Involvement of Bradyrhizobium japonicum denitrification in symbiotic nitrogen fixation by soybean plants subjected to flooding

Denitrification by Bradyrhizobium japonicum bacteroids contributes to nitric oxide (NO) production within soybean nodules in response to flooding conditions. However, the physiological relevance of NO production by denitrification in B. japonicum-Glycine max symbiosis is still unclear. In this work, soybean plants were inoculated with B. japonicum strains lacking the nirK or norC genes which encode the copper-containing nitrite reductase and the c-type nitric oxide reductase enzymes, respectively. 14 days flooding increased nodule number of plants inoculated with the WT and norC strains, but not of plants inoculated with the nirK mutant. However, nodule dry weight was not affected by 14 days flooding regardless of the strain used for inoculation. Supporting this observation, individual nodule growth was significantly higher in plants inoculated with nirK than those inoculated with WT or norC after 14 days flooding. Nodule functioning was strongly inhibited by flooding since leghemoglobin content of the nodules induced by any of the strains was significantly decreased after 7 or 14 days flooding compared to control plants. However, this effect was more relevant in nodules of plants inoculated with the WT or norC mutant than in those inoculated with the nirK mutant. Nitrogen fixation was also estimated by analyzing nitrogen content derived from biological nitrogen fixation in shoots, using the ¹⁵N isotope dilution technique. By using this approach, we observed that the negative effect of 14 days flooding on nitrogen fixation was more pronounced in plants inoculated with the norC mutant. However, nitrogen fixation of plants inoculated with nirK showed the highest tolerance to 14 days flooding. These findings allowed us to demonstrate the previously proposed hypothesis which suggests that NO formed by copper-containing nitrite reductase in soybean nodules, in response to flooding, has a negative effect on nitrogenase activity. We propose that inoculation of soybeans with a B. japonicum nirK mutant, which does not produce NO from nitrate, increases the tolerance of symbiotic nitrogen fixation to flooding.     

Symbiotic effectiveness of Bradyrhizobium japonicum in acid soils can be predicted from their sensitivity to acid soil stress factors in acidic agar media

In acid soil, low pH, reduced availability of nutrients, and toxicity of Al and Mn limit plant growth and the survival and effectiveness of rhizobia. The symbiosis between legumes and rhizobia is particularly sensitive to acid soil stress. A pot experiment evaluated whether Bradyrhizobium japonicum strain growth on acidic agar media would predict ability to colonize the rhizosphere and form effective nodules in acidic soils. Three Indonesian strains of B. japonicum with similar effectiveness at neutral pH in sand culture but with different tolerance of acid soil stress factors in agar media, and an acid-tolerant commercial strain (CB1809) of comparable effectiveness, were tested in three acid soils using the Al tolerant soybean (Glycine max cv PI 416937). At 7 days after inoculation all strains had achieved large rhizosphere populations, but by day 14 the rhizosphere population of the acid-sensitive strain had decreased, while the more acid-tolerant strains increased. The acid-tolerant strains had significantly greater nodulation and symbiotic effectiveness than plants inoculated with the acid-sensitive strain. Laboratory prescreening of B. japonicum for acid, Al and Mn tolerance in acid media successfully identified strains which were symbiotically competent in low pH soils.     

The abundance and efficacy of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

For optimum production, the use of commercial rhizobial inoculant on pea (Pisum sativum L.) at seeding is necessary in the absence of compatible rhizobial strains or when rhizobial soil populations are low or symbiotically ineffective. Multiple site experiments were conducted to characterize the abundance and effectiveness of resident populations of Rhizobium leguminosarum bv. viciae (Rlv) in eastern Canadian prairie soils. A survey of 20 sites across a broad geographical range of southern Manitoba was carried out in 1998 and was followed by more intensive study of five of the sites in 1999 and 2000. Appreciable nodulation of uninoculated pea was observed at all sites which had previously grown inoculated pea. However, uninoculated pea grown at two sites, which had not previously grown pea, had negligible nodulation. Likewise, wild Lathyrus sp. and Vicia sp. plants collected from uncultivated areas adjacent to agricultural sites were poorly nodulated. In the more intensively studied sites, there was a tendency towards higher nodulation in pea plants receiving commercial inoculant containing Rlv strain PBC108 across all site-years (e.g., 4.7% in nodulation and 22% in nodule mass), but the effect was significant at only 2 of 10 site-years. Despite a relatively high range of soil pH (6-8), regression analysis indicated that decreasing soil pH resulted in lower nodulation rates. Likewise, electrical conductivity (EC) was correlated to nodulation levels, however the effect of EC was likely more indicative of the influence of soil texture and organic matter than salinity. As with nodulation, commercial inoculation tended to increase above-ground dry matter (DM) and fixed-N (estimated by the difference method) at the early pod-filling stage, but again the effects were significant at only 2 of 10 site-years. Specifically, above-ground DM and fixed-N levels were up to 29 and 51% greater, respectively, in inoculated compared to non-inoculated treatments at these sites. Addition of N-fertilizer at a rate of 100 kg N ha⁻¹ decreased nodulation at almost all site-years (by as much as 70% at one site), but rarely resulted in increases in above-ground DM compared to inoculated plots. The study indicates for the first time that populations of infective, and generally effective strains of Rlv occur broadly in agricultural soils across the eastern Canadian prairie, but that there is a tendency for increased symbiotic efficiency with the use of commercial inoculant.     

Isotopic fractionation during N2 fixation by four tropical legumes

To quantify the contribution of biological nitrogen fixation (BNF) to legume crops using the ¹⁵N natural abundance technique, it is necessary to determine the ¹⁵N abundance of the N derived from BNF—the B value. In this study, we used a technique to determine B whereby both legume and non-N₂-fixing reference plants were grown under the same conditions in two similar soils, one artificially labelled with ¹⁵N, and the other not. The proportion of N derived from BNF (%Ndfa) was determined from the plants grown in the ¹⁵N-labelled soil and it was assumed that the %Ndfa values of the legumes grown in the two soils were the same, hence the B value of the legumes could be calculated. The legumes used were velvet bean (Mucuna pruriens), sunnhemp (Crotalaria juncea), groundnut (Arachis hypogaea) and soybean (Glycine max) inoculated, or not, with different strains of rhizobium. The values of %Ndfa were all over 89%, and all the legumes grown in unlabelled soil showed negative δ¹⁵N values even though the plant-available N in this soil was found to be approximately +6.0‰. The B values for the shoot tissue (B_s) were calculated and ranged from approximately −1.4‰ for inoculated sunnhemp and groundnut to −2.4 and −4.5‰ for soybean inoculated with Bradyrhizobium japonicum strain CPAC 7 and Bradyrhizobium elkanii strain 29W, respectively. The B (B_wp) values for the whole plants including roots, nodules and the original seed N were still significantly different between the soybean plants inoculated with CPAC 7 (−1.33‰) and 29W (−2.25‰). In a parallel experiment conducted in monoxenic culture using the same soybean variety and Bradyrhizobium strains, the plants accumulated less N from BNF and the values were less negative, but still significantly different for soybean inoculated with the two different Bradyrhizobium strains. The results suggest that the technique utilized in this study to determine B with legume plants grown in soil in the open air, yields B values that are more appropriate for use under field conditions.     

Sampling effects on the assessment of genetic diversity of rhizobia associated with soybean and common bean

Biological nitrogen fixation plays a key role in agriculture sustainability, and assessment of rhizobial diversity contributes to worldwide knowledge of biodiversity of soil microorganisms, to the usefulness of rhizobial collections and to the establishment of long-term strategies aimed at increasing contributions of legume-fixed N to agriculture. Although in recent decades the use of molecular techniques has contributed greatly to enhancing knowledge of rhizobial diversity, concerns remain over simple issues such as the effects of sampling on estimates of diversity. In this study, rhizobia were isolated from nodules of plants grown under field conditions, in pots containing soil, or in Leonard jars receiving a 10⁻² or a 10⁻⁴ serially-diluted soil inoculum, using one exotic (soybean, Glycine max) and one indigenous (common bean, Phaseolus vulgaris) legume species. The experiments were performed using an oxisol with a high population (10⁵ cells g⁻¹ soil) of both soybean rhizobia, composed of naturalized strains introduced in inoculants and of indigenous common-bean rhizobia. BOX-PCR was used to evaluate strain diversity, while RFLP-PCR of the ITS (internally transcribed spacer) region with five restriction enzymes aimed at discriminating rhizobial species. In both analyses the genetic diversity of common-bean rhizobia was greater than that of soybean. For the common bean, diversity was greatly enhanced at the 10⁻⁴ dilution, while for the soybean dilution decreased diversity. Qualitative differences were also observed, as the DNA profiles differed for each treatment in both host plants. Differences obtained can be attributed to dissimilarity in the history of the introduction of both the host plant and the rhizobia (exotic vs. indigenous), to host-plant specificity, rhizobial competitiveness, and population structure, including ease with which some types are released from microcolonies in soil. Therefore, sampling method should be considered both in the interpretation and comparison of the results obtained in different studies, and in the setting of the goals of any study, e.g. selection of competitive strains, or collection of a larger spectrum of rhizobia. Furthermore, effects of sampling should be investigated for each symbiosis.     

Production of growth-promoting substances and high colonization ability of rhizobacteria enhance the nitrogen fixation of soybean when coinoculated with Bradyrhizobium japonicum

Understanding the interaction mechanisms between plant growth-promoting rhizobacteria (PGPR), leguminous crops, and rhizobia is necessary to effectively use PGPR in increasing the biological nitrogen fixation of legumes. We determined the coinoculation effects of Bradyrhizobium japonicum A1017 and a gusA-marked strain of Pseudomonas fluorescens 2137, P. fluorescens WCS365, Azomonas agilis 125, and Azospirillum lipoferum 137 on soybean [Glycine max (L.) Merr] cv. Enrei grown under axenic conditions. The gusA-marked rhizobacteria effectively colonized the root tips and surfaces near the roots tips with a colonization rate ranging from 7.50 to 8.62 log colony forming units (cfu) gfw^-1. P. fluorescens 2137 had the highest colonization activity on soybean roots whether inoculated alone or coinoculated with B. japonicum A1017. Coinoculation of P. fluorescens 2137 and B. japonicum A1017 increased the colonization of B. japonicum A1017 on soybean roots, nodule number, and acetylene reduction activity (ARA) at 10 and 20 days after inoculation. Moreover, the addition of sterile spent medium of P. fluorescens 2137 increased the growth of B. japonicum A1017 in yeast mannitol broth (YMB), indicating that P. fluorescens 2137 may have released substances that increased the rhizobial population. The results of this study suggest that the enhanced nodulation and ARA of soybean due to the high colonization of P. fluorescens on soybean roots could depend on the production of growth-promoting substances that stimulate the growth of B. japonicum. However, coinoculation with P. fluorescens WCS365 decreased the nodule number and ARA, despite its slight stimulation of the growth of B. japonicum on the roots, indicating that coinoculation effects are strain dependent.     

Enhanced soybean (Glycine max L.) plant growth and nodulation by Bradyrhizobium japonicum-SB1 in presence of Bacillus thuringiensis-KR1

Abstract

Nodulation and subsequent nitrogen fixation are important factors that determine the productivity of soybean (Glycine max L.). The beneficial effects of nodulation can be enhanced when rhizobial inoculation is combined with plant-growth-promoting bacteria (PGPB). The PGPB strain Bacillus thuringiensis-KR1, originally isolated from the nodules of Kudzu vine (Pueraria thunbergiana), was found to promote growth of soybean plants (variety VL Soya 2) under Jensen's tube and growth pouch conditions, when co-inoculated with Bradyrhizobium japonicum-SB1. Co-inoculation with Bacillus thuringiensis-KR1 (at a cell density of 10 cfu) provided the highest and most consistent increase in nodule number, shoot weight, root weight, root volume, and total biomass, over rhizobial inoculation and control, under both conditions. The results demonstrate the potential benefits of using nonrhizobial nodule occupants of wild legumes for the co-inoculation of soybean, with Bradyrhizobium japonicum-SB1, in order to achieve plant-growth promotion and increased nodulation.     

Involvement of autoregulation in the interaction between rhizobial nodulation and AM fungal colonization in soybean roots

Soybean plants autoregulate to suppress excessive nodulation. It has been revealed recently that the autoregulation of various legumes controls both nodulation and arbuscular mycorrhizal (AM) fungal colonization. We investigated the involvement of autoregulation in the interaction between rhizobial nodulation and AM fungal colonization. We used a wild-type soybean cv. Enrei and its hypernodulating mutant Kanto100, defective in the autoregulation. We included four different treatments: an uninoculated control, inoculation with rhizobium Bradyrhizobium japonicum alone, inoculation with AM fungus Gigaspora rosea alone, and dual inoculation with rhizobium and AM fungus. In both Enrei and Kanto100, AM fungal colonization enhanced the weight and N₂ fixation of nodules, suggesting that autoregulation of host plant is not involved in the stimulatory effect of AM fungal colonization on rhizobial nodulation. In plants with the AM fungus alone, the AM fungal colonization of Enrei was comparable to that of Kanto100. In plants with dual inoculation, however, this was significantly (P?<?0.05) lower than in Kanto100. To confirm the control of AM fungal colonization by the autoregulation of host plant, a reciprocal grafting experiment was performed between Enrei and Kanto100. In plants with the AM fungus alone, AM fungal colonization was comparable among Enrei (shoot)/Enrei (root), Enrei/Kanto100, Kanto100/Enrei, and Kanto100/Kanto100 grafts. In plants with dual inoculation, however, AM fungal colonization of Enrei/Enrei and Enrei/Kanto100 grafts was significantly (P?<?0.05) lower than that of Kanto100/Enrei and Kanto100/Kanto100. These results indicate that rhizobial nodulation suppresses AM fungal colonization, and the autoregulation of host plant, initiated by nodulation, is involved in this phenomenon.     

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

N natural abundance of biologically fixed N2 in soybean is controlled more by the Bradyrhizobium strain than by the variety of the host plant

The ¹⁵N natural abundance technique is one of those most easily applied ‘on farm’ to evaluate the contribution of biological N₂ fixation (BNF) to legume crops. When proportional BNF inputs are high, the accuracy of this technique is highly dependent on an accurate estimate of the ¹⁵N abundance of the N derived from N₂ fixation (the ‘B’ value). The objective of this study was to determine the influence of soybean variety on ‘B’ value. Plants of five soybean varieties were inoculated separately with two Bradyrhizobium strains (one Bradyrhizobium japonicum and one Bradyrhizobium elkanii) grown in pots of soil virtually free of bradyrhizobia capable of nodulating soybean. The proportion of N derived from BNF (%Ndfa) was estimated in separate pots where a small quantity of enriched ¹⁵N ammonium sulphate was added. The %Ndfa was then used with the ¹⁵N natural abundance data of the nodulated soybean and non-N₂-fixing reference plants, to determine the ‘B’ value for each soybean variety/Bradyrhizobium association. The varieties nodulated by the B. japonicum strain showed significantly greater N content and %Ndfa than those nodulated by the B. elkanii strain, and in all cases the ‘B’ value of the shoot tissue (‘B_s’) was higher. The differences in ‘B_s’ values between varieties nodulated by the same Bradyrhizobium strain were insignificant, indicating that this parameter is influenced much more by the Bradyrhizobium strain than by the variety of the host plant.     

Accumulation of Copper,Lead, and Zinc by In Situ Plants Inoculated with AM Fungi in Multicontaminated Soil

Pot and field experiments were conducted to (1) evaluate bioavailability of copper (Cu), lead (Pb), and zinc (Zn) in contaminated soil and phytoremediation potential by in situ plants, B. pilosa var. radiate and Passiflora foetida var. hispida, as inoculated with arbuscular mycorrhizal (AM) fungi, and (2) compare the results of pot and field experiments. The B. pilosa var. radiate plant inoculated with AM fungi had significantly greater Cu concentrations in the shoots and roots than noninoculated plants. Passiflora foetida var. hispida plant inoculated with AM fungi also had significantly greater Cu and Pb concentrations in the roots than noninoculated plants. As the root dry weight of Passiflora foetida var. hispida inoculated with AM fungi dramatically increased, the root Cu, Pb, and Zn content of Passiflora foetida var. hispida inoculated with AM fungi increased by 9–14 times, as compared with the noninoculated plants. The AM fungi have potential to either promote plant growth or increase heavy‐metal accumulation. The values of element translocation proportion from root to shoot were Zn > Cu > Pb for both plant species in pot and field experiments. For both plant species, the results of the pot and field experiments were significantly different. The concentration values of the pot experiment were greater in comparison to the field experiment, and some values were significantly greater than those in the field experiment.     

Interaction between the soil yeast Rhodotorula mucilaginosa and the arbuscular mycorrhizal fungi Glomus mosseae and Gigaspora rosea

The effect of the soil yeast, Rhodotorula mucilaginosa LBA, on Glomus mosseae (BEG n°12) and Gigaspora rosea (BEG n°9) was studied in vitro and in greenhouse trials. Hyphal length of G. mosseae and G. rosea spores increased significantly in the presence of R. mucilaginosa. Exudates from R. mucilaginosa stimulated hyphal growth of G. mosseae and G. rosea spores. Increase in hyphal length of G. mosseae coincided with an increase in R. mucilaginosa exudates. No stimulation of G. rosea hyphal growth was detected when 0.3 and 0.5 ml per petri dish of yeast exudates was applied. Percentage root length colonization by G. mosseae in soybean (Glycine max L. Merill) and by G. rosea in red clover (Trifolium pratense L. cv. Huia) was increased only when the soil yeast was inoculated before G. mosseae or G. rosea was introduced. Beneficial effects of R. mucilaginosa on arbuscular mycorrhizal (AM) colonization were found when the soil yeast was inoculated either as a thin agar slice or as a volume of 5 and 10 ml of an aqueous solution. R. mucilaginosa exudates (20 ml per pots) applied to soil increased significantly the percentage of AM colonization of soybean and red clover.     

Competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions

This study tested the competitive ability of three locally isolated Cyclopia rhizobia and strain PPRICI3, the strain currently recommended for the cultivation of Cyclopia, a tea-producing legume. Under sterile glasshouse conditions, the three locally isolated strains were equally competitive with strain PPRICI3. In field soils, the inoculant strains were largely outcompeted by native rhizobia present in the soil, although nodule occupancy was higher in nodules growing close to the root crown (the original inoculation area). In glasshouse experiments using field soil, the test strains again performed poorly, gaining less than 6% nodule occupancy in the one soil type. The presence of Cyclopia-compatible rhizobia in field soils, together with the poor competitive ability of inoculant strains, resulted in inoculation having no effect on Cyclopia yield, nodule number or nodule mass. The native rhizobial population did not only effectively nodulate uninoculated control plants, they also out-competed introduced strains for nodule occupancy in inoculated plants. Nonetheless, the Cyclopia produced high crop yields, possibly due to an adequate supply of soil N.     

Nitrogen fixation and CO2 exchange in soybeans (Glycine max L.) inoculated with mixed cultures of different microorganisms

N₂ fixation, photosynthesis of whole plants and yield increases in soybeans inoculated with mixed cultures of Bradyrhizobium japonicum 110 and Pseudomonas fluorescens 20 or P. fluorescens 21 as well as Glomus mosseae were found in pot experiments in gray forest soil carried out in a growth chamber. The effects of pseudomonads and vesicular-arbuscular (VA) mycorrhizal fungus on these parameters were found to be the same. Dual inoculation of soybeans with mixed cultures of microorganisms stimulated nodulation, nitrogenase activity of nodules and enhanced the amount of biological nitrogen in plants as determined by the ¹⁵N dilution method in comparison to soybeans inoculated with nodule bacteria alone. An increased leaf area in dually infected soybeans was estimated to be the major factor increasing photosynthesis. P. fluorescens and G. mosseae stimulated plant growth, photosynthesis and nodulation probably due to the production of plant growth-promoting substances. Increasing phosphorus fertilizer rates within the range of 5–40 mg P 100 g^-1 1:1 (v/v) soil: sand in a greenhouse experiment led to a subsequent improvement in nodulation, and an enhancement of N₂ fixation and yield in soybeans dually inoculated with B. japonicum 110 and P. fluorescens 21. These indexes were considerably higher in P-treated plants inoculated with mixed bacterial culture than in plants inoculated with nodule bacteria alone.     

Accumulation of specific flavonoids in soybean (Glycine max (L.) Merr.) as a function of the early tripartite symbiosis with arbuscular mycorrhizal fungi and Bradyrhizobium japonicum (Kirchner) Jordan

This study is the first report assessing the effect of soil inoculation on the signalling interaction of Bradyrhizobium japonicum, arbuscular mycorrhizal fungi (AMF) and soybean plants throughout the early stages of colonisation that lead to the tripartite symbiosis. In a study using soil disturbance to produce contrasting indigenous AMF treatments, the flavonoids daidzein, genistein and coumestrol were identified as possible signals for regulating the establishment of the tripartite symbiosis. However, it was unclear whether soil disturbance induced changes in flavonoid root accumulation other than through changing the potential for AMF colonization. In this study, soil treatments comprising all possible combinations of AMF and B. japonicum were established to test whether (1) modifications in root flavonoid accumulation depend on the potential for AMF colonization, and (2) synthesis and accumulation of flavonoids in the roots change over time as a function of the early plant-microbial interactions that lead to the tripartite symbiosis. The study was comprised of two phases. First, maize was grown over 3-week periods to promote the development of the AM fungus Glomus clarum. Second, the interaction between soybean, G. clarum and B. japonicum was evaluated at 6, 10, 14 and 40 days after plant emergence. Root colonization by G. clarum had a positive effect on nodulation 14 days after emergence, producing, 30% more nodules which were 40% heavier than those on roots solely inoculated with B. japonicum. The tripartite symbiosis resulted in 23% more N₂ being fixed than did the simpler symbiosis between soybean and B. japonicum. The presence of both symbionts changed accumulation of flavonoids in roots. Daidzein and coumestrol increased with plant growth. However, development of the tripartite symbiosis caused a decrease in coumestrol; accumulation of daidzein, the most abundant flavonoid, was reduced in the presence of AMF.     

Nickel-tolerant Brevibacillus brevis and arbuscular mycorrhizal fungus can reduce metal acquisition and nickel toxicity effects in plant growing in nickel supplemented soil

The growth of clover (Trifolium repens ) and its uptake of N, P and Ni were studied following inoculation of soil with Rhizobium trifolii, and combinations of two Ni-adapted indigenous bacterial isolates (one of them was Brevibacillus brevis) and an arbuscular mycorrhizal (AM) fungus (Glomus mosseae). Plant growth was measured in a pot experiment containing soil spiked with 30 (Ni I), 90 (Ni II) or 270 (Ni III) mg kg⁻¹ Ni-sulphate (corresponding to 11.7, 27.6 and 65.8 mg kg⁻¹ available Ni on a dry soil basis). Single inoculation with the most Ni-tolerant bacterial isolate (Brevibacillus brevis) was particularly effective in increasing shoot and root biomass at the three levels of Ni contamination in comparison with the other indigenous bacterial inoculated or control plants. Single colonisation of G. mosseae enhanced by 3 fold (Ni I), by 2.4 fold (Ni II) and by 2.2 fold (Ni III) T. repens dry weight and P-content of the shoots increased by 9.8 fold (Ni I), by 9.9 fold (Ni II) and by 5.1 fold (Ni III) concomitantly with a reduction in Ni concentration in the shoot compared with non-treated plants. Coinoculation of G. mosseae and the Ni-tolerant bacterial strain (B. brevis) achieved the highest plant dry biomass (shoot and root) and N and P content and the lowest Ni shoot concentration. Dual inoculation with the most Ni-tolerant autochthonous microorganisms (B. brevis and G. mosseae) increased shoot and root plant biomass and subtantially reduced the specific absorption rate (defined as the amount of metal absorbed per unit of root biomass) for nickel in comparison with plants grown in soil inoculated only with G. mosseae. B. brevis increased nodule number that was highly depressed in Ni I added soil or supressed in Ni II and Ni III supplemented soil. These results suggest that selected bacterial inoculation improved the mycorrhizal benefit in nutrients uptake and in decreasing Ni toxicity. Inoculation of adapted beneficial microorganisms (as autochthonous B. brevis and G. mosseae) may be used as a tool to enhance plant performance in soil contaminated with Ni.     

Lime pelleting inoculated serradella (Ornithopus spp.) increases nodulation and yield

Lime pelleting of the inoculated seed is recommended for most pasture legume species to improve survival of the rhizobia on the seed and to counter deleterious effects of soil or fertiliser acidity on rhizobial numbers. Except for New South Wales, lime pelleting is specifically not recommended for serradella (Ornithopus spp.). Our objectives were to evaluate effects of lime pelleting on bradyrhizobial numbers on seed, and nodulation and growth of the serradella plants. Three experiments are reported at two acid-soil sites in northern New South Wales involving four cultivars of yellow serradella (Ornithopus compressus) and Bradyrhizobium sp. (Lupinus) strains WSM471 (current inoculant strain) and WU425 and WSM480. Lime pelleting increased bradyrhizobial numbers on seed, 24 h after inoculation, by an average of 90%. Similarly, lime pelleting increased nodulation and shoot dry matter of the inoculated plants by an average of 57 and 28%, respectively. The three strains were similar in effects on plant growth. Relative values for shoot dry weight, averaged over sites, were 100 for WSM471 and 98 for both WU425 and WSM480. Our results confirmed previous research that lime pelleting inoculated serradella seed was not deleterious to survival of the bradyrhizobial inoculum, and showed that it could result in enhanced symbiotic activity of the inoculum in some instances. We recommend lime pelleting of serradella and that WSM471 remain the inoculant strain.     

Nodulation competition among Bradyrhizobium japonicum strains as influenced by rhizosphere bacteria and iron availability

Summary Bacteria isolated from the root zones of field-grown soybean plants [Glycine max (L.) Merr.] were examined in a series of glasshouse experiments for an ability to affect nodulation competition among three strains of Bradyrhizobium japonicum (USDA 31, USDA 110, and USDA 123). Inocula applied at planting contained competing strains of B. japonicum with or without one of eleven isolates of rhizosphere bacteria. Tap-root nodules were harvested 28 days after planting, and nodule occupancies were determined for the bradyrhizobia strains originally applied. Under conditions of low iron availability, five isolates (four Pseudomonas spp. plus one Serratia sp.) caused significant changes in nodule occupancy relative to the corresponding control which was not inoculated with rhizosphere bacteria. During subsequent glasshouse experiments designed to verify and further characterize these effects, three fluorescent Pseudomonas spp. consistently altered nodulation competition among certain combinations of bradyrhizobia strains when the rooting medium did not contain added iron. This alteration typically reflected enhanced nodulation by USDA 110. Two of these isolates produced similar, although less pronounced, effects when ferric hydroxide was added to the rooting medium. The results suggest that certain rhizosphere bacteria, particularly fluorescent Pseudomonas spp., can affect nodulation competition among strains of R. japonicum. An additional implication is that iron availability may be an important factor modifying interactions involving the soybean plant, B. japonicum, and associated microorganisms in the host rhizosphere.Paper No. 10648 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601, USA     

Use of aliette,a basipetally translocated antimicrobial compound,to enhance rhizophere colonization and nodulation by root-nodule bacteria

Summary A method was developed to improve the colonizing ability of inoculated strains of root-nodule bacteria using aliette (aluminum tris-O-ethyl phosphonate), a basipetally translocated fungicide. Aliette applied to seeds of alfalfa inoculated with an aliette-resistant strain of Rhizobium meliloti increased the numbers of R. meliloti in the rhizosphere after 3 but not 37 days, increased the number of nodules, and with some seed treatments, increased the growth of alfalfa. The enhanced colonization by R. meliloti as a result of seed treatment with aliette lasted for at least 31 days for alfalfa, although plant weights did not increase, Colonization by R. meliloti was further enhanced if seeds and foliage were treated with the fungicide. Coating seeds or sparaying the foliage with aliette also increased the number and weight of nodules and nitrogenase activity in soybeans inoculated with an aliette-resistant strain of Bradyrhizobium japonicum. The stimulation of B. japonicum in the rhizosphere and of nodulation was evident with successive plantings of soybeans if the seeds for each planting were treated with the chemical, but aliette did not increase the yield of inoculated soybeans in the subsequent plantings. With only the seeds of the first planting of inoculated soybeans treated with aliette, the numbers of B. japonicum in the rhizosphere of subsequent plantings were only occasionally greater and the numbers of nodules on the later plantings were not increased. We suggest that root colonization, nodulation, and N₂ fixation by Rhizobium and Bradyrhizobium may be enhanced by the use of basipetally translocated antimicrobial compounds together with root-nodule bacteria that are resistant to those compounds.     

Enhanced root nodulation of subterranean clover (Trifolium subterraneum) byRhizobium leguminosarium biovartrifolii in the presence of the earthwormAporrectodea trapezoides (Lumbricidae)

In a greenhouse study, the effect of the earthwormAporrectodea trapezoides on root nodulation in seedlings of subterranean clover (Trifolium subterraneum) was examined in the presence and absence of addedRhizobium leguminosarium biovartrifolii (strain NA 30). WhenR. trifolii NA 30 was inoculated into dung and placed on the soil surface, the total number of root nodules was five times greater (P<0.001) in the presence of earthworms than without earthworms and the number of nodules on the primary root of the plants 2–8 cm below the soil surface was 4 to 6 times greater (P<0.001) in the presence of earthworms. The additional nodulation did not affect plant growth or foliar N. When NA30 was dispersed through the soil at the beginning of the experiment, the presence of earthworms did not influence the level of root nodulation. The presence of earthworms increased root dry weight by 20–30%, plant top weight by up to 125% (P<0.001), and foliar N by 5–25% (P<0.001). Surface-applied dung increased the dry weight of plant tops (2-to 3-fold,P<0.001) but did not affect the concentration of foliar N (P<0.005).