Homologous Rhizobia Research Articles

The Role of Flavonoids in Nodulation Host-Range Specificity: An Update.

Flavonoids are crucial signaling molecules in the symbiosis between legumes and their nitrogen-fixing symbionts, the rhizobia. The primary function of flavonoids in the interaction is to induce transcription of the genes for biosynthesis of the rhizobial signaling molecules called Nod factors, which are perceived by the plant to allow symbiotic infection of the root. Many legumes produce specific flavonoids that only induce Nod factor production in homologous rhizobia, and therefore act as important determinants of host range. Despite a wealth of evidence on legume flavonoids, relatively few have proven roles in rhizobial infection. Recent studies suggest that production of key “infection” flavonoids is highly localized at infection sites. Furthermore, some of the flavonoids being produced at infection sites are phytoalexins and may have a role in the selection of compatible symbionts during infection. The molecular details of how flavonoid production in plants is regulated during nodulation have not yet been clarified, but nitrogen availability has been shown to play a role.

Características simbióticas e fenotípicas de Azorhizobium doebereinerae, microissimbiote de Sesbania virgata

Foram realizados quatro experimentos em casa de vegetação. O primeiro e o segundo experimentos tiveram por objetivo verificar a capacidade de nodulação e a eficiência simbiótica de Sesbania virgata com estirpes de rizóbio homólogas (isoladas de nódulos da mesma espécie inoculada com amostras de solos da floresta amazônica e do Sudeste brasileiro, identificadas como Azorhizobium doebereinerae) e com estirpes de rizóbio isoladas de outras espécies de leguminosas. No terceiro e quarto experimentos, o objetivo foi analisar se a presença de A. doebereinerae em solos de diferentes ecossistemas, em dois municípios do Sul de Minas Gerais, estava relacionada com a presença de S. virgata. Em todos os experimentos foram adicionados três tratamentos- controle para verificar a ausência de contaminação e adequação das condições experimentais para expressão da eficiência simbiótica, além servirem de referências para comparação dos demais tratamentos. Dois controles foram sem inoculação: um sem nitrogênio mineral e outro em que foi adicionado nitrogênio mineral (35 mg.L-1 N- NH4 NO3). No terceiro controle, foi inoculada a estirpe BR 5401T, recomendada como inoculante para S. virgata e também estirpe tipo de A. doebereinerae. Os tratamentos foram aplicados sete dias após a repicagem das sementes para os frascos de vidro contendo 800 mL de solução nutritiva sem nitrogênio, onde permaneceram durante 50 dias. Objetivou-se, ainda, estudar a diversidade cultural e fenotípica de isolados de A. doebereinerae de solos da Amazônia e dos isolados obtidos nos experimentos III e IV. No primeiro e segundo experimentos, verificou-se que S. virgata somente nodulou quando foi inoculada com estirpes de rizóbio homólogas. No terceiro e quarto experimentos, observou-se que S. virgata nodulou somente com a inoculação de amostras de solos coletadas próximas de S. virgata. Concluiu-se que os isolados de A. doebereinerae de solos da Amazônia e os isolados dos experimentos III e IV, de solos de Minas Gerais, apresentaram alta similaridade simbiótica e fenotípica entre si e com a estirpe BR 5401T e a simbiose entre S. virgata e A. doebereinerae, independentemente da estirpe, foi altamente eficiente.

Interaction of pea ( Pisum sativum L.) lectins with rhizobial strains

Lectins from two varieties (PG-3 and LFP-48) of pea have been purified by affinity chromatography on Sephadex G-50. The specific activity increased by 23 and 25 folds, respectively. These lectins from both the varieties were found to be specific for mannose. The purified fluorescein isothiocyanate (FITC)-labelled lectins showed binding reaction with homologous as well as heterologous strains of Rhizobium spp. The results revealed that pea lectins are not highly specific to their respective rhizobia. Moreover, these lectins showed a greater stimulatory effect on homologous Rhizobium leguminosarum strains.

A new peroxidase cDNA from white clover: its characterization and expression in root tissue challenged with homologous rhizobia, heterologous rhizobia, or Pseudomonas syringae.

Temporal reverse transcription-polymerase chain reaction (RT-PCR) expression analyses were performed on Trprx2, a new white clover peroxidase, with roots challenged with homologous rhizobia, heterologous rhizobia, and a pathogen, Pseudomonas syringae. Low levels of Trprx2 expression were evident in all rhizobial treatments but in P.syringae-treated clover background expression was dramatically reduced within 1 h and was undetectable in treatments inoculated for more than 3 h. Spraying 4 mM salicylic acid onto seedlings increased Trprx2 expression. These data suggest a defensive role for Trprx2 in white clover and indicate active defense suppression by the pathogen.

Enhanced production of ethylene by soybean roots after inoculation with Bradyrhizobium japonicum

The production of ethylene by soybean ( Glycine max (L.) Merr.) roots during nodulation was investigated. The production of ethylene by roots was stimulated by inoculation with Bradyrhizobium japonicum. The stimulation of the production of ethylene by the roots of inoculated plants was maximal 3 days after inoculation and then the rate of production of ethylene fell to that in the roots of uninoculated plants. No enhanced production of ethylene after inoculation was detected with the roots of a non-nodulating soybean mutant or after inoculation with a heterologous rhizobium, Rhizobium leguminosarum bv. viciae. Treatment of roots of wild-type soybean with an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine (AVG), prevented the enhanced production of ethylene. However, the number of nodules formed on the roots was barely affected by the treatment with AVG. The results indicate that the production of ethylene by soybean roots is transiently enhanced by infection with the homologous rhizobium.

Phenology, growth, and yield of field-grown soybean and bush bean as a function of varying modes of N nutrition

In field trials conducted at four sites in Hawaii, soybean ( Glycine max) and bush bean ( Phaseolus vulgaris) were either inoculated with homologous rhizobia, fertilized at high rates with urea, or left unamended. Crop phenology was assessed every few days. Rates of biomass and N accumulation and components of yield were measured five times during each crop cycle to assess the extent to which: (i) crops relying on soil, symbiotic, or fertilizer N differed in their growth characteristics; (ii) mode of N nutrition affected the timing of developmental stages; and (iii) effects of N nutrition on crop growth and development were related to final yield. While all measured variables differed significantly between sites, the effect of changing N source on these variables, in N limited environments, was consistent across sites. Rate and extent of node production, crop growth and yield were increased in symbiotic and N-fertilized crops as compared to unamended, non-fixing crops, while reproductive development was protracted. Extended time required to reach reproductive maturity was attributable to an increase in seed fill duration as time to flowering was not affected. Development and yield of N 2-fixing crops were similar but not equivalent to those of N-fertilized crops. To produce reliable yield estimates, legume growth simulation models must be able to accurately simulate crop growth and phenology. The present data indicate that information relating to source and supply of N must be incorporated before such models can be used to generate reliable yield estimations. Results of these trials also provide a valuable dataset for calibrating model subroutines for inorganic nitrogen uptake and nitrogen fixation in soybean and bush bean growth under field conditions and adjusting model coefficients for tropical environments.

Nodulation studies on legumes exotic to Australia: symbiotic relationships between Chamaecytisus palmensis (tagasaste) and Lotus spp

Strains of rhizobia were isolated from soil around the roots of tagasaste (Chamaecytisus palmensis) growing at 15 widely separated locations in south-eastem Australia. A further collection of strains of both Rhizobium loti and Bradyrhizobium sp. (Lotus) was assembled from 18 legumes including Lotus and other species symbiotically related to Lotus. The strains were used to inoculate tagasaste and 4 species of Lotus in experiments conducted under bacteriologically controlled conditions in a temperature-controlled glasshouse. Tagasaste formed nodules and fixed N2 with all of its homologous rhizobia but there was a wide range of effectiveness among the 15 strains. Tagasaste also formed nodules with each of the 18 strains from other species but fixed N2 with only 10. Four species of Lotus were inoculated with 3 tagasaste strains. One strain nodulated each species and fixed N2 with L. conimhricensis and L. corniculatus but not with L. parviflorus or L. pedunculatus. A second tagasaste strain formed nodules with all 4 Lotus spp. but did not fix N2, while the third nodulated only L. pedunculatus but did not fix N2. A pattern analysis based on the nodulating ability of the host plants in association with 21 strains showed that tagasaste and L. corniculatus formed 1 symbiotic group, and the other 3 Lotus species formed a third group. The pattern analysis procedure based on nodulating capacity of 21 rhizobial strains in association with the 5 host species indicated substantial symbiotic diversity within the collection, with the strains comprising 8 different symbiotic groups. No strain was highly effective on both tagasaste and any of the 4 species of Lotus. Data were insufficient to classify the root-nodule bacteria of tagasaste as either Rhizobium loti or Bradyrhizobium sp. (Lotus).

Environmental effects on competition for nodule occupancy between introduced and indigenous rhizobia and among introduced strains

Understanding the impact of environmental variables on interstrain competition is important to ensure the successful use of rhizobial inoculant. In eight inoculation trials conducted at five diverse sites on Maui, Hawaii, equal numbers of three serologically distinct strains of effective, homologous rhizobia in a peat-based inoculant were applied to seeds of soybean, bush bean, cowpea, lima bean, peanut, leucaena, clover, and tinga pea. We studied the influence of environmental variables on interstrain competition between applied and indigenous rhizobia and among the three strains comprising the inoculum. Although temperature and soil fertility were correlated with nodule occupancy by inoculant strains in a few cases, the most significant environmental variable controlling their competitive success was the size of the indigenous rhizobial population. Nodule occupancy was best described (r2 = 0.51, p < 0.001) by the equation y = 97.88 − 15.03(log10(x + 1)), where y is percent nodule occupancy by inoculant rhizobia and x is the most probable number of indigenous rhizobia per gram soil. For each legume, one of the three inoculant strains was a poor competitor across sites. Competition between the other two strains varied between sites, but was infrequently related to environmental variables. Results indicated that competitive strains could be selected that perform well across a range of environments. Key words: competition, rhizobial ecology, inoculation response, competitiveness index.

12] Site-directed Tn5 and transplacement mutagenesis: Methods to identify symbiotic nitrogen fixation genes in slow-growing Rhizobium

Publisher Summary This chapter discusses how site-directed Transposase (Tn5) mutagenesis is used in the laboratory to identify genes essential for symbiotic nitrogen fixation in slow-growing rhizobium species. This chapter has experimented with several methods for this kind of mutagenesis and will discuss the protocol that has worked most successfully in conjunction with these organisms. The chapters also describe a method for site-directed transplacement of in vitro altered DNA sequences that should be applicable to all gram-negative bacteria. Tn5 is stably maintained in the chromosome throughout the life cycle of the rhizobium cell in the nodules of the plant. Maintenance of an extrachromosomal plasmid, such as plasmid (pVK100) under these same nonselective conditions, however, is sometimes a problem. Moreover, false-positive complementation results can occur between two Tn5 insertions that are actually within the same transcriptional unit as a result of marker exchange between the homologous Rhizobium DNA contained on the plasmid and in the chromosome. Although the advancement of the genetics of the slow-growing Rhizobium species has preceded more slowly than that of the fast-growing Rhizobium species, the techniques for mutagenesis described in this chapter should facilitate progress in this field.

Evaluation of Diverse Antisera, Conjugates, and Support Media for Detecting Bradyrhizobium japonicum by Indirect Enzyme-Linked Immunosorbent Assay

We evaluated three antisera and four enzyme conjugates for the detection of Bradyrhizobium japonicum by an indirect enzyme-linked immunosorbent assay in microtiter plates. Nitrocellulose membrane sheets were then evaluated as an alternative support medium by using some combinations. Partially purified immunoglobulin G (IgG) or unpurified antisera to strain USDA 110 raised in rabbits, goats, or sheep was reacted in microtiter plates with alkaline phosphatase conjugated to protein A, goat anti-rabbit (GAR), sheep anti-rabbit (SAR), or rabbit anti-goat (RAG) IgG. Cultures or nodules containing homologous rhizobia were detected with equal sensitivity when protein A, GAR, or SAR was reacted with 5 mug of protein IgG per ml or a 1:800 titer of antisera from rabbits, but not goats or sheep. RAG reacted with IgG or antisera from goats or sheep. The detection limit was 2 x 10 rhizobia per well. Rhizobia were spotted on nitrocellulose sheets as an alternative support medium, followed by soaking in 5 mug of protein per ml as IgG and 1:4,000 dilutions of protein A or GAR conjugate. Rhizobia in serogroup 110 were detected with the dye combination Nitro Blue Tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP), and rhizobia in serogroup 122 were detected with fast red-naphthol phosphate (FR-NP). At the conclusion of the 5-h assay, purple (NBT-BCIP) or red (FR-NP) spots were visible in positive reactions. The sensitivity of detection was about 1,000 rhizobial cells or 3 mug of nodules tissue.

Symbiotic incompatibility between two forage species of Hedysarum, grown in Morocco, and their homologous rhizobia

Symbiotic experiments in the glasshouse demonstrated that two species of sulla, Hedysarum coronarium and Hedysarum flexuosum, grown in Morocco were mutually incompatible in their requirements for effectively nitrogen-fixing strains of rhizobia: nitrogen-fixing Rhizobium strains isolated from H. coronarium nodulated H. flexuosum but did not fix nitrogen, conversely strains from H. flexuosum were ineffective for H. coronarium. The agronic implications are discussed.

Symbiotic incompatibility between two forage species ofHedysarum, grown in Morocco, and their homologous rhizobia

Abstract Symbiotic experiments in the glasshouse demonstrated that two species of sulla, Hedysarum coronarium and Hedysarum flexuosum , grown in Morocco were mutually incompatible in their requirements for effectively nitrogen-fixing strains of rhizobia: nitrogen-fixing Rhizobium strains isolated from H. coronarium nodulated H. flexuosum but did not fix nitrogen, conversely strains from H. flexuosum were ineffective for H. coronarium . The agronic implications are discussed.

Competition Among Rhizobium leguminosarum Strains for Nodulation of Lentils ( Lens esculenta )

Thirty-one cultures of Rhizobium leguminosarum were screened for effectiveness (C(2)H(2) reduction) on lentils (Lens esculenta). Fluorescent antibodies prepared against three of the most effective strains (Hawaii 5-0, Nitragin 92A3, and Nitragin 128A12) exhibited a high degree of strain specificity; the antibodies reacted strongly with their homologous rhizobia in culture and with bacteroids in nodules. They did not cross-react with one another, and only weakly with 5 of the 47 other R. leguminosarum cultures tested. In competition studies in the growth chamber, whenever strain Nitragin 92A3 was included in the inoculum mixture, it consistently (but not always significantly, P = 0.05) occupied the majority of nodules on all four cultivars used. However, some degree of strain X cultivar interaction was apparent: Hawaii 5-0 was of equal competitiveness (P = 0.05) with Nitragin 92A3 on three of the varieties (Commercial, Tekoa, and Benewah), but inferior (P = 0.01) on the Chilean variety; Nitragin 92A3 completely dominated (P = 0.01) Nitragin 128A12 on all cultivars; and Hawaii 5-0 was of equal competitiveness (P = 0.05) to Nitragin 128A12 on the Chilean variety and more competitive (P = 0.01) on the commercial variety and less so on the other two varieties. In field experiments, Hawaii 5-0 proved of equal competitiveness (P = 0.01) with Nitragin 92A3 in one soil (an Inceptisol) and superior (P </= 0.05) to it in another (an Oxisol). Incidence of double-strain occupancy of nodules varied from 0 to 36% in vermiculite, depending on the strains in the mixture and the host variety, and from 0 to 38% in the field, depending on the strains in the mixture and the soil type. The results suggest a close relationship between the competitiveness of a strain and its occurrence in doubly infected nodules.

Populations ofRhizobium japonicum associated with the surfaces of soil-grown roots

Populations of nativeRhizobium japonicum 123 in the rhizospheres of field and pot grown plants as determined by immunofluorescence were calculated on the basis of root surface area. The density ofR. japonicum 123 on the root fluctuated between a few hundred to over a thousand per square centimeter of root surface. As root volume expanded rapidly, the Rhizobium density fell to less than one hundred per unit area. There was no appreciable effect due to different plant, nitrogen amendment, or addition of another strain ofR. japonicum, on the surface density of the nativeR. japonicum population on roots. Nor did the native population influence the added strain. Direct examination of root surface segments revealed that naturalized rhizobia existed sparsely on root surfaces in the form of short rods. They were observed to be attached sideways or in a polar manner on root hairs, epidermal cells, and at junctions of tap and lateral roots. There was no evidence of specific stimulation of the homologous Rhizobium by the host plant as a prelude to nodulation.

Associative effect of inoculation of different strains of Azotobacter and homologous rhizobium on the yield of moong (Vigna radiata), soybean (Glycine max) and pea (Pisum sativum)

It has been observed that in the case ofVigna radiata andGlycine max incorporation of suitable strain of Azotobacter gave higher yield than obtained by the use of Rhizobium as inoculant. In the case ofVigna radiata even a strain of Azotobacter isolated from the rhizosphere of berseem gave similar yields as Rhizobium. In the case ofPisum sativum association of Rhizobium with a strain ofAzotobacter chroococcum isolated from the rhizosphere of pea gave numerically higher yield than Rhizobium alone. It may be possible that statistically higher yield may be obtained when a suitable strain of Azotobacter is used after screening a large number of strains of Azotobacter from the rhizosphere of pea.

Studies on the Interaction of Legume Root Callus with Rhizobium

Root calli of three legumes — Arachis hypogaea, Cajanus cajan and Cicer arietinum were inoculated with homologous rhizobia. Measurable nitrogenase activity was detected when inoculated calli Arachis , Cajanus and Cicer were tested. Light microscopic observations of transections oe inoculated calli revealed bacterial penetration in the form of infection thread-like cells. In Cicer , characteristics noduar outgrowths were seen whose cells where also filled with bacteria. None of these observations were made in uninoculated calli indicating the features of possible symbiosis in legume root calli upon inoculation with Rhizobium .

Biochemical Changes in the Roots of Legumes upon Inoculation

Summary Lucerne ( Medicago sativa L.) and urid ( Phaseolus mungo L.) plants were grown under axenic culture by inoculating the plants with both homologous and heterologous rhizobia in pots, containing acid-washed sand. The roots were collected at various intervals and analyzed for total and o.D. phenols, reducing and non-reducing sugars, and amino and total nitrogen. The results run parallel to the observation made in plant-parasite interactions. From the results, it may possibly be argued that the biochemical changes in the plant body upon inoculation are responsible for differentiating the resistance of a host nodulation by a heterologous Rhizobium and its susceptibility to infection by a homologous Rhizobium , as observed in host-parasite interactions.

Pectin methyl esterase activity of root exudates of legumes in relation to Rhizobia

Seven different legumes were grown aseptically and in the presence of homologous and heterologous rhizobia in the root region. Samples of root exudates were tested for pectin methyl esterse activity. Root exudates of aspetically grown legumes possesed demonstrable pectin methyl esterase activity. In six legumes, the presence of homologous rhizobia in the root region stimulated the enzyme activity of root exudates while the presence of heterologous rhizobia often decreased the enzyme activity of the exudate. However, the results obtained withTrigonella foenum-graecum did not lend themselves easily to any generalization.