Symbiotic Performance Research Articles

Genetic variation in two cultures of Bradyrhizobium japonicum 110 differing in their ability to impart drought tolerance to soybean.

The polymerase chain reaction with arbitrary primers (RAPD) discriminated between two separately maintained cultures of Bradyrhizobium japonicum USDA 110 differing in symbiotic performance under drought conditions. Since strain 110 is used in inoculum production, the use of RAPD to monitor inoculum cultures could help to preserve their genetic composition and prevent the loss of important symbiotic properties. The use of RAPD could also be extended to other B. japonicum strains currently used in inoculum production.

Evaluation of N 2 fixation and agroforestry potential in selected tree legumes for sustainable use in South Africa

Seven tree legumes with agroforestry potential were tested for their capacity to nodulate with indigenous rhizobia or bradyrhizobia in soils collected from different ecological zones in South Africa. Our findings show that all the tested species, except Acacia senegal, could effectively nodulate with root-nodule bacteria native to Venda and Klipspruit soils. Since Acacia seyal, Acacia galpinii, Albizia lebbeck and Acacia auriculiformis exhibited, in that order, a greater level of growth and symbiotic performance, they manifest themselves as suitable species for agroforestry programs in Venda and Klipspruit soils. One indigenous species, Acacia ataxancantha, was assessed to be a non-nodulating legume even when inoculated with a promiscuous strain of Rhizobium NGR234.

Comparative efficieny of Rhizobium/Bradyrhizobium spp. strains in nodulating Cajanus cajan in relation to characteristic metabolic enzyme activities

The comparative symbiotic properties of Rhizobium spp. and Bradyrhizobium spp. strains infecting pigeon pea were evaluated. Bradyrhizobium strains (Cajanus) were found to be superior to Rhizobium strains (Cajanus) and the superiority was ascertained to be due to the higher enzyme activity of the tricarboxylic acid (TCA) cycle in comparison to Rhizobium spp. strains. Moreover, metabolic superiority or rapid growth rate does not necessarily correlate with symbiotic effectiveness. The symbiotic performance of isolates varied with the host cultivar. The dry matter accumulation could be correlated with the total acetylene reduction activities rather than nodule number or nodule fresh weight per plant.

Evaluation of the recA-based containment system in Rhizobiummeliloti GR4

Rhizobium meliloti recA strains have been reported as safe hosts for deliberate release experiments due to their reduced survival in soil microcosms. Using R. meliloti integration vectors, two GUS-positive RecA + and RecA − isogenic derivatives of R. meliloti strain GR4 were constructed. In contrast to previously reported RecA − strains, the RecA − strain GR4KLR showed similar doubling times as its isogenic RecA + strain GR4KLC. Accordingly, GR4KLR showed normal growth competition capacity when coinoculated with the parental strain in sterile soil microcosms. In addition, the recA mutant showed delayed nodulation on axenically grown alfalfa plants, and its competitive nodulation ability was severely affected. This defect could be corrected by a genetic manipulation based on the use of a multicopy nifA-expressing plasmid. Another novel phenotype associated with the recA mutation in R. meliloti GR4 was an increased rate of plasmid loss. When inoculated in non-sterile soil microcosms, RecA + and RecA − strains established at high cell densities and no significant differences in their survival were found after 150 days incubation. Our results show that symbiotic performance of a R. meliloti GR4 recA strain may be significantly altered whereas its relative survival in a test soil where it was to be released was not significantly reduced with respect to RecA + strains.

Persistence and effectiveness of rhizobia nodulating promiscuous soybeans in moist savanna zones of Nigeria

The symbiotic performance of promiscuous soybeans depends upon the population size, effectiveness and survival of indigenous or introduced rhizobia in the field. A pot experiment was conducted using soils collected from 13 farmers' fields located in two agroecological zones (northen and southern Guinea savanna) in the moist savanna of Nigeria to determine the relationships between growth response to previous rhizobial inoculation and the indigenous rhizobial populations. At each farmer's field, soil was collected in plots which were planted the previous year (1993) to: (1) maize, (2) soybean cv. Bossier, (3) soybean cv. TGX 1456-2E and (4) soybean cv. TGX 1660-19F. The soybean cultivars TGX 1660-19F and TGX 1456-2E were either uninoculated or inoculated with an enriched population of a mixture of local rhizobial strains. Four test plants: soybean cultivars TGX 1660-19F (slightly promiscuous), TGX 1456-2E (highly promiscuous), Bossier (non-promiscuous) and cowpea (typically promiscuous) were planted in pots containing soils from the above field treatments. Previous inoculation increased shoot dry matter production by an average of 32% over the uninoculated controls in 94 of the 312 (30%) legume inoculations and farmers' field combinations while the indigenous rhizobia were more effective than the introduced ones in 103 combinations (33%) with an average of 20% increase compared to previous inoculation treatments. Previous inoculation increased biomass yield of both promiscuous and non-promiscuous soybean varieties. The response to previous inoculation treatments was farmers' fields dependent and inversely related to the numbers of rhizobia in the soil. Soil rhizobial population ranged from 0 to > 400 cells g −1 soil and response to inoculation often occurred when numbers of indigenous rhizobia were fewer than 10 cells g −1 soil. Numbers of indigenous rhizobia were generally lower or below detection limit in soils previously cropped to maize. These results indicate a relationship between rhizobia cell counts and promiscuous soybean responses, which may be used to indicate under which conditions inoculation will be beneficial to a farmer.

Rhizobium etli cytochrome mutants with derepressed expression of cytochrome terminal oxidases and enhanced symbiotic nitrogen accumulation

A method to isolate mutants with derepressed expression of cytochrome oxidases and better symbiotic performance is presented. A mutant of Rhizobium etli, CFN030, isolated by its azide-resistant phenotype, was obtained by transposon Tn5 -mob mutagenesis. This mutant has a derepressed expression of cytochrome aa3, higher respiratory activities when cultured microaerobically and an improved symbiotic nitrogen fixation capacity. This phenotype was similar to the previously described mutant CFN037, which was isolated by its increased capacity to oxidize N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) [Soberon M et al. (1990) J Bacteriol 172:1676–1680]. We show here that although both mutants have a similar symbiotic phenotype, they are affected in different genes. Strain CFN030 has the Tn5 inserted in the chromosome while in strain CFN037 the transposon was located in plasmid b. Cytochrome spectral analysis of both mutant strains in the post-exponential phase of growth, showed the expression of an additional terminal oxidase (cbb3) that is not expressed in the wild-type strain.

Growth and symbiotic performance of faba bean inoculated withRhizobium leguminosarumbiovar.viciaestrains tolerant to salt

Abstract Strain GRA19 of Rhizobium leguminosarum biovar. viciae was found to be tolerant to low levels of salt (50 mM) by comparing growth under stress conditions to that in the absence of stress. Growth and symbiotic N2 fixation (acetylene reduction activity) under saline conditions of the faba bean (Vicia faba L.) cultivar Alameda inoculated with GRA19 were reduced. Salinity significantly decreased shoot and root dry weight, nodule dry weight, and average individual nodule dry weight. Nitrogen fixation was more severely affected by salinity than plant growth and nodulation. Evidence presented here suggests the need for selecting faba bean cultivars that are tolerant to salt stress for breeding programs in order to improve the symbiotic performance of R. leguminosarum biovar. viciae.

Rhizobium plasmids in bacteria-legume interactions

The functional analysis of plasmids in Rhizobium strains has concentrated mainly on the symbiotic plasmid (pSym). However, genetic information relevant to both symbiotic and saprophytic Rhizobium life cycles, localized on other 'cryptic' replicons, has also been reported. Information is reviewed which concerns functional features encoded in plasmids other than the pSym: biosynthesis of cell surface polysaccharides, metabolic processes, the utilization of plant exudates, aromatic compounds and diverse sugars, and features involved symbiotic performance. In addition, factors which affect plasmid evolution through their influence on structural features of the plasmids, such as conjugative transfer and genomic rearrangements, is discussed. Based on the overall data, we propose that together the plasmids and the chromosome constitute a fully integrated genomic complex, entailing structural features as well as saprophytic and cellular functions.

Assessment of strains of Bradyrhizobium sp. (Lupinus) for serradellas (Ornithopus spp.)

Several strains of Bradyrhizobium sp. (Lupinus) were compared, in the glasshouse and field, with the Australian commercial inoculant strain, WU42.5, for symbiotic performance on serradella. Strains WSM471 and USDA3709 were more effective in nitrogen fixation (12 and 8% respectively) than WU42.5 across 10 serradella lines, although WU42.5 generally displayed good levels of effectiveness. WSM471 was a more saprophytically competent strain than WU425 in mildly acidic sandy soils, resulting in a 2.8-fold increase in the nodule score of serradella sown 32 cm from a line of bradyrhizobia established the previous year. Although WSM471, when compared with WU42.5, increased the nodulation and early production of an established sward of serradella, it did not improve total annual production of the sward. The possible replacement of WU42.5 with WSM471 in serradella inoculants is discussed.

Influence of host genotypes on growth, symbiotic performance and nitrogen assimilation in faba bean (Vicia faba L.) under salt stress

Fifteen genotypes of faba bean (Vicia faba L.) were inoculated with salt-tolerant Rhizobium leguminosarum biovar. viciae strain GRA 19 in solution culture with 0 (control) and 75 mM NaCl added immediately after transplanting. Genotypes varied in their tolerance of high levels of NaCl. Physiological parameters (dry weight of shoot and root, number and dry weight of nodules) were not affected by salinity in lines VF46, VF64 and VF112. Faba bean line VF60 was sensitive to salt stress. Host tolearance appeared to be a major requisite for nodulation and N2 fixation under salt stress. Tolerant line VF112 sustained nitrogen fixation under saline conditions. Activity of the ammonium assimilation enzymes glutamine synthetase and glutamate synthase, and soluble protein content, were reduced by salinity in all genotypes tested. Evidence presented here suggests a need to select faba bean genotypes that are tolerant to salt stress.

Symbiotic properties of 5-methyltryptophan-resistant mutants of Bradyrhizobium japonicum

The effect that resistance to 5-methyltryptophan (MT) has on the symbiotic properties of B. japonicum was examined in a survey of fourteen clones. Resistance to MT often involves a mutational alteration in the regulation of tryptophan biosynthesis. Resistant clones (MTR) were isolated from agar plates containing MT. In the selection process care was taken to avoid pigmented clones that are likely to accumulate large amounts of indole compounds or show increased tryptophan catabolism. Wild-type control clones (WTc) were isolated from plates containing no selective agent. In greenhouse studies. Tracy-M soybean plants were inoculated with the two types of clones. After six weeks, plants which were inoculated with the MT resistant clones showed a much greater range of symbiotic effectiveness than did plants that received the control clones. While most MT-resistant clones were poor symbionts or unchanged in their symbiotic performance, one clone was obtained that had significantly improved symbiotic properties. The procedure may offer a way of selecting for clones with improved symbiotic performance. These results also indicate a link between tryptophan biosynthesis and symbiotic effectiveness.

Signal exchange in the Bradyrhizobium-soybean symbiosis

Rhizobium, Bradyrhizobium and Azorhizobium are able to infect and establish an N 2-fixing symbiosis with a variety of leguminous plants. The result of this infection process is the formation of a novel plant organ, the nodule, in which the bacteria reside. This nodulation process is controlled, at least in part, by the exchange of diffusable signals between the bacterial symbiont and plant host. Understanding this signaling process in plant-microbe interactions may lead to agronomic benefit. The bacterial nodulation genes are essential for the infection of the host root and the establishment of the nodule. The expression of these genes requires the NodD regulatory protein and the presence of specific flavonoids released from the host plant roots. Our research on B. japonicum, symbiont of soybean, has shown that, in addition to these factors, nod gene expression requires NodVW, members of the large family of two-component regulatory proteins. In addition to positive regulators, nod gene expression is controlled by a repressor, NolA. The proteins encoded by the nod genes encode the biosynthesis of substituted lipo-chitin molecules (so-called nod factors). B. japonicum strains produce a large variety of nod factors but the common and most active factor is a pentamer of ca. 1–4 linked N-acetylglucosamine acylated at the non-reducing end with vaccenic acid and substituted at the reducing, terminal sugar with 2- O-methylfucose. This purified molecule induces root hair curling and cortical cell division in soybean roots when applied at n m concentrations or lower. The products of the nodABC genes apparently encode for the synthesis of the acylated lipo-chitin backbone while specific modifications to this molecule are made by proteins encoded by the host-specific nodulation genes. In B. japonicum, NodZ encodes for the fucosylation of the terminal, reducing sugar. The diversity of regulatory responses and signal molecules involved in legume nodulation is an important determinant of host species and genotype specificity. In the latter case, the specificity shown by plant genotypes for particular rhizobial strains may be of importance in bacterial interstrain competition, an important agronomic problem for the enhancement of symbiotic N 2 fixation. Understanding the signaling pathways between rhizobia and their host plants may allow modifications of this interaction to improve symbiotic performance.

Comparative growth and symbiotic performance of four Acacia mangium provenances from Papua New Guinea in response to the supply of phosphorus at various concentrations

Plant growth performance, the P content in root and nodule tissues, and nodulation and N2-fixing ability were studied in four provenances of Acacia mangium from Papua New Guinea following different levels of P fertilizer application. A. mangium did not seem to need high levels of P for growth and N2 fixation. The response by this leguminous tree to the P supply varied significantly according to provenance and to P concentrations in the culture solution. The provenances of A. mangium were classified into three types according to their P response: (1) Growth performance, nodulation, and N2 fixation of plants were stimulated as concentrations of P increased (provenance PH 482); (2) the maximal effect of P on plant growth was found only at P concentrations higher than 500 μM (provenance PH 484); and (3) the plant response to P fertilization was low, even with nutrient solutions containing P concentratins higher than 500 μM (provenances PH 483 and PH 485). Provenance PH 483 was distinguished by its low nodulating ability. However, this provenance grew well, probably because of its high N2 fixation efficiency as expressed by specific acetylene reduction activity and its high P content in nodule tissues. Therefore, in certain cases, these two parameters may be useful criteria in selecting leguminous plants for field use. Statistical analyses of the study results showed that the effect of the factor P supply on N2 fixation efficiency and nodule development was only significant at P concentrations lower than 250 μM whereas the effect of the factor plant provenance was significant regardless of the P concentration used. This observation emphasizes the value of provenance screening in the identification of plants for use in a wide range of soil types.

The construction, detection and use of bioluminescent Rhizobium leguminosarum biovar trifolii strains

A. CRESSWELL, L. SKOT AND A.R. COOKSON. 1994. The gene encoding the firefly luciferase enzyme (luc) was introduced to Rhizobium leguminosarum biovar trifolii strains with a view to using the resulting bioluminescent strains to study the survival of genetically-engineered rhizobia in soil microcosms. The genetically-engineered micro-organisms (GEMs) behaved similarly to their parent strains with respect to growth rate in laboratory media and in their symbiotic performance with their host plants. No gene transfer could be detected in laboratory mating experiments. When inoculated onto a non-sterile soil the population of the GEM declined sharply from an initial cell density of 2 times 1077 g-1 soil to approach a stable cell density of approximately 3 times 102 g-1 after 150 d. Direct photography of bioluminescent rhizobia enabled the detection of colonies as small as 0.1 mm in diameter without the need for transferring colonies onto filter paper. When a Rhizobium strain carrying the luc marker on a plasmid was used as inoculant it was possible to visualize differences in colonization of the rhizosphere of white clover and ryegrass by contact print and colour transparency films. The photographic detection methods described here demonstrate the possibilities of using bioluminescent rhizobia for assessing their survival in soil, and for looking at rhizosphere populations which may be an important site for potential gene transfer.

Soil acidity in relation to groundnut-Bradyrhizobium symbiotic performance

Effects of soil acidity on groundnut-Bradyrhizobium symbiotic performance were studied in a potted, sandy soil in a glasshouse in Zimbabwe. The soil was limed to soil-pH levels of 5.0 and 6.5. Soil acidity negatively affected plant development, measured as leaf area and plant dry weight, while nodulation was enhanced. This acidity-enhanced nodulation was most evident when nodulation was caused by the indigenousBradyrhizobium population. Effects of soil acidity differed between groundnut cultivars andBradyrhizobium spp. strains, the former having greater importance. TwoArachis hypogaea L. Spanish-type cultivars, Falcon and Plover, performed equally well at neutral soil pH, but Falcon was more acid tolerant. Comparison of the symbiotic performance in neutral versus acid soil of twoBradyrhizobium spp. strains, MAR 411 (3G4b20) and MAR 1510 (CB 756), showed that MAR 411 performed superiorly in neutral soil, but MAR 1510 in acid soil. The indigenousBradyrhizobium population was more effective than was inoculation with strains MAR 411 or MAR 1510. Comparison of twelveBradyrhizobium spp. strains for their symbiotic performance in acid soil showed that some strains were totally ineffective under acidity stress (MAR 253, MAR 967 and MAR 1506), while others performed well.Bradyrhizobium spp. strain MAR 1576 (32 H1) ranked highest for nitrogen accumulation, plant dry weight and leaf area, with strains MAR 1555 (TAL 11) and MAR 1510 following closely. Nitrate fertilisation of groundnut plants led to soil alkalinisation, while nitrogen fixation resulted in soil acidification. Soil acidity in combination with soil sterilisation gave rise to symptoms associated with Al and Mn toxicity.

Genotypic variability of nitrogen metabolism enzymes in nodulated roots of Vicia faba

Twenty-nine genotypes of Vicia faba were grown in the presence or absence of NO 3 − to study variability in root and nodule nitrate reductase (NR, EC 1.6.6.1) and nodule cytosolic glutamine synthetase (GS, EC 6.3.1.2) activities. These V. faba lines apparently lack constitutive root NR activity (NRA), whereas inducible activity was detected in all of them except line VF17. Although a marked genotypic variability was found for this activity ( F-value = 400), the major factor affecting its expression was nitrate (95% of the total effect observed). Most of the lines, however, showed appreciable rates of constitutive NRA in their nodules, that further declined with 4 mM NO 3 −. High genotypic variability was also found for the level of GS activity from the plant fraction of V. faba nodules. This activity, little affected by 2 mM NO 3 −, was generally declined by 4 m m for a week at the end of the experiment. No correlation was found between the activities measured here and measurements of symbiotic performance. Nodule NR, however, correlated inversely with both GS and nodule protein content.

A survey of proportional dependence of subterranean clover and other pasture legumes on N2 fixation in south-west Australia utilizing 15N natural abundance

In an attempt to understand why pasture production in southern Australia has declined markedly in recent years a survey of the symbiotic performance of the legume component of annual pastures on 81 farms (243 sites) was undertaken in the southern coastal region of Western Australia. The 15N natural abundance technique was used to determine the percentage of plant nitrogen derived from the atmosphere (%Ndfa) using capeweed (Arctotheca calendula) as principal non-fixing reference species. %Ndfa values were then related to edaphic and management information, e.g. soil total nitrogen, soil pH, stocking rates and cropping history of the sites. The principal legume species encountered exhibited similar mean %Ndfa values but substantial variation in symbiotic performance was evident across the sites, viz, Trifolium subterraneum 72%Ndfa (n = 184, range of values encountered 0-100%), Medicago spp. 7l%Ndfa (n = 24, range 7-l00%), Lotus spp. 8l%Ndfa (n = 15, range 1-l00%), Ornithopus compressus 76%Ndfa (n = 15, range 25-100%) and Trifolium balansae 69%Ndfa (n = 7, range 0-100%). In the case of subterranean clover, the most widely occurring species, almost one third (29%) of sites surveyed recorded %Ndfa values within the range 0-65%, suggesting that symbiotic performance might well be quite widely limiting to herbage production in the study region. Of the 24 edaphic and management factors evaluated, only one, %A1 in shoot, DM showed a significant relationship with %Ndfa, with 40% of the pastures surveyed deemed at risk in terms of acidity related aluminium toxicity. Correlations of %Ndfa with soil pH and soil total N produced examples of high values %Ndfa for sub-clover being associated with very low soil pH or high soil N, suggesting possible adaptation of symbiotic partnerships to acidity or high mineral N.

Symbiotic nitrogen fixation and physiological performance of bean (Phaseolus vulgarisL.) plants as affected byRhizobiuminoculum position and bean rugose mosaic virus infection

Bean (Phaseolus vulgaris L. var. Tacarigua) plants were grown in sterilized Leonard jars under controlled conditions. Before sowing, 1 g of gamma irradiated peat containing the Rhizobium tropici strain CIAT899 was placed at either 2 or 10 cm below the sand surface. Mechanical infection of bean rugose mosaic virus (BRMV) was carried out in 3-d-old seedlings. Thus, the early events of nodulation occurred before the arrival of virus particles to roots. Rhizobium inoculation at 2 cm deep resulted in the formation of nodule clusters close to the crown, in contrast to the homogeneous nodulation along the roots observed in plants inoculated with Rhizobium at a depth of 10 cm

Genetic diversity among Rhizobium strains from Cicer arietinum L.

Following bacteriological cloning and determination of their symbiotic performance, 15 representative, diverse strains of chickpea rhizobia were genetically analysed for restriction fragment length polymorphisms. Analyses of genomic DNAs showed several different groups. Almost half (7) of the strains examined were very similar and clearly represented a single species. There was a related group of four strains which could be a subspecies. There was also one distinct group of four strains which were apparently unrelated to the reference strain 3377. This latter group may constitute a separate species. Phenotypic differences should be investigated further.

Effects of Bradyrhizobium strain and host genotype, nodule dry weight and leaf area on groundnut (Arachis hypogaea L. ssp. fastigiata) yield

Effects of inoculating four Arachis hypogaea ssp. fastigiata cultivars with 17 Bradyrhizobium spp. strains were studied in a glasshouse experiment using a sandy soil devoid of an indigenous Bradyrhizobium population. Firstly, a wide range of parameters, indicative of symbiotic performance, were assessed for their influence on seed yield, by correlation and statistical analyses. It was found that nodule dry weight and leaf area were relevant parameters concerning seed yield. Secondly, the effects of host and strain genotype on those parameters were described.