Improvement of biological nitrogen fixation in Egyptian winter legumes through better management of Rhizobium

The diversity of rhizobia nodulating common bean ( Phaseolus vulgaris), berseem clover (Trifolium alexanderinum) and lentil (Lens culinaris) was assessed using several characterization techniques, including nitrogen fixation efficiency, intrinsic antibiotic-resistance patterns (IAR), plasmid profiles, serological markers and rep-PCR fingerprinting. Wide diversity among indigenous rhizobial populations of the isolates from lentil, bean and clover was found. Strikingly, a large percentage of the indigenous rhizobial population was extremely poor at fixing nitrogen. This emphasizes the need to increase the balance of highly efficient strains within the rhizobial population. Use of high-quality inocula strains that survive and compete with other less-desired and less-efficient N2-fixing rhizobia represents the best approach to increase biological nitrogen fixation of the target legume. In field-grown lentils, the inoculant strains were not able to outcompete the indigenous rhizobia and the native lentil rhizobia occupied 76–88% of the total nodules formed on inoculated plants. Nitrogen fixation by lentils, estimated using the ¹⁵N isotope dilution technique, ranged between 127 to 139 kg ha-1 in both inoculated and un-inoculated plants. With berseem clover, the inoculant strains were highly competitive against indigenous rhizobia and occupied 52–79% of all nodules. Inoculation with selected inocula improved N2 fixation by clover from 162 to 205 kg ha-1 in the three cuts as compared with 118 kg ha-1 in the un-inoculated treatment. The results also indicated the potential for improvement of N2 fixation by beans through the application of efficient N2-fixing rhizobia.     

Comparison of N(2) Fixation and Yields in Cajanus cajan between Hydrogenase-Positive and Hydrogenase-Negative Rhizobia by In Situ Acetylene Reduction Assays and Direct N Partitioning

Pigeon peas [Cajanus cajan (L.) Millsp.] were grown in soil columns containing ¹⁵N-enriched organic matter. Seasonal N₂ fixation activity was determined by periodically assaying plants for reduction of C₂H₂. N₂ fixation rose sharply from the first assay period at 51 days after planting to a peak of activity between floral initiation and fruit set. N₂ fixation (acetylene reduction) activity dropped concomitantly with pod maturation but recovered after pod harvests. Analysis of ¹⁵N content of plant shoots revealed that approximately 91 to 94% of plant N was derived from N₂ fixation. The effect of inoculation with hydrogenase-positive and hydrogenase-negative rhizobia was examined. Pigeon peas inoculated with strain P132 (hydrogenase-positive) yielded significantly more total shoot N than other inoculated or uninoculated treatments. However, two other hydrogenase-positive strains did not yield significantly more total shoot N than a hydrogenase-negative strain. The extent of nodulation by inoculum strains compared to indigenous rhizobia was determined by typing nodules according to intrinsic antibiotic resistance of the inoculum strains. The inoculum strains were detected in almost all typed nodules of inoculated plants.

Gas samples were taken from soil columns several times during the growth cycle of the plants. H₂ was never detected, even in columns containing pigeon peas inoculated with hydrogenase-negative rhizobia. This was attributed to H₂ consumption by soil bacteria. Estimation of N₂ fixation by acetylene reduction activity was closest to the direct ¹⁵N method when ethylene concentrations in the gas headspace (between the column lid and soil surface) were extrapolated to include the soil pore space as opposed solely to measurement in the headspace. There was an 8-fold difference between the two acetylene reduction assay methods of estimation. Based on a planting density of 15,000 plants per hectare, the direct ¹⁵N fixation rates ranged from 67 (noninoculated) to 134 kilograms per hectare, while grain yields ranged from 540 to 825 kilograms per hectare. Grain yields were not increased with N fertilizer.

     

Peanut (Arachis hypogaea) response to inoculation with Bradyrhizobium sp. in soils of Argentina

Soil bacteria (rhizobia) of the genus Bradyrhizobium form symbiotic relationships with peanut root cells and fix atmospheric nitrogen by converting it to nitrogenous compounds. Inoculation of peanut with rhizobia can enhance the plant’s ability to fix nitrogen from the air and thereby reduce the requirement for nitrogen fertiliser. We evaluated three Bradyrhizobium sp. strains for effect on root nodulation and on pod yield of peanut in Argentina soils, using laboratory and field experiments. Of these, strain C‐145 was the most effective in laboratory studies. In‐furrow inoculation with this strain produced increased nodule number, relative to seed inoculation. However, pod yield was not increased significantly by either type of inoculation. In view of the inconsistent response of peanut to inoculation, we examined the effect of indigenous strains of bradyrhizobia. The high degree of nodulation and nitrogen fixation produced by indigenous rhizobia were sufficient for maximal yield under the field and inoculation conditions used in this study. The data are important for future investigation of alternative inoculant strains and conditions for improving peanut production.     

Influence of different Sinorhizobium meliloti inocula on abundance of genes involved in nitrogen transformations in the rhizosphere of alfalfa (Medicago sativa L.)

Inoculation of leguminous seeds with selected rhizobial strains is practised in agriculture to ameliorate the plant yield by enhanced root nodulation and nitrogen uptake of the plant. However, effective symbiosis between legumes and rhizobia does not only depend on the capacity of nitrogen fixation but also on the entire nitrogen turnover in the rhizosphere. We investigated the influence of seed inoculation with two indigenous Sinorhizobium meliloti strains exhibiting different efficiency concerning plant growth promotion on nitrogen turnover processes in the rhizosphere during the growth of alfalfa. Quantification of six target genes (bacterial amoA, nirK, nirS, nosZ, nifH and archaeal amoA) within the nitrogen cycle was performed in rhizosphere samples before nodule formation, at bud development and at the late flowering stage. The results clearly demonstrated that effectiveness of rhizobial inocula is related to abundance of nifH genes in the late flowering phase of alfalfa. Moreover, other genes involved in nitrogen turnover had been affected by the inocula, e.g. higher numbers of amoA copies were observed during flowering when the more effective strain had been inoculated. However, the respective gene abundances differed overall to a greater extent between the three plant development stages than between the inoculation variants.     

A rhizobia strain isolated from root nodule of gymnosperm Podocarpus macrophyllus

Symbiotic nitrogen fixation of rhizobia and leguminous plants is considered as the most important biologic nitrogen fixation system on earth. Symbiotic nodulation of gymnosperm Podocarpus macro-phyllus and rhizobia has never been reported. In this study, 11 endophytic bacteria strains were isolated from root nodules of P. macrophyllus and its variation P. macrophyllus var. maki. The plant infection tests on these strains indicated that the isolated strains could be nodulated on P. macrophyllus plants, and weak nitrogenase activity of nodules was found in acetylene reduction method. According to the physiological and biochemical characteristics of the 11 strains, GXLO 02 was selected as the representative strain. 16S rDNA full-length sequence analysis of GXLO 02 confirmed that the representative strain GXLO 02 belongs to Rhizobium sp.     

Survival of Bradyrhizobium sp. (Arachis) on fungicide-treated peanut seed in relationship to plant growth and yield

Survival and viability of Bradyrhizobium inoculant on fungicide-treated peanut seed and the resulting effects on nitrogen fixation, plant growth and seed yield were determined. Vitavax and Benomyl had the most and least lethal actions against Bradyrhizobium strains grown on YEM medium containing a fungicide, respectively, while Thiram and Captan effects were intermediate. Survival of Bradyrhizobium USDA 3384 and USDA 3456, as single strain peat inoculants, on peanut (Arachis hypogaea L. var. Florunner) seeds treated with Benomyl or Vitavax at the rate of 3g/kg seed was also examined. Both fungicides inhibited the growth and affected the survival of strain USDA 3384 on peanut seed. Vitavax killed the inoculant in 9 h. In contrast, USDA 3456 resisted both fungicides, and survived for up to 72h. Nodule formation on greenhouse-grown plants inoculated with USDA 3384 was inhibited by all fungicides. Shoot dry weight and plant nitrogen content significantly decreased as compared to controls. Fungicides, except Vitavax, had a slight effect on nodulation and plant growth when USDA 3456 was used as inoculant. The agronomic importance of fungicide-inoculant interaction was examined in field experiments conducted in Egypt in soil free of peanut-nodulating Bradyrhizobium, where seeds were treated with a combination of two fungicides and a single strain peat inoculant of either USDA 3384 or USDA 3456. All fungicides decreased nodulation, nitrogen fixation, plant growth and seed yield, especially with USDA 3384 as inoculant. Fungicides inhibited viability and survival of Bradyrhizobium on peanut seeds which decreased nodule formation leading to reduced peanut seed yield.     

A rhizobia strain isolated from root nodule of gymnosperm Podocarpus macrophyllus

Symbiotic nitrogen fixation of rhizobia and leguminous plants is considered as the most important biologic nitrogen fixation system on earth. Symbiotic nodulafion of gymnosperm Podocarpus macrophyllus and rhizobia has never been reported. In this study, 11 endophytic bacteria strains were isolated from root nodules of P. macrophyllus and its variation P. macrophyllus var. maki. The plant infection tests on these strains indicated that the isolated strains could be nodulated on P. macrophyllus plants, and weak nitrogenase activity of nodules was found in acetylene reduction method. According to the physiological and biochemical characteristics of the 11 strains, GXLO 02 was selected as the representative strain. 16S rDNA full-length sequence analysis of GXLO 02 confirmed that the representative strain GXLO 02 belongs to Rhizobium sp.     

Inoculation Response of Legumes in Relation to the Number and Effectiveness of Indigenous Rhizobium Populations

The response of legumes to inoculation with rhizobia can be affected by many factors. Little work has been undertaken to examine how indigenous populations or rhizobia affect this response. We conducted a series of inoculation trials in four Hawaiian soils with six legume species (Glycine max, Vigna unguiculata, Phaseolus lunatus, Leucaena leucocephala, Arachis hypogaea, and Phaseolus vulgaris) and characterized the native rhizobial populations for each species in terms of the number and effectiveness of the population for a particular host. Inoculated plants had, on average, 76% of the nodules formed by the inoculum strain, which effectively eliminated competition from native strains as a variable between soils. Rhizobia populations ranged from less than 6 × 10⁰/g of soil to 1 × 10⁴/g of soil. The concentration of nitrogen in shoots of inoculated plants was not higher than that in uninoculated controls when the most probable number MPN counts of rhizobia were at or above 2 × 10¹/g of soil unless the native population was completely ineffective. Tests of random isolates from nodules of uninoculated plants revealed that within most soil populations there was a wide range of effectiveness for N₂ fixation. All populations had isolates that were ineffective in fixing N₂. The inoculum strains generally did not fix more N₂ than the average isolate from the soil population in single-isolate tests. Even when the inoculum strain proved to be a better symbiont than the soil rhizobia, there was no response to inoculation. Enhanced N₂ fixation after inoculation was related to increased nodule dry weights. Although inoculation generally increased nodule number when there were less than 1 × 10² rhizobia per g of soil, there was no corresponding increase in nodule dry weight when native populations were effective. Most species compensated for reduced nodulation in soils with few rhizobia by increasing the size of nodules and therefore maintaining a nodule dry weight similar to that of inoculated plants with more nodules. Even when competition by native soil strains was overcome with a selected inoculum strain, it was not always possible to enhance N₂ fixation when soil populations were above a threshold number and had some effective strains.     

Competitiveness of a <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. Strain in Soils Containing Indigenous Rhizobia

The success of rhizobial inoculation on plant roots is often limited by several factors, including environmental conditions, the number of infective cells applied, the presence of competing indigenous (native) rhizobia, and the inoculation method. Many approaches have been taken to solve the problem of inoculant competition by naturalized populations of compatible rhizobia present in soil, but so far without a satisfactory solution. We used antibiotic resistance and molecular profiles as tools to find a reliable and accurate method for competitiveness assay between introduced Bradyrhizobium sp. strains and indigenous rhizobia strains that nodulate peanut in Argentina. The positional advantage of rhizobia soil population for nodulation was assessed using a laboratory model in which a rhizobial population is established in sterile vermiculite. We observed an increase in nodule number per plant and nodule occupancy for strains established in vermiculite. In field experiments, only 9% of total nodules were formed by bacteria inoculated by direct coating of seed, whereas 78% of nodules were formed by bacteria inoculated in the furrow at seeding. In each case, the other nodules were formed by indigenous strains or by both strains (inoculated and indigenous). These findings indicate a positional advantage of native rhizobia or in-furrow inoculated rhizobia for nodulation in peanut.     

The effect of Rhizobium inoculation and nitrogen fertiliser on nitrogen fixation and seed yield of dry beans (Phaseolus vulgaris)

The potential benefit to be derived from seed inoculation of Phaseolus vulgaris beans with effective strains of Rhizobium phaseoli, was investigated in field experiments over three years on a site low in soil nitrogen and lacking indigenous effective strains of R. phaseoli. Inoculation with R. phaseoli (strain RCR 3644) produced significant increases in nodulation, nitrogenase activity and plant growth in all experiments. In trials in 1978 and 1979, with cv. Seafarer, inoculation, in the absence of nitrogen fertiliser doubled seed yields. In 1978, the seed yields from inoculated beans without nitrogen fertiliser (1–6 t/ha) were not significantly different from those obtained with uninoculated beans receiving the optimum nitrogen fertiliser treatment of 120 kg N/ha (1–75 t/ha). In 1979, with lower rainfall favouring more efficient utilisation of nitrogen fertiliser, inoculation gave seed yields (1–88 t/ha) equivalent to those obtained with 60 kg N/ha (1–70 t/ha) but significantly less than with 120 kg N/ha (2–88 t/ha). More precise estimates from nitrogen response curves showed that inoculation supplied the fertiliser equivalent of 105 and 70 kg N/ha in 1978 and 1979 respectively. In both years, significant benefits were also obtained by the combination of inoculation and nitrogen fertiliser. In a separate experiment in 1979, with four R. phaseoli strains inoculated onto eight bean cultivars, three were highly effective nitrogen fixers on all cultivars. Two strains (RCR 3644 and NVRS 963A) each increased mean yields, in the absence of nitrogen fertiliser, from 1–39 t/ha uninoculated to c. 2–5 t/ha inoculated whilst strain RCR 3622 was outstanding with a mean yield of 3-0 t/ha. An analysis of the nitrogen content of seed showed that gains from nitrogen fixation were 37–57 kg N/ha/growing cycle for the combination RCR 3644 with cv. Seafarer. However, 106 kg N/ha/growing cycle was recorded for the combination RCR 3622 and cv. Aurora.     

Ureide assay for measuring nitrogen fixation by nodulated soybean calibrated by N methods

We report experiments to quantify the relationships between the relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap, and hot water extracts of stems and petioles of nodulated soybean (Glycine max [L.] Merrill cv Bragg) and the proportion of plant N derived from nitrogen fixation. Additional experiments examined the effects of plant genotype and strain of rhizobia on these relationships. In each of the five experiments reported, plants of cv Bragg (experiment 1), cv Lincoln (experiments 3, 4, 5), or six cultivars/genotypes (experiment 2) were grown in a sand:vermiculite mixture in large pots in a naturally lit, temperature-controlled glasshouse during summer. Pots were inoculated at sowing with effective Bradyrhizobium japonicum CB1809 (USDA 136) or with one of 21 different strains of rhizobia. The proportions of plant N derived from nitrogen fixation were determined using ¹⁵N dilution. In one experiment with CB1809, plants were supplied throughout growth with either N-free nutrients or with nutrients supplemented with 1, 2, 4, or 8 millimolar ¹⁵N-nitrate and harvested on eight occasions between V6 and R7 for root-bleeding sap, vacuum-extracted sap, stems (including petioles), and whole plant dry matter. Analyses of the saps and stem extracts for ureides (allantoin plus allantoic acid), α-amino-N, and nitrate, and of dry matter for N and ¹⁵N, indicated a positive effect of nitrate supply on concentrations of nitrate in saps and extracts and a negative effect on ureides and on the proportion of plant N derived from nitrogen fixation. The relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap (100 [ureide-N]/[ureide-N+ α-amino-N + nitrate-N]) and stem extracts (100 [ureide-N]/[ureide-N + nitrate-N]) and the proportion of plant N, derived from nitrogen fixation between successive samplings were highly correlated (r = 0.97-1.00). For each variable, two standard curves were prepared to account for the shifts in the compositions of N solutes of xylem saps and extracts after flowering which were not related to a change in nitrogen fixation. Relationships between relative ureide-N and the proportion of plant N derived from nitrogen fixation were not affected by plant genotype or by strain of rhizobia. Therefore, assessment of nitrogen fixation by soybean using the ureide technique should now be possible with the standard curves presented, irrespective of genotype or strain of rhizobia occupying the nodules.     

Response of Leucaena/Rhizobium symbiosis to mineral nutrients in Southwestern Nigeria

Pot and field experiments carried out at the International Institute of Tropical Agriculture (IITA) and at Fashola, Southwestern Nigeria, examined the effect of inoculation and N, P and micronutrients on nodulation and growth ofLeucaena leucocephala (Lam.) de Wit. In pot studies all parameters measured, except the percentage of nitrogen in shoots, were improved by inoculation, nitrogen and phosphorus. Micronutrients increased only nitrogen and allantoin contents. Interactions between inoculation and P, N and micronutrients on nodulation and growth of leucaena were observed. The effect of inoculation and fertilization with phosphorus or micronutrients was further investigated in field experiments. Establishment of uninoculated and unfertilized leucaena was poor at both locations due to low soil fertility and the presence of only a few native leucaena rhizobia. At one site, only inoculated plants were nodulated, while at the other, all plants produced nodules. Shoot dry weight, total nitrogen and phosphorus of inoculated plants were statistically equal to nitrogen-fertilized plants. Uninoculated plants were stunted. Generally, micronutrients did not influence nodulation, total nitrogen or growth of leucaena. They had only a positive effect on nitrogenase activity. Phosphorus increased total nitrogen and phosphorus uptake and plant growth. A 75% increase in shoot dry weight was obtained when 80 kg P ha^–1 was applied to inoculated leucaena with Rhizobium strain IRc 1045. Inoculated plants contained more allantoins than uninoculated ones but no significant correlation was found between these compounds and other parameters of N fixation.     

Modeling Symbiotic Performance of Introduced Rhizobia in the Field by Use of Indices of Indigenous Population Size and Nitrogen Status of the Soil

The ability to predict the symbiotic performance of rhizobia introduced into different environments would allow for a more judicious use of rhizobial inoculants. Data from eight standardized field inoculation trials were used to develop models that could be used to predict the success of rhizobial inoculation in diverse environments based on indices of the size of indigenous rhizobial populations and the availability of mineral N. Inoculation trials were conducted at five diverse sites on the island of Maui, Hawaii, with two to four legumes from among nine species, yielding 29 legume-site observations. The sizes of indigenous rhizobial populations were determined at planting. Soil N mineralization potential, total soil N, N accumulation and seed yield of nonnodulating soybean, and N derived from N₂ fixation in inoculated soybean served as indices of available soil N. Uninoculated, inoculated, and fertilizer N treatments evaluated the impact of indigenous rhizobial populations and soil N availability on inoculation response and crop yield potential. The ability of several mathematical models to describe the inverse relationship between numbers of indigenous rhizobia and legume inoculation responses was evaluated. Power, exponential, and hyperbolic functions yielded similar results; however, the hyperbolic equation provided the best fit of observed to estimated inoculation responses (r² = 0.59). The fact that 59% of the observed variation in inoculation responses could be accounted for by the relationship of inoculation responses to numbers of indigenous rhizobia illustrates the profound influence that the size of soil rhizobial populations has on the successful use of rhizobial inoculants. In the absence of indigenous rhizobia, the inoculation response was directly proportional to the availability of mineral N. Therefore, the hyperbolic response function was subsequently combined with several indices of soil N availability to generate models for predicting legume inoculation response. Among the models developed, those using either soil N mineralization potential or N derived from N₂ fixation in soybean to express the availability of mineral N were most useful in predicting the success of legume inoculation. Correlation coefficients between observed and estimated inoculation responses were r = 0.83 for the model incorporating soil N mineralization potential and r = 0.96 for the model incorporating N derived from N₂ fixation. Several equations collectively termed “soil N deficit factors” were also found to be useful in estimating inoculation responses. In general, models using postharvest indices of soil N were better estimators of observed inoculation responses than were those using laboratory measures of soil N availability. However, the latter, while providing less precise estimates, are more versatile because all input variables can be obtained through soil analysis prior to planting. These models should provide researchers, as well as regional planners, with a more precise predictive capability to determine the inoculation requirements of legumes grown in diverse environments.     

Engineering the nifH promoter region and abolishing poly-beta-hydroxybutyrate accumulation in Rhizobium etli enhance nitrogen fixation in symbiosis with Phaseolus vulgaris

Rhizobium etli, as well as some other rhizobia, presents nitrogenase reductase (nifH) gene reiterations. Several R. etli strains studied in this laboratory showed a unique organization and contained two complete nifHDK operons (copies a and b) and a truncated nifHD operon (copy c). Expression analysis of lacZ fusion demonstrated that copies a and b in strain CFN42 are transcribed at lower levels than copy c, although this copy has no discernible role during nitrogen fixation. To increase nitrogenase production, we constructed a chimeric nifHDK operon regulated by the strong nifHc promoter sequence and expressed it in symbiosis with the common bean plant (Phaseolus vulgaris), either cloned on a stably inherited plasmid or incorporated into the symbiotic plasmid (pSym). Compared with the wild-type strain, strains with the nitrogenase overexpression construction assayed in greenhouse experiments had, increased nitrogenase activity (58% on average), increased plant weight (32% on average), increased nitrogen content in plants (15% at 32 days postinoculation), and most importantly, higher seed yield (36% on average), higher nitrogen content (25%), and higher nitrogen yield (72% on average) in seeds. Additionally, expression of the chimeric nifHDK operon in a poly-beta-hydroxybutyrate-negative R. etli strain produced an additive effect in enhancing symbiosis. To our knowledge, this is the first report of increased seed yield and nutritional content in the common bean obtained by using only the genetic material already present in Rhizobium.     

Ineffective utilization of nitrate by soybean during pod fill

The relative effectiveness of nitrate, allantoin, or nitrate plus allantoin as sources of nitrogen for the indeterminate soybean plant [ Glycine max (L.) Merr cv. Harper] was studied throughout vegetative and reproductive growth. All plants were provided with 3.0 m M nitrogen and were grown hydroponically in growth chambers. During vegetative and early reproductive growth, plants given nitrate or nitrate plus allantoin grew faster than plants provided allantoin only. However, during pod fill, plants provided with allantoin or allantoin plus nitrate gained weight more rapidly than plants receiving just nitrate. More importantly, at maturity plants that had been provided with allantoin or allantoin plus nitrate during pod fill were 30% heavier in total dry weight, 50% higher in nitrogen content, and 50% higher in seed yield than plants that had received just nitrate. At full bloom, all plants were inoculated with the same culture of Bradyrhizobium japonicum , and twice each week throughout pod fill each plant was assayed for nitrogen fixation (acetylene reduction). Correlation coefficients obtained by linear regression analysis show a strong positive correlation between the measured rate of nitrogen fixation and maximum plant fresh weight (r = 0.83), total plant nitrogen (r = 0.81), or seed yield (r = 0.76). The fact that nitrogen fixation during pod fill stimulates plant growth and seed yield, coupled with the facts that nitrate blocks nodulation and is not used efficiently during pod fill by the soybean plant, may explain why seed yield of field-grown soybeans usually does not respond to added fertilizer nitrogen. Thus, it is suggested that enhanced nitrogen fixation may be the key factor in improving soybean seed yield.     

Effect of inoculation and leaf litter amendment on establishment of nodule-forming Frankia populations in soil

High-N(2)-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [(15)N]NO(3) and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N(2) fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N(2) fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% +/- 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% +/- 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% +/- 6%) than by group IV (81% +/- 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N(2) fixation rates by (15)N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N(2)-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N(2)-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

Enhancing the growth of Vicia faba plants by microbial inoculation to improve their phytoremediation potential for oily desert areas

Two experiments were conducted to investigate the effect of inoculating Vicia faba plants (broad beens) raised in clean and oily sand with nodule-forming rhizobia and plant-growth-promoting rhizobacteria (PGPR) on growth of these plants in sand and to test whether this can improve the phytoremediation potential of this crop for oily desert areas. It was found that crude oil in sand at concentrations < 1.0% (w/w) enhanced the plant heights, their fresh and dry weights, the total nodule weights per plant, and the nitrogen contents of shoots and fruits. Similar enhancing effects were recorded when roots of the young plants were inoculated with nodule bacteria alone, PGPR alone, or a mixture of one strain of nodule bacteria and one of the PGPR. Such plant growth effects were associated with a better phytoremediation potential of V. faba plants for oily sand. The total numbers of oil-utilizing bacteria increased in the rhizosphere and more hydrocarbons were eliminated in sand close to the roots. The nodule bacteria tested were two strains of Rhizobium leguminosarum and the PGPR were Pseudomonas aeruginosa and Serratia liquefaciens. The four strains were found to use crude oil, n-octadecane, and phenanthrene as sole sources of carbon and energy. It was concluded that coinoculation of V. faba plant roots in oily sand with nodule bacteria and PGPR enhances the phytoremediation potential of this plant for oily desert sand through improving plant growth and nitrogen fixation.     

Combined inoculation with Glomus intraradices and Rhizobium tropici CIAT899 increases phosphorus use efficiency for symbiotic nitrogen fixation in common bean (Phaseolus vulgaris L.)

This study compared the response of common bean (Phaseolus vulgaris L.) to arbuscular mycorrhizal fungi (AMF) and rhizobia strain inoculation. Two common bean genotypes i.e. CocoT and Flamingo varying in their effectiveness for nitrogen fixation were inoculated with Glomus intraradices and Rhizobium tropici CIAT899, and grown for 50 days in soil–sand substrate in glasshouse conditions. Inoculation of common bean plants with the AM fungi resulted in a significant increase in nodulation compared to plants without inoculation. The combined inoculation of AM fungi and rhizobia significantly increased various plant growth parameters compared to simple inoculated plants. In addition, the combined inoculation of AM fungi and rhizobia resulted in significantly higher nitrogen and phosphorus accumulation in the shoots of common bean plants and improved phosphorus use efficiency compared with their controls, which were not dually inoculated. It is concluded that inoculation with rhizobia and arbuscular mycorrhizal fungi could improve the efficiency in phosphorus use for symbiotic nitrogen fixation especially under phosphorus deficiency.     

Nodule ultrastructure and initial growth of Anadenanthera peregrina (L.) Speg. var. falcata (Benth.) altschul plants infected with rhizobia

The anatomy and ultrastructure of root nodules of Anadenanthera peregrina var. falcata (Leguminosae-Mimosoideae) were analysed, as was plant growth. To ensure that nodules developed, seedlings were inoculated with a mixture of six strains of rhizobia. Nodules were produced that differed in appearance-and probably also effectiveness-but their structure was similar and they showed characteristics typical of indeterminate nodules, such as persistent meristematic tissue and a gradient of cells at different stages of development. Many starch grains were present in inner cortex cells and interstitial cells of infected tissue. Infected cells were densely packed with bacteroids, which contained many poly-beta-hydroxybutyrate granules. The high incidence of these granules, together with high levels of starch accumulation in interstitial cells, suggested low N2-fixation efficiency of the rhizobia isolates used for inoculation. In the symbiosomes of early-senescent infected cells, reticulum-like structures, small vesicles and a fibrillar material were observed; these may be related to bacteroid degradation. In the cytoplasm of late-senescent infected cells, many vesicles and membrane-like structures were observed, probably associated with membrane degradation of bacteroids and peribacteroids. The total biomass of plants inoculated with rhizobia was low and their xylopodia and shoots had low levels of N compared with noninoculated plants fertilized with ammonium nitrate. However, inoculated plants did not show N-deficiency symptoms and grew better than non-inoculated plants without N fertilization. These growth results, together with ultrastructural observations of nodules, suggest that nitrogen fixation of rhizobia isolates associated with Anadenanthera peregrina var. falcata roots is poor.     

Effect of the VAM fungus Glomus sp. on the growth and yield of soybean inoculated with Bradyrhizobium japonicum

The effect of two Bradyrhizobium japonicum strains (D344 and Urbana), on the frequency and intensity of infection by a VAM fungal Glomus sp. and the effect of VAM on biomass production by nodulating plants were tested in soybean growing in a soil containing low levels of accessible P and N. During the initial stage of vegetative growth, mycorrhiza frequency in roots inoculated with the two rhizobial strains did not differ. However, during flowering it was 178% higher in roots with the strain D344 than in the presence of the strain Ubrana. At final harvest (green pods) the VAM frequency did not differ in the presence of either strain. VAM positively affected biomass production, foliar concentrations of P, Zn and Cu, and number and dry matter yield of pods, but did not increase concentrations of total N and K. In nonmycorrhizal plants total nitrogenase activity (not nodule mass) and growth were higher with the rhizobial strain Urbana. The greatest nitrogenase activity, growth and yield occurred in the presence of the VAM fungus, and did not differ for plants with different strains of rhizobia.