Factors in the physical and biological environment affecting nodulation and nitrogen fixation by legumes

Summary The effects of root temperature on the four main stages of nodule formation and function are reviewed. Compared with results obtained under optimal conditions, lower root temperatures retard root hair infection more than they affect nodule initiation, nodule development (including bacteroid tissue development and degeneration), or nitrogen assimilation. Higher root temperatures upset the formation of bacteroid tissue and hasten its degeneration. Tropical and subtropical legumes have higher minimum temperatures for nodule formation than temperate species. Low and high shoot temperatures affect nodulation and nitrogen fixation, but the effect is less severe than that of similar root temperatures. Various approaches to minimise adverse environmental effects are considered. These include the selection of appropriate biological material (both host plants and bacterial strains) for the prevailing conditions, and the adoption of management practices designed to utilise the environment or to minimise its adverse effects. The importance of increase in bacteroid volume in relation to increase in rate of nitrogen fixation is considered, and the concept of compensation in nodule production and activity is examined. The limited information on defoliation effects on the nodulation of both temperate and tropical legumes is reviewed and aspects requiring examination are discussed.     

Agrobacterium rhizogenes transformed soybean roots differ in their nodulation and nitrogen fixation response to genistein and salt stress

We evaluated response differences of normal and transformed (so-called ‘hairy’) roots of soybean (Glycine max L. (Merr.), cv L17) to the Nod-factor inducing isoflavone genistein and salinity by quantifying growth, nodulation, nitrogen fixation and biochemical changes. Composite soybean plants were generated using Agrobacterium rhizogenes-mediated transformation of non-nodulating mutant nod139 (GmNFR5α minus) with complementing A. rhizogenes K599 carrying the wild-type GmNFR5α gene under control of the constitutive CaMV 35S promoter. We used genetic complementation for nodulation ability as only nodulated roots were scored. After hairy root emergence, primary roots were removed and composite plants were inoculated with Bradyrhizobium japonicum (strain CB1809) pre-induced with 10 μM genistein and watered with NaCl (0, 25, 50 and 100 mM). There were significant differences between hairy roots and natural roots in their responses to salt stress and genistein application. In addition, there were noticeable nodulation and nitrogen fixation differences. Composite plants had better growth, more root volume and chlorophyll as well as more nodules and higher nitrogenase activity (acetylene reduction) compared with natural roots. Decreased lipid peroxidation, proline accumulation and catalase/peroxidase activities were found in ‘hairy’ roots under salinity stress. Genistein significantly increased nodulation and nitrogen fixation and improved roots and shoot growth. Although genistein alleviated lipid peroxidation under salinity stress, it had no significant effect on the activity of antioxidant enzymes. In general, composite plants were more competitive in growth, nodulation and nitrogen fixation than normal non-transgenic even under salinity stress conditions.     

Biochar improved nodulation and nitrogen metabolism of soybean under salt stress

To investigate salt stress and biochar application effects on nodulation and nitrogen metabolism of soybeans (Glycine max cv. M7), an experiment was conducted under the control condition. The treatments comprised three biochar rates (non, 50 and 100 g kg^?1 soil) and three salinities (0, 5 and 10 dS m^?1 NaCl), with four replications of treatments. Salt stress diminished the number of nodules and their weights in the soybean roots. Nitrogen content and metabolism decreased in nodules, roots and shoots, while reducing the activity of glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamine oxoglutarate aminotransferase (GOGAT) and nitrate reductase (NR). Also, salinity brought down root and shoot weight, total plant biomass, chlorophyll content, leaf area (LA) and rubisco activity in the soybean. On the other hand, application of biochar improved nodulation, nitrogen content, rubisco activity, GDH, GS, GOGAT and NR activities in different parts of the soybean and nodules under salt stress, and consequently improved chlorophyll content, LA, root and shoot weight. Both the 50 and 100 g kg^?1 biochar rates showed similar effects in improving nitrogen metabolism and plant performance under salt stress. Generally, biochar increased nodulation and nitrogen metabolism of the soybean under saline conditions.     

Cysteine proteases in nodulation and nitrogen fixation

The cysteine proteinases or cysteine endopeptidases (EC 3.4.22) are known to occur widely in plant cells. They are involved in almost all aspects of plant growth and development including germination, circadian rhythms, senescence and programmed cell death. They are also involved in mediating plant cell responses to environmental stress (such as water stress, salinity, low temperature, wounding, ethylene, and oxidative conditions) and plant-microbe interactions (including nodulation). In the development and function of legume root nodules, cysteine proteases could be involved in several important processes:-(i) a defence response to root invasion by microorganisms; (ii) protein turnover required during the formation of new tissue; (iii) cellular homeostasis and metabolism; (iv) adaptation of host cells to physiological stresses; (v) control of nodule senescence. Because of their central importance to plant physiology, cysteine proteases could serve as important targets for the study of nodule development and functioning at the molecular level. Because of their widespread occurrence in nodulating plants they could also serve as candidate genes for targeted plant breeding programmes.     

Effect ofAzospirillum inoculation on nitrogen fixation and growth of several winter legumes

Summary Inoculation of naturally nodulatedPisum sativum L. (garden pea) withAzospirillum in the greenhouse caused a significant increase in nodule numbers above controls. Field inoculation of garden peas in the winter 1981–1982 andCicer arietinum L. (chick pea), in winter 1982–1983, withAzospirillum one week after plant emergence, produced a significant increase in seed yield, but did not affect plant dry matter yield. ForVicia sativa L. (vetch) grown in soil in the greenhouse and in the field for forage, winter 1980–1981, inoculation significantly increased dry matter yield, %N, N-content, and acetylene reduction (nitrogen fixation) activity. InHedysarum coronarium L. (sulla clover), winter 1981–1982, inoculated with both its specificRhizobium (by the slurry method) andAzospirillum, 7 days after emergence, there was an increase in acetylene reduction above controls inoculated withRhizobium alone. These results suggest that it is possible, under conditions tested in this work, to increase nodulation, nitrogen fixation, and crop yields of winter legumes by inoculation withAzospirillum.     

根瘤菌共生固氮能力的进化模式

根瘤菌-豆科植物共生固氮体系对农业的可持续性发展至关重要,也是研究原核与真核生物互利共生的模式体系之一。长期以来,根瘤菌共生固氮相关研究主要集中在结瘤因子与固氮酶合成及调控等少数关键基因,但仅获得这些关键基因却不能保证细菌获得结瘤固氮能力。随着比较和功能基因组学的快速发展和应用,越来越多的研究发现根瘤菌使用了很多系统发育分支特异的遗传机制与豆科植物建立有效的共生关系,进一步揭示了双方互利共生的复杂性。本综述总结了近年来比较基因组学、遗传学以及实验进化等方面的相关研究进展,在此基础上讨论根瘤菌共生固氮能力的进化模式。     

Interaction of yeasts and some nitrogen fixing bacteria on nodulation of legumes

Summary Combined inoculation ofRhizobium trifolii withSaccharomyces cerevisiae and other yeasts generally enhanced the number of nodules, length of plants and dry weight of Egyptian clover (Trifolium alexandrinum) seedlings grown on agar slopes. Similar effects were observed when seedlings were inoculated withR. trifolii in the presence of dialyzed culture filtrate ofS. cerevisiae.     

A comparison of the effect of combined nitrogen on nodulation in non-legumes and legumes

Summary 1. The effect of the presence of combined (ammonium) nitrogen in the rooting medium on nodule development has been investigated inAlnus glutinosa andMyrica gale, the plants being grown by a water culture technique with provision for the control of the level of combined nitrogen and of pH. For purposes of comparison a similar experiment with a legume (Ulex europaeus) has been included.2. In some instances the differential treatment with combined nitrogen was commenced and inoculation effected at an early stage in plant development, while in others the plants were grown on to a larger size before these treatments were applied.3. In the two non-legumes the presence of combined nitrogen led to an increase in the weight of nodulus formed per plant, at least at the lower levels of nitrogen. Relative to the enhanced growth of the plant as a whole, nodule development was continuously depressed as the level of combined nitrogen was increased.4. InUlex when the nitrogen status of the plants at the time of inoculation was similar to that in the corresponding non-legume experiment, the above responses were also shown by the legume. It is concluded thatAlnus andMyrica resemble legumes closely in their responses to combined nitrogen.5. The data show that inAlnus the effect of a given level of combined nitrogen on nodulation depends on the nitrogen status of the plant at the time of inoculation, the adverse effect tending to be stronger if the plant is initially relatively high in nitrogen. This suggests that as in legumes the effect of the combined nitrogen is exerted internally in the plant tissues.     

Field experiments on nitrogen fixation by nodulated legumes

Summary Phosphate increased nitrogen uptake by lucerne appreciably on a saline soil. Nitrogenous fertiliser or inoculation with an effective strain ofRhizobium meliloti did not increase the yield significantly. In soils where indigenousRhizobium japonicum was absent inoculation increased soybean yields and the additional fixed nitrogen removed by soybeans amounted to 40 to 120 kg ha⁻¹. Gram and groundnut also responded to Rhizobium inoculation in field trials.     

Effect of light and atmospheric carbon dioxide concentration on nitrogen fixation by herbage legumes

Summary Lucerne, red clover and white clover were grown at two atmospheric concentrations of CO₂ (300 and 1000 μl l⁻¹) and the effects on N₂ fixation, nodule mass/number and root/shoot dry matter production determined. Pea plants were similarly evaluated as a comparison with grain legumes. CO₂ enrichment increased N₂ fixation activity in all cases but activity/unit nodule mass was significantly increased only in the pea. The enhancement of N₂ fixation in herbage legumes by CO₂ enrichment reflected an increase in nodule mass which in turn was attributed to increased nodule number, and results show that under the experimental conditions obtaining here photosynthate supply did not limit nodule N₂ fixation in these plants though it was limiting in the case of peas. White clover growing in a 6 and 14 hour photoperiod was studied for response of the N₂ fixing system to light. Long photoperiod (14 hour) plants assayed at constant temperature (20°C) did not show a significant response to light at the end of the dark period either in terms of fixation per plant or per unit nodule mass, in contrast with short photoperiod (6 hour) plants which showed significant responses. Short photoperiod plants compensated for reduced photosynthates by maintaining only half the root nodule mass and fixation activity of 14 hour photoperiod plants though plants in both systems supported similar rates of N₂ fixation per unit mass of nodule during the photoperiod. Comparison of N₂ fixation activities in whole and decapitated plant systems indicates the importance of shoot reserves for sustaining nitrogenase activity in white clover during short-term interruption of photosynthesis. These results support the conclusion of the CO₂ enrichment studies, that herbage legumes have the potential for supplying their nodule photosynthate requirements for sustaining optimum rates of N₂ fixation and excess carbon supply is used solely to promote further nodulation. Nodules of short photoperiod white clover plants were less efficient in N₂ fixation in that they evolved more H₂ relative to N₂ (C₂H₂) reduced than did long photoperiod plants.     

Effect of night temperature and photoperiod on growth,nodulation and nitrogen fixation inLotus tenuis

Summary Lotus tenuis was inoculated withRhizobium spp. and grown at three different night temperatures (17, 21 and 27°C) and at two different photoperiods for each temperature: short days (SD) of 8-h light and long days (LD) of 16-h light. Night temperature exerted a stronger effect on dry-matter partitioning than of total dry-matter accumulation. Leaf area increased under LD though less N per leaf area was found. A greater number of leaves under SD might be linked to a day-length effect upon apical dominance and hence on branching, thus resulting in a greater crown growth. Nodulation and nitrogen fixation was greater under SD though specific nodule activity was the same regardless of the environmental conditions of growth. It can be concluded that the best planting time forLotus tenuis would be in the autumn when the days are shortening and the temperature is lower.

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Effet de la température nocturne et de la photo-période sur la croissance nodulaire et la fixation d'azote chez Lotus tenuis

Résumé Lotus tenuis a été inoculé avecRhizobium spp. et mis à croître aux trois températures nocturnes, de 17, 21, et 27°C et à deux photo-périodes pour chaque température: des jours courts (SD) de 8 heures de lumière et des jours longs (LD) de 16 heures de lumière. La température nocturne exerce un effet davantage marqué sur la répartition de la matière sèche que sur l'accumulation totale de la matière sèche. La surface foliaire augmente dans le cas des LD bien que l'on trouve moins d'azote par unité de surface foliaire. Dans le cas des SD, de plus grands nombres de feuilles pourraient être liés à un effet de longueur de jours sous dominance apicale, c'est-à-dire en banchement, résultant donc dans la croissance d'une plus grande couronne. La nodulation et la fixation d'azote sont plus grandes dans le cas des SD bien que l'activité spécifique des nodules soit la même, indépendamment des conditions environnementales de croissance. On peut en conclure que le meileur moment de plantation pourLotus tenuis serait l'automne lorsque les jours raccourcissent et que la température est plus basse.

     

Influence of lime on nitrogen fixation by tropical and temperate legumes

Summary A nitrogen deficient Oxisol which had been fertilized with P, K, Zn and Mo received CaCO₃ at rates which increased continuously from zero to 22 tons/ha. Liming produced a range of pH in the saturation paste from 4.7 to 7.1; a range of calcium in the saturation extract from 0.3 to 3 meq/l; and a significant decline in available manganese. Responses of 9 tropical and 7 temperate legumes were compared across the pH variable.For Stylosanthes spp. nodule numbers and weight and plant yield declined above pH 5.5. Arachis hypogaea and Vigna sinensis, whose yield increased by only 30%, showed no clear improvement in nodulation or nodule effectiveness (acetylene reduction rate per unit nodule weight).Increased nodule effectiveness could account for most of the growth increase in Dolichos axillaris and Glycine max var. Kahala. Both the number and effectiveness of nodules increased for Desmodium intortum, Glycine wightii, Medicago sativa, and Trifolium subterraneum. Nodule size and effectiveness increased for G. max var. Kanrich. Only in one species, Coronilla varia, could increase in nodule numbers alone account for the increased growth associated with liming, although increased numbers of nodules probably accounted for much of the response by Lotus corniculatus, and by Desmodium canum and D. intortum up to pH 5.3.Increased nodulation and plant N contents were consistent with the conclusion that for most species improved N₂-fixation was the cause of growth improvement associated with liming. However, percent N was high in leaves of Vigna and Phaseolus vulgaris at all lime levels. In Phaseolus, variation in nodulation and growth were unrelated. The growth improvement was associated with decline in leaf N, suggesting that something other than N nutrition was limiting.Journal Series No. 1957 of the Hawaii Agricultural Experiment Station.Department of Soils and Plant Nutrition, University of California, Davis.Department of Agronomy and Soil Science, University of Hawaii, Honolulu, Hawaii.     

Methods for measuring biological nitrogen fixation in grain legumes

To assure proper management and fully realize the benefits of the legume-Rhizobium symbiosis it is necessary to be able to quantify the amount of nitrogen fixed. Having measured the effectiveness of atmospheric N₂ fixation the macro- or micro-symbionts as well as agronomic factors can be manipulated with the objective to maximize biological nitrogen fixation. A suitable method to quantify nitrogen fixation is therefore necessary in any programme aiming at increasing N₂ fixation, like the one being reported in this volume. There are several methods available to quantify nitrogen fixation and most of the commonly used ones are described in the present paper listing their advantages and disadvantages.     

Effect of composted textile sludge on growth, nodulation and nitrogen fixation of soybean and cowpea

The effect of composted textile sludge on growth, nodulation and nitrogen fixation of soybean and cowpea was evaluated in a greenhouse experiment. The compost was incorporated into soil at 0, 9.5, 19 and 38 t ha(-1) (bases upon the N requirement of the crops, i.e., 0, 50, 100 and 200 kg available N ha(-1)). Growth, nodulation and shoot accumulation of nitrogen were evaluated 36 and 63 days after plant emergence. Nodule glutamine synthetase (GS) activity and leghemoglobin content were evaluated 63 days after emergence. Composted textile sludge did not show negative effects on nodule number and weight, nodule GS activity and leghemoglobin content. Nitrogen accumulation in shoot dry matter in soybean and cowpea was higher than other treatments with application of 19 t ha(-1) of compost. Composting can be an alternate technology for the management of solid textile mill sludge. This study verifies that the composted textile sludge was not harmful to growth, nodulation and nitrogen fixation of soybean and cowpea.     

Effect of phosphorus and zinc on nodulation and nitrogen fixation in chickpea (Cicer arietinum L.)

A green house study was conducted on the effect of P and Zn on nodulation and N fixation in chickpea (Cicer arietinum L.) in a loamy sand (Typic Torripsamments) using treatment combinations of five levels of P (0, 25, 50, 100 and 250 ppm), and six levels of Zn (0, 5, 10, 20, 40 and 100 ppm). The number, dry matter and leghaemoglobin content of nodules, and amount of N fixed generally increased with Zn alone upto 19 ppm and P alone upto 50 ppm, and decreased with their higher levels. Application of 25 to 50 ppm P and 5 to 10 ppm Zn counteracted to a greater extent the adverse effect of 40 and 100 ppm Zn, and 250 ppm P, resp. Maximum nodulation and N fixation (91 to 145% over zero P and Zn, at maturity) was recorded with 25 to 50 ppm P applied along with 5 to 10 ppm Zn. At 64 days, depletion in soil-N was noted, particularly when P was applied, whereas at maturity there was a gain in soil-N, ranging from 10.5 to 44.5 kg/2×10⁶ kg soil depending upon P and Zn treatments. The increase in nodulation and N fixation with balanced P and Zn nutrition might be attributed to an increase in leghaemoglobin, and K and Fe concentration in nodules, and increased plant growth, resulting into enhanced activity of N fixing organisms. The results showed that balanced P and Zn nutrition is essential not only for plant growth but also for maximum activity of Rhizobium for N fixation. Work done at Harvana Agricultural University, Hissar, India.