis and comparison of sequences resulting from DNA amplification with standard isolates within NCBI to detect new bacterial isolates

We have investigated the effectiveness of a new soybean fertilization technique, named crack fertilization, which involves the application of nodule bacteria on biochar to soybean roots through cracks formed between planting rows during midterm tillage. In the present study, the factors and timing of crack fertilization were investigated in an upland field converted from a paddy. The variables investigated were: 1) crack formation without the application of any agricultural materials, and 2) the application of biochar or 3) nodule bacteria on biochar into cracks. The treatment periods were: 1) before sowing, 2) during midterm tillage, and 3) during both periods. The combination of crack fertilization and reduced tillage was also tested. The method of crack fertilization that increased yield was the combination of crack formation and the application of biochar, and the most effective period for the treatment was before sowing in the reduced tillage field. Seed yields in conventional and reduced tillage fields were comparable in the upland field converted from the paddy. These results suggest that the application of biochar into cracks after scratching the soil surface to remove weeds before sowing is a practical method of increasing soybean yield in upland fields converted from paddies.

Vigna riukiuensis plant – a rare type of vigna, found only in Taiwan and the islands of Okinawa prefecture, Japan – possesses intrinsic property of high level of salt and heat tolerance. To underst...

For the first time, bacteria were isolated and identified from the root nodules of a wild-growing medicinal legume plant Thermopsis lanceolata , originated from Mongolia. The taxonomic position of 14 isolates obtained was determined using of sequencing of the 16S rRNA ( rrs ) and atpD genes. It was shown a significant biodiversity of the isolates from T. lanceolata , which belonged to three genera of the order Rhizobiales : Phyllobacterium (family Phyllobacteriaceae ), Rhizobium (family Rhizobiaceae ) and Bosea (family Bradyrhizobiaceae ). Six isolates belonged to the species Phyllobacterium zundukense and Phyllobacterium trifolii (100 и 99,9% rrs similarity with the type strains P. zundukense Tri-48 T and P. trifolii PETP02 T , respectivelly), three isolates were identified as Rhizobium anhuiense (99,8% rrs similarity with the type strain R. anhuiense CCBAU 23252 T ). Two slow-growing isolates of the genus Bosea Tla-534 and Tla-545 may potentially belong to new species, since their rrs -similarity to the closest type strains B. massiliensis LMG 26221 T , B. lathyri LMG 26379 T and B. vaviloviae Vaf18T was 98,5-99,0%. Non-rhizobial strains were not isolated. The isolation and future investigation of the rhizobial microsymbionts of the valuable medicinal legume Thermopsis lanceolata is one of the necessary prerequisites for its industrial cultivation.

The symbiotic system of Legumes and root-nodule bacteria is a significant process for enriching barren and dry soils and supporting plant growth. The aim of this study was to isolate root-nodule bacteria from Medicago falcate and to select high activity strains with resistance to environmental stress. As a result, only MSZ-25 strain grew in the medium containing 0.8M NaCl, with pH4.8-9.8 and at temperatures of 4-45°С. All rhizobial isolates were tolerant to the concentrations of 0.01-0.1% Pb(NO3)2, 0.01% ZnSO4, CdCI2, CuCI2 and NiCI2. The strains were also resisted to penicillin 10μ/ml, erythromycin 10μ/ml, and chlorampenicol 10μ/ml but the growths of all isolates were inhibited by kanamycin 30μ/ml, novobiocin 30μ/ml and tetracycline 10μ/ml.

Strigolactones (SLs), a group of plant secondary metabolites, play an important role as a host recognition signal for symbiotic arbuscular mycorrhizal (AM) fungi in the rhizosphere. SLs promote symbioses with other beneficial microbes, including root nodule bacteria. Root parasitic weeds also take advantage of SLs as a clue to locate living host roots. In plants, SLs function as plant hormones regulating various growth and developmental processes including shoot and root architectures. Plants under nutrient deficiencies, especially that of phosphate, promote SL production and exudation to attract symbionts and to optimize shoot and root architecture.

Waterlogging is the constraint for soybean growth and yield, because soybean is often cultivated in upland fields converted from paddy in Japan. However, efficient cultivation techniques for alleviating the adverse effects have not been developed. We have proposed the new soybean cultivation technique named crack fertilization which enables yield increase due to enhancing new root growth and N acquisition by increasing nodulation. Waterlogging induces N deficiency due to the suppression of nutrient uptake by the inhibition of root growth and nodule activity. Thus, it is hypothesized that crack fertilization would be effective to alleviate the inhibition of soybean growth and yield. The soybean cultivar of Sachiyutaka was planted in 1/5000 a Wagner pots and root boxes. Two separate waterlogging treatments were imposed to soybean plants at different growth stages, V1 and R4, and crack fertilization was done at V3. After these treatments, soybean plants were sampled at R5 in 2012 and 2013 experiments, respectively. Waterlogging at V1 and R4 inhibited the growth and yield of soybean and nodule growth, and the decreases in physiological parameters of soybean such as photosynthesis, chlorophyll content, and xylem sap exudation rate were observed. The adverse effects of waterlogging at V1 were alleviated by crack fertilization at V3, whereas crack fertilization could not alleviate the adverse effects of waterlogging at R4. Thus, crack fertilization after waterlogging at early vegetative stage would be the cultivation technique that enables to alleviate the adverse effects.

Amyloids are protein fibrils with a highly ordered spatial structure called cross-β. To date, amyloids were shown to be implicated in a wide range of biological processes, both pathogenic and functional. In bacteria, functional amyloids are involved in forming biofilms, storing toxins, overcoming the surface tension, and other functions. Rhizobiales represent an economically important group of Alphaproteobacteria, various species of which are not only capable of fixing nitrogen in the symbiosis with leguminous plants but also act as the causative agents of infectious diseases in animals and plants. Here, we implemented bioinformatic screening for potentially amyloidogenic proteins in the proteomes of more than 80 species belonging to the order Rhizobiales. Using SARP (Sequence Analysis based on the Ranking of Probabilities) and Waltz bioinformatic algorithms, we identified the biological processes, where potentially amyloidogenic proteins are overrepresented. We detected protein domains and regions associated with amyloidogenic sequences in the proteomes of various Rhizobiales species. We demonstrated that amyloidogenic regions tend to occur in the membrane or extracellular proteins, many of which are involved in pathogenesis-related processes, including adhesion, assembly of flagellum, and transport of siderophores and lipopolysaccharides, and contain domains typical of the virulence factors (hemolysin, RTX, YadA, LptD); some of them (rhizobiocins, LptD) are also related to symbiosis.

Strain CCMM B554, also known as FSM-MA, is a soil dwelling and nodule forming, nitrogen-fixing bacterium isolated from the nodules of the legume Medicago arborea L. in the Maamora Forest, Morocco. The strain forms effective nitrogen fixing nodules on species of the Medicago, Melilotus and Trigonella genera and is exceptional because it is a highly effective symbiotic partner of the two most widely used accessions, A17 and R108, of the model legume Medicago truncatula Gaertn. Based on 16S rRNA gene sequence, multilocus sequence and average nucleotide identity analyses, FSM-MA is identified as a new Ensifer meliloti strain. The genome is 6,70 Mbp and is comprised of the chromosome (3,64 Mbp) harboring 3574 predicted genes and two megaplasmids, pSymA (1,42 Mbp) and pSymB (1,64 Mbp) with respectively 1481 and 1595 predicted genes. The average GC content of the genome is 61.93%. The FSM-MA genome structure is highly similar and co-linear to other E. meliloti strains in the chromosome and the pSymB megaplasmid while, in contrast, it shows high variability in the pSymA plasmid. The large number of strain-specific sequences in pSymA as well as strain-specific genes on pSymB involved in the biosynthesis of the lipopolysaccharide and capsular polysaccharide surface polysaccharides may encode novel symbiotic functions explaining the high symbiotic performance of FSM-MA. Electronic supplementary material The online version of this article (doi: 10.1186/s40793-017-0298-3) contains supplementary material, which is available to authorized users.

Eleven extra-slow-growing strains were isolated from nodules of the relict legume Vavilovia formosa growing in North Ossetia (Caucasus) and Armenia. All isolates formed a single rrs cluster together with the type strain Tardiphaga robiniae LMG 26467(T), while the sequencing of the 16S-23S rDNA intergenic region (ITS) and housekeeping genes glnII, atpD, dnaK, gyrB, recA and rpoB divided them into three groups. North Ossetian isolates (in contrast to the Armenian ones) were clustered separately from the type strain LMG 26467(T). However, all isolates were classified as T. robiniae because the DNA-DNA relatedness between them and the type strain LMG 26467(T) was 69.6 % minimum. Two symbiosis-related genes (nodM and nodT) were amplified in all isolated Tardiphaga strains. It was shown that the nodM gene phylogeny is similar to that of ITS and housekeeping genes. The presence of the other symbiosis-related genes in described Tardiphaga strains, which is recently described genus of rhizobia, as well as their ability to form nodules on any plants are under investigation.

Soils in different parts of the world are generally being depleted of nitrogen (N), and this has now become a huge challenge to food production and security. Different sources of nutrients for enriching the soil have been evaluated in the past years especially the use of chemical fertilizers, but its usage is gradually dwindling as a result of numerous constraints, among which are environmental pollution, health challenges, and the negative impact of climate change. Better alternative strategies of replacing depleted soil N have been researched which include biological N fixation (BNF) using leguminous crops. Leguminous crops planted as cover crops, together with the symbiotic activities between root nodule bacteria and legumes, are the source of biologically fixed N. Because of the genetic diversity in legumes, there are so many underutilized leguminous crops whose potentials have not been fully tapped to understand their functionalities within the realm of BNF. This chapter brings to the limelight some of these legumes for biotechnological purpose in a bid to find a solution to soil infertility using the available cropping systems.

This study aims to evaluate the phenotypical characteristics of bacterial isolates from mulungu (Erythrina velutinaWilld.) nodules and determinate their Box-PCR fingerprinting. All bacteria were evaluated by the following phenotypic characteristics: growth rate, pH change, colony color and mucus production. The bacterial isolates able to re-nodulate the original host were also evaluated regarding its tolerance to increased salinity and different incubation temperatures, ability to growth using different carbon sources, intrinsic antibiotic resistance and “in vitro” auxin biosynthesis. The molecular fingerprints were set up using the Box-PCR technique and the isolates were clustered by its profiles. Among the 22 bacterial isolates obtained, eight were able to re-nodulate the original host. Among the nodule inducing isolates, some were tolerant to 1% of NaCl and 39° C and all of them metabolized the maltose, fructose, glucose, sucrose and arabinose, were resistant to rifampicin and produced auxin. The bacteria showed low genetic similarity among them and reference strains, which indicates the great genetic variability of the isolates. The results of this work are the first reports about the bacterial isolates able to nodulate this endemic specie. A more deep study of these bacteria may reveal the existence of isolates tolerant to environmental stresses and suitable as a future mulungu inoculant., O objetivo desse estudo foi avaliar as características fenotípicas dos isolados de nódulos de mulungu (Erythrina velutinaWilld.) e determinar seufingerprintingmolecular por meio de Box-PCR. Todas as bactérias foram avaliadas pelas seguintes características fenotípicas: tempo de crescimento, alteração de pH, cor da colónia e produção de muco. As bactérias isoladas hábeis em renodular o hospedeiro original também foram avaliadas em relação à tolerância ao aumento da salinidade e da temperatura de incubação, capacidade de crescimento utilizando diferentes fontes de carbono, resistência intrínseca aos antibióticos e biossíntese de auxina “in vitro”. Ofingerprintmolecular foi estabelecido usando a técnica Box-PCR e os isolados foram agrupados pelos perfis obtidos. Dos 22 isolados obtidos, oito foram hábeis em renodular o hospedeiro original. Entre eles, alguns foram tolerantes a 1% de NaCl e a 39° C e todos metabolizaram as fontes maltose, frutose, glicose, sacarose e arabinose, resistiram à rifampicina e produziram auxina. As bactérias isoladas mostraram baixa similaridade genética entre elas e as estirpes de referência, o que indica a grande variabilidade genética dos isolados. Os resultados deste trabalho constituem o primeiro relato sobre bactérias capazes de nodular esta espécie endémica. O estudo mais aprofundado destas bactérias poderá revelar a existência de isolados tolerantes a stresses ambientais e adequados a serem utilizados como futuros inoculantes de mulungu.

The ability of wild indigenous legumes to form root nodules capable of biological nitrogen (N 2) fixation has rarely been demonstrated for species in natural ecosystems in large parts of Europe. In ...

Background and aims Legumes of the genus Lessertia have recently been introduced to Australia in an attempt to increase the range of forage species available in Australian farming systems capable of dealing with a changing climate. This study assessed the diversity and the nodulation ability of a collection of Lessertia root nodule bacteria isolated from different agro-climatic areas of the Eastern and Western Capes of South Africa.

Thirty two rhizobial isolates were obtained from different bioclimatic regions of Tunisia using as trap plants, Medicago sativa, Medicago ciliaris, Medicago polymorpha and Medicago minima. To study their diversity and characterize them in relation to Mediterranean conditions, abiotic stress resistance, symbiotic properties and genetic diversity in terms of 16S rRNA and nodA sequences were assessed. Five isolates from M. sativa, three from M. ciliaris and three from M. minima could grow at 45°C. Only two isolates from M. sativa grew at 4% NaCl. The most stress tolerant isolates were obtained from arid soils. A phylogenetic analysis of 16S rRNA genes revealed 29 isolates to be closely related to Ensifer including one (Pl.3-9) that showed a 16S rRNA sequence similar to that of Ensifer meliloti and nodA sequence similar to that of Ensifer medicae. However, three isolates were categorized into Agrobacterium containing the nodA of Ensifer. Furthermore, these isolates developed nodules on original hosts. The results for the four isolates suggest horizontal gene transfer between the species.

Using a sand pouch-plant infection method, populations of several species of root-nodule bacteria (rhizobia) were enumerated in eighty soils collected throughout Poland. Rhizobium leguminosarum bv. viciae (symbionts of pea, faba bean, vetch) and R. leguminosarum bv. trifolii (symbionts of clover) were detected in 77 and 76 soils, respectively. Most of these soils contained moderate and high numbers of these species of the rhizobia. Symbionts of beans, R. leguminosarum bv. phaseoli, were assessed in 76 soils; of this number 15 soils had no detectable populations of bean rhizobia and in 40 soils high or moderate numbers of these bacteria were found. Bradyrhizobium sp. (Lupinus), root-nodule bacteria of lupine and serradella, were absent in 19 soils, out of 80 tested, and 34 soils were colonised by high or moderate populations of bradyrhizobia. Sinorhizobium meliloti, rhizobia nodulating alfalfa, were sparse in the examined soils; with 56 soil containing no detectable numbers of S. meliloti and only 6 soils harbouring high or moderate populations of this species. The estimated numbers of the rhizobia in the studied soils were also related to some physical and chemical properties of these soils.

Symbiotic nitrogen fixation is the main route for sustainable input of nitrogen into ecosystems. Nitrogen fixation in agriculture can be improved by inoculation of legume crops with suitable rhizobia. Knowledge of the biodiversity of rhizobia and of local populations is important for the design of successful inoculation strategies. Soybeans are major nitrogen-fixing crops in many parts of the world. Bradyrhizobial inoculants for soybean are very diverse, yet classification and characterization of strains have long been difficult. Recent genetic characterization methods permit more reliable identification and will improve our knowledge of local populations. Forage legumes form another group of agronomically important legumes. Research and extension policies valorizing rhizobial germplasm diversity and preservation, farmer training for proper inoculant use and legal enforcement of commercial inoculant quality have proved a successful approach to promoting the use of forage legumes while enhancing biological N 2 fixation. It is worth noting that taxonomically important strains may not necessarily be important reference strains for other uses such as legume inoculation and genomics due to specialization of the different fields. This article points out both current knowledge and gaps remaining to be filled for further interaction and improvement of a rhizobial commons.

The changes in the contents of protein and free amino acids in pea plants inoculated with Rhizobium leguminosarum were studied taking into account the susceptibility of roots to root nodule bacteria. The content of cytoplasmic protein during infection increased in the actively growing root zone (0–5 mm) and decreased in the root zones susceptible to rhizobia (5–20 mm from the root tip). The quantitative composition of free amino acids changed essentially upon inoculation of pea seedlings with R. leguminosarum.

Dr Phillip Nutman was a microbiologist and plant physiologist, distinguished for his research into the infection of roots of legumes by root nodule bacteria of the genus Rhizobium. This is a subject that is truly symbiotic, involving both leguminous host plants and free–living soil bacteria, which join in a complex, often specific interaction to produce symbiotic, nodulated, nitrogen–fixing plants. His research pre–dated the molecular genetics now available to modern researchers and used the techniques of plant physiology and Mendelian genetics to explore the mechanisms of infection, subsequent nodule development and the symbiotic fixation of atmospheric nitrogen. The research took place in the laboratories and fields of Rothamsted Experimental Station, Harpenden, Hertfordshire, where the study of nitrogen fixation by nodulated legumes first developed in the UK (Russell 1966). There were components of related research in Australia, 1953–57 (Bergersen 2001), and also in other countries during the International Biological Programme of 1966–73, in which Nutman was an active participant. He was a well–respected leader in active research during a period in which the subject underwent rapid development. In many ways his work became the basis on which more recent research has developed. He was a modest, self–effacing, scrupulously honest man who became increasingly impatient with the methods of research management that were emerging within the Agriculture and Food Research Council towards the end of his career at Rothamsted. After his retirement, in his Personal Record, he wrote critically about these matters.

Indigenous root-nodule bacteria isolated from the acid sands of the Cape using Aspalathus linearis, Aspalathus hispida, Aspalathus carnosa, Aspalathus capensis and Amphithalea ericifolia as trap hosts showed considerable tolerance to low pH. Isolates from A. ericifolia and A. carnosa could even grow in YMB medium at pH 3. Although, all strains grew well at pH 4, 5 and 6, the isolates from A. carnosa exhibited the highest growth rate at each of the three pH regimes. The isolates from A. linearis subsp. linearis, A. capensis and A. carnosa grew significantly better when re-cultured from pH 3 in pH 5 or same pH 3 medium as compared to first-time culture in pH 3. With isolates from A. capensis and A. linearis, growth of cells from pH 3 re-cultured in pH 5 or same pH 3 was not significantly different. Except for isolates from A. carnosa, which showed a marked increase and A. capensis with a major decrease, no differences were observed in bacterial growth when cells from pH 5 were re-cultured in pH 3. Providing 0.5% of root metabolites from A. linearis subsp. linearis to its microsymbiont at pH 3 significantly reduced cell growth from 0.8 to less than 0.1 OD units. At pH 5, however, bacterial growth was neither inhibited nor promoted by the addition of root extract.Key words: Bacterial isolates, acid soils, optical density, Western Cape.

In Africa, arid and semi-arid soils are characterized by a diverse occurrence of plants including nodulated legumes which are adapted to the prevailing and often harsh environmental conditions. Such soils also harbour a large diversity of indigenous rhizobial symbionts (comparable to legume diversity) and other bacterial endophytes that have a potential to be developed into elite strains to be used as inoculum. However, very few studies have investigated the diversity of root-nodule bacteria on African soils, the main reason being that funds for research are often lacking. Discoveries of new rhizobial species are to be expected from different soil ecologies that are likely to be inhabited by various types of microorganisms. It is concluded that the semi-arid and arid African soils have rhizobia that is possibly as equally diverse as their host legumes. Research in these areas is likely to find novel species with a great potential for use in agricultural systems.

The aim of these studies was to investigate whether residual toxic effects of exposing soybean root nodule bacteria to Al in a solid defined media (SDM) alter tolerance to Al, survival, sensitivity to antibiotics, N2 fixation effectiveness and genetic diversity of Bradyrhizobium strains. After being exposed four times to Al, strains showed variation in Al tolerance but there was no evidence of change in their original Al tolerance, sensitivity to the antibiotics or genetic diversity. Exposure of Bradyrhizobium strains to SDM plus Al did not alter biological N2 fixation effectiveness of five strains. Strain SEMIA 587 showed a reduction in its N2 fixation effectiveness but it seems that it was just a superficial toxic effect because one single passage through the plant eliminated this effect. Residual Al did not cause increases in Al tolerance and reductions in the survival and N2 fixation effectiveness of Bradyrhizobium strains USDA 143, SEMIA 586, SEMIA 5019, SEMIA 5039 and SEMIA 5073. It also did not alter the resistance to antibiotics of strains USDA 143, SEMIA 5039 and SEMIA 5073, and the genetic diversity of the strains SEMIA 587 and SEMIA 5019. O objetivo deste trabalho foi investigar se os efeitos tóxicos residuais do Al afetam a tolerância Al, sobrevivência, sensibilidade a antibióticos, eficiência de fixação de nitrogênio e diversidade genética de estirpes de Bradyrhizobium. Quando os nódulos de bactérias da raiz de soja foram expostos ao Al, em meio sólido por quatro vezes consecutivas, as estirpes de Bradyrhizobium mostraram variações na tolerância ao Al. Não houve alterações nos níveis originais de tolerância ao Al, sobrevivência, sensibilidade aos antibióticos testados e diversidade genética. Cinco das estirpes expostas ao Al não alteraram a eficiência de fixação de nitrogênio. A estirpe SEMIA 587, por sua vez, mostrou uma redução da sua eficiência de fixação de N2, mas apenas superficial, porque uma simples passagem através da planta eliminou esse efeito tóxico. Sucessivas exposições de estirpes de Bradyrhizobium ao Al não aumentam a tolerância ao Al, não reduzem a sobrevivência e a eficiência de fixação simbiótica do N2 das estirpes USDA 143, SEMIA 586, SEMIA 5019, SEMIA 5039 e SEMIA 5073, não alteram a tolerância a antibióticos das estirpes USDA 143, SEMIA 5039 e SEMIA 5073 e não alteram a diversidade genética das estirpes SEMIA 587 e SEMIA 5019.

The Rhizobiaceae are a bacterial family of enormous agricultural importance due to the ability of its members to fix atmospheric nitrogen in an intimate relationship with plants. Their survival as naturally occurring soil bacteria in agricultural soils as well as popular seed inocula is affected directly by drought and salinity. Survival after desiccation in the presence of NaCl is enabled by underlying genetic mechanisms in the model organism Sinorhizobium meliloti 1021. Since salt stress parallels a loss in water activity, the identification of NaCl-responsive loci may identify loci involved in survival during desiccation. This approach enabled identification of the loci asnO and ngg by their reduced ability to grow on increased NaCl concentrations, likely due to their inability to produce the osmoprotectant N-acetylglutaminylglutamine (NAGGN). In addition, the mutant harboring ngg ::Tn 5luxAB was affected in its ability to survive desiccation and responded to osmotic stress. The desiccation sensitivity may have been due to secondary functions of Ngg (N-acetylglutaminylglutamine synthetase)-like cell wall metabolism as suggested by the presence of a d -alanine- d -alanine ligase (dAla-dAla) domain and by sensitivity of the mutant to β-lactam antibiotics. asnO ::Tn 5luxAB is expressed during the stationary phase under normal growth conditions. Amino acid sequence similarity to enzymes producing β-lactam inhibitors and increased resistance to β-lactam antibiotics may indicate that asnO is involved in the production of a β-lactam inhibitor.

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 microM added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2-5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron. All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 microM added iron). Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 microM added iron) during the stationary phase of growth.

In this review, we consider how the nitrogen-fixing root nodule bacteria, the 'rhizobia', acquire various metals, paying particular attention to the uptake of iron. We also review the literature pertaining to the roles of molybdenum and nickel in the symbiosis with legumes. We highlight some gaps in our knowledge, for example the lack of information on how rhizobia acquire molybdenum. We examine the means whereby different metals affect rhizobial physiology and the role of metals as signals for gene regulation. We describe the ways in which genetics has shown (or not) if, and how, particular metal uptake and/or metal-mediated signalling pathways are required for the symbiotic interaction with legumes.

The Sinorhizobium-legume interaction is sensitive to a number of environmental factors: one of the important stresses is soil acidity. Low pH imposes severe constraints on legume productivity. In the Sinorhizobium-Medicago partnership, prokaryotic partner is the most acid-sensitive. The isolation of acid-tolerant Sinorhizobium strains from the eastern and southern Mediterranean rim have allowed the successful development of medic pastures on over I million ha of land in Western Australia, posing two important questions: • How do acid-tolerant strains differ from acid-sensitive counterparts? • How do agriculturally important root nodule bacteria respond to low pH? Knowledge of the genetic circuits required and activated by the cell in acidic conditions is vital to understand the response and survival of bacteria in these conditions. The molecular basis of acid tolerance has been investigated with several approaches (Glenn et al. 1999).

Quantification of acidity tolerance in the laboratory may be the first step in rhizobial strain selection for the Amazon region. The present method evaluated rhizobia in Petri dishes with YMA medium at pH 6.5 (control) and 4.5, using scores of 1.0 (sensitive, "no visible" growth) to 4.0 (tolerant, maximum growth). Growth evaluations were done at 6, 9, 12, 15 and 18 day periods. This method permits preliminary selection of root nodule bacteria from Amazonian soils with statistical precision. Among the 31 rhizobia strains initially tested, the INPA strains 048, 078, and 671 presented scores of 4.0 at both pHs after 9 days of growth. Strain analyses using a less rigorous criterion (growth scores higher than 3.0) included in this highly tolerant group the INPA strains 511, 565, 576, 632, 649, and 658, which grew on the most diluted zone (zone 4) after 9 days. Tolerant strains still must be tested for nitrogen fixation effectiveness, competitiveness for nodule sites, and soil persistence before their recommendation as inoculants.

Symbiotic instability occurs in some elite strains of Rhizobium leguminosarum bv. viciae during continued cultivation and preparation as inoculants resulting in loss of nodulation ability. This project aims to understand the symbiotic instability of these strains and identify criteria for the selection of stable elite inoculant strains of pea root nodule bacteria.

Root nodule bacteria infect and form nodules in the roots on various leguminous plant hosts. The bacteria undergo morphological changes in the cells of the nodules; from free-living to bacteroidal form. The bacteroidal form is classified to two types: Rhizobium and Bradyrhizobium. The bacteroid morphology is determined by rhizobial genera, the host plant and some unknown mechanisms.

Native s-glucuronidase activity is absent in the root nodule bacteria. The Escherichia coli gusA gene (conferring s-glucuronidase activity) can be used as a tool for the genetic analysis of these bacteria. The gusA was modified by positioning stop codons in all three reading frames (before the RBS of gusA) to prevent translational fusions.

One of the most interesting aspects of Rhizobium and Bradyrhizobium ecology is the competitivity for nodulation between strains. Native rhizobial populations, often poorly effective for symbiotic nitrogen fixation, are well adapted to soil conditions and are able to occupy most of the nodules formed in the presence of bacterial strains applied to seeds. This is a cause of the common fail of attempts carried out to improve productivity in agricultural fields by inoculation with superior strains of root nodule bacteria.

A plasmid-cured derivative of Rhizobium loti strain NZP2037, strain PN4010, was significantly more effective in N2 fixation and showed an improved capacity to compete with other root nodule bacteria for nodulation of Lotus pedunculatus, relative to that of strain NZP2037. Reintroduction of the plasmid pRlo2037 into PN4010 resulted in a return to the NZP2037 level of symbiotic effectiveness and competitiveness. The enhanced effectiveness and competitiveness of PN4010 for L. pedunculatus was associated with its increased capacity to form nodules. Strain PN4010 did not differ from NZP2037 in its growth characteristics in pure culture, in its resistance to L. pedunculatus root flavolan or in its ability to multiply in the L. pedunculatus rhizosphere in the presence of a competing strain of R. loti. An examination of 35 R. loti strains (including 25 field isolates) revealed two strains (NZP2014 and NZP2042) that did not contain a single large indigenous plasmid; both were significantly more effective in N2 fixation with L. pedunculatus than was NZP2037 or two other Nod+ Fix+ plasmid-containing strains of R. loti. Transfer of pRlo2037 into NZP2014 and NZP2042 reduced their effectiveness for L. pedunculatus.

Summary Cross-inoculation group specificity in Cicer arietinum L. (chickpea) was studied. Seventy-one strains of root nodule bacteria of C. arietinum were examined for nodulation on 87 species of legumes. These species represent all the known cross-inoculation groups and were selected from various tribes and genera of family Fabaceae and Mimosaceae. In a reciprocal cross-inoculation study, 287 strains of root nodule bacteria from 52 of the 87 species, were examined on various genotypes of C. arietinum. C. arietinum and its root nodule bacteria did not show cross inoculation affinity with any of the members of the known cross-inoculation groups: alfalfa, clover, pea, bean, soybean, lupin-lotus and cowpea miscellany; except for some loose non-reciprocal kinship with Sesbania, which in its turn has strong affinity with the cowpea miscellany. However, under natural conditions in soil, even the loose non-reciprocal kinship between Cicer and Sesbania was rare. For practical reasons, it is suggested that C. arietinum and its root nodule bacteria should be considered in a separate cross-inoculation group. ‘Cross-inoculation host specificity’ appears to be of some help in the revision of the taxonomy of order Leguminosales and supports the placement of Cicer in a monogeneric tribe Cicereae.

Leakage of materials reacting with the fluorescent probe 1-anilino-8-naphthalcne sulphonate (ANS) from root-nodule bacteria subjected to desiccation and rehydration suggests that death of these bacteria is associated with changes in membrane permeability. The slow-growing rhizobia and the fast-growing Rhizobium meliloti differ from other fast-growing bacteria of the R. leguminosarum group in that they exhibit little fluorescence in the presence of ANS. This probably reflects differences between these groups of bacteria in the polar/non-polar nature of the outer cell envelopes. Following desiccation and rehydration, all groups of root-nodule bacteria fluoresced strongly in the presence of ANS. Repair of desiccation-induced damage to the cell envelopes of R. japonicum QA372. R. trifolii SU297/32B and R. meliloti CC131 was not observed using a range of screening agents. However, repair of significant numbers of lysozyme-sensitive bacteria was observed with R. leguminosarum TA101 within an hour of rehydration.