In vitro studies on the effects of herbicides on the growth of rhizobia

AIMS: To study the possible adverse effect of herbicides on nodulation and nitrogen fixation in legumes by affecting the nitrogen-fixing rhizobia. METHODS AND RESULTS: Experiments were conducted to study the effect of four herbicides (terbutryn/terbuthylazine, trietazine/simazine, prometryn and bentazone) on the growth of nitrogen-fixing pea rhizobia (Rhizobium leguminosarum) in vitro by measuring optical density. Terbutryn/terbuthylazine, trietazine/simazine and prometryn had an adverse effect on the growth of rhizobia whereas bentazone was safe to rhizobia. CONCLUSIONS: The above herbicides could be used in pea at the recommended rates. SIGNIFICANCE AND IMPACT OF THE STUDY: The adverse effects of herbicides on rhizobia were observed at concentrations not normally expected to occur under field conditions. Further, previously observed adverse effects of these herbicides on nodulation and nitrogen fixation of peas were, possibly, not due to their effects in rhizobia but to their adverse effects on the plant growth itself.     

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.     

Competition between soil- and seed-borneRhizobium trifolii in nodulation of introducedTrifolium subterraneum

Summary Native rhizobia associated withTrifolium albopurpureum, T. bifidum, T. ciliolatum, T. depauperatum, T. dichotomum, T. flavulum, T. melanthum, T. microcephalum, T. microdon, T. oliganthum andT. tridentatum were found in Northern California range soils. These rhizobia nodulate subterranean clover but are ineffective in nitrogen fixation with this host. Native rhizobia compet with those in commercial inoculants to form nodules. To ensure effective nodulation by nitrogen fixing rhizobia, commercial inoculants should be applied at rates greater than those recommended by the manufacturerse Effective nodulation was achieved by an application of 7.5×10⁴ rhizobia per seed, four times the recommended rate.     

Effect of manganese toxicity on growth and N2 fixation in groundnut, Arachis hypogaea

The development of manganese (Mn) toxicity symptoms and its effects on the growth, nodulation, and nitrogen fixation of groundnut genotypes were examined using a quartz-sand/solution culture system. The 11 genotypes tested all accumulated considerable concentrations of manganese (1.04–3.07 mg g^-1dry matter) when supplied with 15 μg Mn ml^-1of nutrient solution daily. Toxicity symptoms differed between genotypes: some showed no visual effects, some produced marginal leaf spots, and others developed marginal leaf spots coupled with an inward rolling of the margins of the younger leaves. The growth of one genotype (ICG 5394) grown with inorganic nitrogen as its source of N was more severely affected by Mn toxicity than when dependent on symbiotic fixation for its nitrogen.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Effect of methabenzthiazuron on growth and nitrogenase activity in Vicia faba

The effects of the herbicide methabenzthiazuron (175 and 220 g ha^-1) on vegetative and reproductive growth, nodulation and nitrogenase activity of Vicia faba were studied in the field under Mediterranean conditions. Nitrogenase activity of excised nodules was estimated using the acetylene reduction assay four times during the developmental period. Leaf area index, dry weight and nitrogen content of the different parts of the plants were measured. Methabenzthiazuron-treated plants showed an increase in nodulation, nitrogenase activity and vegetative growth at early pod fill. Methabenzthiazuron also caused an increase in leaf N content and fruits. These were transient effects found during early and mid pot fill. Nevertheless, plants treated with these sublethal doses of herbicide improved seed production and nitrogen content of seeds at harvest time. The stimulatory effect of methabenzthiazuron on N₂ fixation and vegetative growth seems not be related with the transient stimulatory effect on photosynthetic capacity, also caused by the herbicide, since the stimulatory effect on N₂ fixation was apparent during pod fill, when photosynthetic capacity declined and was not modified by methabenzthiazuron.     

Ontogenic variation in nitrogen assimilation,nodulation and nitrogen fixation in groundnut (Arachis hypogaea) genotypes*

Seasonal pattern of acetylene reduction (AR) and shoot nitrogen accumulation was studied in nine groundnut cultivars. Shoot N accumulation by all the cultivars was maintained until shortly before maturity and it occurred faster over the reproductíve growth phase than over the earlier phases. In all cultivars plant AR (PAR) did not reflect this pattern of N accumulation, being greater over the vegetative and pod initiation phases. This suggests that the commonly observed low PAR values for groundnut over the reproductive growth phase may be the result of factors other than sink competition. There were significant interactions of cultivar with stage of crop growth for PAR, nodule mass, and specific nitrogenase activity (SNA). Virginia types generally showed better nodulation, higher N₂-fixing capacity (both PAR and SNA) than valencias, and significant differences were observed between cultivars within a botanical type.     

The physiology and biochemistry of cultivar-strain interactions in the white clover-Rhizobium symbiosis

Two white clover cultivars were inoculated with two Rhizobium leguminosarum bv. trifolii strains in a factorial series of experiments. Plants were grown in axenic conditions in nitrogen free nutrient solution in a controlled environment room. Variations in nitrogen fixation were dependent partly upon general strain effects, partly upon general cultivar effects but there were also substantial differences attributable to precise interactions between specific combinations. The physiological and biochemical basis of these differences was examined. There were variations in the onset of nodulation and nitrogenase (acetylene reduction) activity. The rate at which nitrogenase activity developed also differed between associations as did the average size and number of nodules but none of these effects correlated well with differences in plant dry matter accumulation. Studies on nodule biochemistry revealed that the major nitrogen fixation enzymes were present in all four associations. Nodule protein content and enzyme activity (on a g nodule fresh weight basis) were substantially greater in associations formed by the more effective strain but cannot explain the interactive effect on dry matter accumulation. The relevance of these data to our understanding of factors regulating variations in nitrogen fixation is discussed.     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Nonparametric approach to multitrait selection for yield in groundnut (Arachis hypogaea L.)

Summary Eight characters related to nitrogen fixation and pod development measured 30 days after flowering were evaluated for their correct grading of the relative yield performance of 17 genetically diverse lines of groundnut (Arachis hypogaea L.). Each line was assigned a high or low yield status based on its pod yield, shelling percentage, and 100-kernel weight. Seventeen character combinations were examined for their relative merit in correct identification of the yield status of lines. The character sets, nitrogenase activity alone or in combination with nitrogen percent or shoot weight identified the status of 77% of lines correctly. The extent to which various characters accounted for the variation in pod yield was also checked by multiple regression analysis. While the character combination, nitrogen percent plus leaf area explained 75% of variation in pod yield, nodule mass, nitrogenase activity, and leaf area occurred in some other combinations that explained yield variation to a lesser extent. These analyses point to the profitability of involving crop physiological traits such as leaf area and nitrogen percent in selecting for relative yield performance in groundnut.IARI Regional Station, Tutikandi, Simla 171004, IndiaNational Research Center for Groundnut, Timbawadi, Junagadh 362015, India     

Inoculation of pea (Pisum sativum L.) by Rhizobium leguminosarum bv. viceae preincubated with naringenin and hesperetin or application of naringenin and hesperetin directly into soil increased pea nodulation under short season conditions

In short season areas, low soil temperature is the major limiting factor for symbiotic nitrogen fixation of legume. One greenhouse and four field experiments were conducted in 1999 to determine whether the pre-incubation of Rhizobium leguminosarum bv. viceae with hesperetin and naringenin or application of these compounds onto the seed surface or into the seed furrow at the time of planting can increase pea nodulation and final grain yield. The results from these experiments clearly indicated that application of naringenin and hesperetin by either pre-incubating R. leguminosarum bv. viceae prior to inoculation of plant or directly applying onto the seed surface or into seed furrow at the time of planting can increase pea nodulation, and plant pod numbers. Interactions existed between symbiotic signal compounds and pea cultivars or R. leguminosarum bv. viceae strains. However, there was no impact on the final grain yield by the treatments from the field experiments. The effects of these treatments on the final grain yield have to be farther tested.     

Comparative Proteomic Analysis of Soybean Nodulation Using a Supernodulation Mutant,SS2-2

Legumes have the ability to form root nodules that fix atmospheric nitrogen through a symbiotic interaction with nitrogen-fixing bacteria. As a first step in dissecting the molecular process of nodulation, proteome reference maps of soybean roots and nodules were constructed. Time course analysis revealed that the transition from root to nodule was accompanied with downregulation of defense-response related proteins, including Mn-superoxide dismutase, peroxidase (Prx), PR10, and stress-induced protein, leading to the initiation of a symbiotic interaction between the two partners. Following nitrogenase biosynthesis, the host plant cooperated with the rhizobia to fix atmospheric nitrogen under microaerobic conditions via expression of leghemoglobins and antioxidant proteins. Comparative proteome analysis indicated lower expression of malate dehydrogenase (MDH), leghemoglobins and nitrogenase in the nodule development of the supernodulation mutant, SS2-2, as compared to the wild type, indicating that SS2-2 forms functionally immature nodules in higher numbers with the lower activity of nitrogen fixation.     

N2 fixation by Acacia species increases under elevated atmospheric CO2

In the present study the effect of elevated CO₂ on growth and nitrogen fixation of seven Australian Acacia species was investigated. Two species from semi‐arid environments in central Australia (Acacia aneura and A. tetragonophylla) and five species from temperate south‐eastern Australia (Acacia irrorata, A. mearnsii, A. dealbata, A. implexa and A. melanoxylon) were grown for up to 148 d in controlled greenhouse conditions at either ambient (350 µmol mol^?1) or elevated (700 µmol mol^?1) CO₂ concentrations. After establishment of nodules, the plants were completely dependent on symbiotic nitrogen fixation. Six out of seven species had greater relative growth rates and lower whole plant nitrogen concentrations under elevated versus normal CO₂. Enhanced growth resulted in an increase in the amount of nitrogen fixed symbiotically for five of the species. In general, this was the consequence of lower whole‐plant nitrogen concentrations, which equate to a larger plant and greater nodule mass for a given amount of nitrogen. Since the average amount of nitrogen fixed per unit nodule mass was unaltered by atmospheric CO₂, more nitrogen could be fixed for a given amount of plant nitrogen. For three of the species, elevated CO₂ increased the rate of nitrogen fixation per unit nodule mass and time, but this was completely offset by a reduction in nodule mass per unit plant mass.     

Nodulation and biological nitrogen fixation of 80 soybean cultivars in symbiosis with indigenous rhizobia

Eighty soybean cultivars were assessed for their potential for nodulation and nitrogen fixation with indigenous rhizobia in a Nigerian soil. Seventy-six days after planting (DAP) 87%, 3% and 10% of the soybean cultivars had from 0 to 30, 31 to 60 and over 61 nodules/plant, respectively. Only 8% had a nodule dry weight of 600 to 1100 mg/plant. At 84 DAP the proportion of nitrogen derived from the atmosphere (Ndfa) ranged from 0 to 65% 16% of the cultivars derived 51 to 65% of their N₂ from the atmosphere. The diversity of soybean germplasm and the variation in nodulation and N₂ fixation permitted the selection of the five best cultivars in terms of their compatibility with indigenous rhizobia, % Ndfa and the amount of N₂ which they fixed.     

Elevated CO2 stimulates associative N2 fixation in a C3 plant of the Chesapeake Bay wetland

In this study, the response of N₂ fixation to elevated CO₂ was measured in Scirpus olneyi, a C₃ sedge, and Spartina patens, a C₄ grass, using acetylene reduction assay and ¹⁵N₂ gas feeding. Field plants grown in PVC tubes (25 cm long, 10 cm internal diameter) were used. Exposure to elevated CO₂ significantly (P < 0·05) caused a 35% increase in nitrogenase activity and 73% increase in ¹⁵N incorporated by Scirpus olneyi. In Spartina patens, elevated CO₂ (660 ± 1 μ mol mol ^{− 1}) increased nitrogenase activity and ¹⁵N incorporation by 13 and 23%, respectively. Estimates showed that the rate of N₂ fixation in Scirpus olneyi under elevated CO₂ was 611 ± 75 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} compared with 367 ± 46 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} in ambient CO₂ plants. In Spartina patens, however, the rate of N₂ fixation was 12·5 ± 1·1 versus 9·8 ± 1·3 ng ¹⁵N fixed plant ^{− 1} h ^{− 1} for elevated and ambient CO₂, respectively. Heterotrophic non-symbiotic N₂ fixation in plant-free marsh sediment also increased significantly (P < 0·05) with elevated CO₂. The proportional increase in ¹⁵N₂ fixation correlated with the relative stimulation of photosynthesis, in that N₂ fixation was high in the C₃ plant in which photosynthesis was also high, and lower in the C₄ plant in which photosynthesis was relatively less stimulated by growth in elevated CO₂. These results are consistent with the hypothesis that carbon fixation in C₃ species, stimulated by rising CO₂, is likely to provide additional carbon to endophytic and below-ground microbial processes.     

Effect of different levels of mineral nitrogen on nodulation and N2 fixation of two cultivars of common bean (Phaseolus vulgaris L.)

An experiment was conducted under greenhouse conditions to evaluate the effect of mineral nitrogen on N₂ fixation of two cultivars of Phaseolus vulgaris L., Puebla 152 and Negro Argel. Nitrogen application was 0, 2.5, 12.5 and 25 mg N Kg^–1 of a vermiculite-sand-mixture at planting time. Shoot and root growth were elevated by nitrogen application at all growth stages. During vegetative growth (V 5) nodule dry weight and nitrogenase activity (acetylene reducing activity) per plant were reduced by nitrogen supply in both cultivars, but less in Negro Argel than in Puebla 152. At later stages nodulation in nitrogen-treated Puebla 152 did not differ from that in non-treated plants, whereas increased nodule number was found in Negro Argel at high nitrogen levels. The influence of mineral N on the total amount of nitrogen fixed in the two bean cultivars was only slightly different.     

Co-inoculation with Glomus intraradices and Rhizobium tropici CIAT899 increases P use efficiency for N2 fixation in the common bean (Phaseolus vulgaris L.) under P deficiency in hydroaeroponic culture

Common bean (Phaseolus vulgaris L.) genotypes CocoT and Flamingo were inoculated with Rhizobium tropici CIAT899 and Glomus intraradices (Schenck & Smith) and grown under sufficient versus deficient phosphorus supply for comparing the effects of double inoculation on growth, nodulation, mycorrhization of the roots, phosphorus use efficiency and total nitrogen. Although the double inoculation induced a significant increase in all parameters whatever the phosphorus supply in comparison to control, significant differences were found among genotypes and treatments. Nevertheless, the highest phosphorus use efficiency and plant total nitrogen were found under P deficiency in combination with arbuscular mycorrhizal fungi. It is concluded that inoculation with rhizobia and arbuscular mycorrhizal fungi could improve symbiotic nitrogen fixation even under phosphorus deficiency.     

Regulation of nodule formation in soybean-Bradyrhizobium symbiosis is controlled by shoot or/and root signals

Two strains of Bradyrhizobium japonicum wereevaluated with five commercial cultivars of soybean(Clark, Crauford, Davis, Centaur, and Nessen) and onehypernodulating mutant NOD1-3. The hypernodulatingNOD1-3 produced 30–50 times more nodules thancommercial cultivars either inoculated with B.japonicum strain USDA 123 or RCR 3409. The currentexperiments were extended to determine if therestricted nodulation of commercial cultivars could be overcome by grafting them to a hypernodulated shoot (NOD1-3). Grafting of NOD1-3 shoots to Clark and Davis roots induced hypernodulation on roots of Clark and Davis but did not enhance nodulation when grafted onto the roots of Crauford, Centaur, and Nessen. The shoots of Clark, Davis, Centaur and Nessen significantlyinhibited nodule formation on the root of NOD1-3,while Crauford shoots did not alter nodule formationon the roots of NOD1-3 as compared with self-grafts ofNOD1-3. It appears that the shoot of NOD1-3 has theability to alter autoregulatory control of nodulationof Clark and Davis cultivars, but did not withCrauford, Centaur and Nessen. The results suggestedthat the regulation of nodulation in soybean cultivarsClark and Davis is controlled by the shoot factors,while the Crauford was root controlled.Reciprocal-grafts between NOD1-3 and Centaur or Nessenindicate that both shoot and root factors involved inregulation of nodulation and the regulation ofnodulation did not depend on bradyrhizobial strains. Isoflavonoid analyses from extracts of grafted plantsshowed that NOD1-3 shoots had markedly higher rootisoflavonoid concentrations in roots of both Clark andNOD1-3. The shoot control of hypernodulation may becausally related to differential root isoflavonoidlevels, which are also controlled by the shoot. Thecurrent work was extended to investigate the effect ofapplication of an isoflavonoid (daidzein) on nodulationand nitrogen fixation of soybean cultivars Clark andCentaur as well as in vitro growth of Bradyrhizobium japonicum. Application of theisoflavonoid (daidzein) significantly enhanced thenodulation and nitrogenase activity of Clark but notof Centaur indicating that this character is notrelated to isoflavonoids. Therefore, autoregulationin Clark and Centaur plants may be separate events inlegume-rhizobia symbiosis and regulated by differentkinds of signals. Addition of daidzein to yeastmannitol broth medium promoted the growth of B.japonicum strain USDA 123 and RCR 3409. It seemsthat this compound is able to help the nodulation ofsoybean cv Clark by a Bradyrhizobium strain. Understanding the signaling pathways between rhizobiaand their host plants may allow modifications of thisinteraction to improve symbiotic performance.     

Response of Groundnut (Arachis hypogaea L.) to inoculation withRhizobium strains isolated from wild arboreal legumes

Summary The rhizobial strain isolated from wild arboreal legumes viz.Butea monosperma, Glyricidia sepium andAcacia nilotica effectively increased nodulation, shoot weight, pod weight, nitrogenase activity and also showed high competitive ability as compared to the homologous strains NC92 and G2 as well as chemical and absolute controls in groundnut. Thus, the findings of present study open a new field for exploring superior rhizobia from wild legumes, which can be used for improving the yield of cultivated legumes.

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Réponse de l'arachide (Arachis hypogaea L.) à l'inoculation par des souches de Rhizobium isolées de légumes arboricoles sauvages

Résumé Les souches rhizobiennes isolées de légumes sauvages arboricoles commeButea monosperma,Glyricidia sepium etAcacia nilotica ont effectivement augmenté la nodulation, le poids de la racine, le poids des cosses, l'activité de nitrogénase. Elles ont aussi montré une aptitude à la forte compétition par comparaison avec les souches homologues NC92 et G2 de même qu'un contrôle chimique et absolu de l'arachide. Aussi, les résultats de la présente étude ouvrent un nouveau champ pour l'exploration des rhizobia supérieurs de légumes sauvages, qui peuvent être utilisés pour l'amélioration du rendement de légumes cultivés.

     

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.