Effect of inoculation with Azotobacter chroococcum on nitrogenase activity of Zea mays roots grown in agricultural soils under aseptic and non-sterile conditions

Summary Inoculated and non-inoculated seedlings of Zea mays were grown in agricultural soils under aseptic and non-sterile conditions. Acetylene reduction activity and microbial counts were determined after 7 and 30 days of growth. Irrespective of the soil type Azotobacter spp. were commonly isolated under maize cultivation. Inoculation of agricultural soils with a suspension of A. chroococcum led to an increase in Azotobacter numbers, although this effect diminished with time. Nitrogenase activity was detected on maize roots and increased in response to the inoculation with A. chroococcum, showing that this associative growth could be of primary importance for the plant. The results of assays for acetylene reduction activity indicated that the nitrogenase activity was associated only with the root systems.     

A bradyrhizobial-<Emphasis Type="Italic">Penicillium</Emphasis> spp. biofilm with nitrogenase activity improves N<Subscript>2</Subscript> fixing symbiosis of soybean

A bradyrhizobial-fungal biofilm (i.e. Bradyrhizobium elkanii SEMIA 5019-Penicillium spp.) developed in vitro was assayed for its nitrogenase activity and was evaluated for N₂-fixing symbiosis with soybean under greenhouse conditions. The biofilm showed nitrogenase activity, but the bradyrhizobial strain alone did not. Shoot and root growth, nodulation and N accumulation of soybean increased significantly with an inoculum developed from the biofilm. This study concludes that such biofilmed inoculants can improve N₂-fixing symbiosis in legumes, and can also directly contribute to soil N fertility in the long term. Further studies should be conducted to investigate the performance of these inoculants under field conditions.     

N2-fixation by ammonium-excreting Azospirillum brasilense in auxine-induced root tumours of wheat (Triticum aestivum L.)

Summary Wheat seedlings, treated with the auxine 2,4-dichlor-phenoxy acetic acid (2,4-D) during germination developed only a residual root system. Root elongation was extremely restricted and root tips were deformed to thick club-shaped tumours. When 2,4-D was added in a later stage of plant growth the plants developed additional nodule-like knots along primary roots. Root and shoot dry-matter production was slightly repressed in all 2,4-D treatments and N translocation from roots to shoots was repressed as well. When transferred to an auxine-free growth medium, the 2,4-D-affected roots were not capable of complete recovery. In plants inoculated gnotobiotically with Azospirillum brasilense, either with the wild type or with the NH ₄ ⁺ -excreting mutant strain C3, a 2,4-D addition increased rhizosphere acetylene-reduction activity at pO₂ 1.5 kPa. The O₂ sensitivity of root-associated nitrogenase activity tended to be reduced. The number of root-colonizing bacteria, at approximately 10⁸ colony-forming units (cfu) per g dry root, was similar in the 2,4-D treatments and untreated controls. Plant treatment with high concentrations of the chemical isomer 3,5-dichlor-phenoxy acetic acid (3,5-D) did not have comparable effects, either on plant development or on rhizosphere-associated nitrogenase activity. Root-tumour tissue inhabited by A. brasilense showed purple staining when subjected to a tetrazolium chloride solution, which may indicate intensive local nitrogenase activity in this tissue. Exposed to an ¹⁵N₂-enriched atmosphere, plants treated with 2,4-D and with A. brasilense incorporated significantly higher amounts of ¹⁵N than untreated controls. In all cases the highest values of ¹⁵N enrichment were found following inoculation with the NH ₄ ⁺ -excreting mutant strain C3. Present address: F. A. Janssens Laboratory of Genetics, Catholic University of Leuven, Willem de Croylan 42, B-3001 Heverlee, Belgium     

Nitrogenase Activity and Nodule Biomass of Cowpea (Vigna unguiculata L. Walp.) Decrease in Cover Crop Mixtures

Legume cover crops are often used to build soil nitrogen (N) fertility and there is increasing interest in cover crop mixtures. The objective of this mechanistic greenhouse study was to determine the effect of cover crop community diversity and soil fertility on nitrogenase activity and nodule biomass of cowpea. Cover crops were grown for 42–53 days, aboveground biomass was harvested, and nitrogenase activity was estimated with the acetylene reduction assay. Roots were then excavated to determine nodule and root biomass. Nitrogenase activity and nodule biomass per plant were greatest in cowpea monoculture and reduced by 71–98 percent in four-species mixtures. Reduced capacity for N₂ fixation was partially driven by lower cowpea biomass in mixtures. The ratio of root nodule / shoot biomass increased by 81–297 percent in low-fertility relative to high-fertility soils, which contributed to increased nitrogenase activity. Results suggest cowpea monocultures in low-fertility soils have the greatest potential for N₂ fixation.     

Using the spermosphere model technique to describe the dominant nitrogen-fixing microflora associated with wetland rice in two Egyptian soils

Summary This study is an attempt to describe the dominant N₂-fixing microflora associated with the roots of wetland rice. Rice cultivar Giza 171 was grown in a phytotron on two alluvial Egyptian soils for 8 days, a stage when the nitrogenase activity of undisturbed plants reached a level of 245 × 10^–6 mol C₂H₄ h^–1 g^–1 dry weight of leaf. The roots and rhizosphere soils were then used for counting and isolating dominant diazotrophs. Counts and initial enrichment steps were carried out on a selective medium made of an axenic rice plantlet, the spermosphere model, incubated under 1 % acetylene. The counts were very high, exceeding 10⁸ bacteria g^–1 dry weight of rhizosphere soil. Enterobacteriaceae were dominant; most isolates were Enterobacter cloacae belonging to different biotypes in the two soils. Enterobacter agglomerans, Citrobacter freundii and Klebsiella planticola were also present as members of the dominant microflora. Azospirillum brasilense and Azospirillum lipoferum were present as well, but less abundant.     

Effect of salt stress on interaction between lentil (Lens culinaris) genotypes and Rhizobium spp. strains: symbiotic N2 fixation in normal and sodic soils

 Following screening, selection, characterization and examination of their symbiotic N₂ fixation, only two Rhizobium strains (ND-16 and TAL-1860) and four lentil genotypes (DLG-103, LC-50, LC-53 and Sehore 74-3) were found to be suited to sodic soils. Interactions between salt-tolerant lentil genotypes and Rhizobium strains were found to be significant, and resulted in greater nodulation, N₂ fixation (nitrogenase activity), total nitrogen, plant height, root length and grain yield in sodic soils under field conditions compared to uninoculated controls. Significantly more nodulation, nitrogenase activity, glutamine synthetase (GS) and NADH-dependent glutamate synthase (NADH-GOGAT) activities were found in normal soil as compared to the soil supplemented with 4% and 8% NaCl. Salt stress inhibited nitrogenase, GS and NADH-GOGAT activities. However, nitrogenase activity in nodules was more sensitive to salt stress than GS and NADH-GOGAT activities (NH₄ ⁺ assimilation). The relevance of these findings for salt-tolerant symbionts is discussed. Received: 14 November 1997     

Effect of nitrogen levels and Rhizobium strains on symbiotic N2 fixation and grain yield of Phaseolus vulgaris L. genotypes in normal and saline-sodic soils

Summary Following screening, selection, characterization, and symbiotic N₂ fixation with 12,5, 25.0, and 40.0 mg N kg^–1 in normal and saline-sodic soils, only two Phaseolus vulgaris genotypes (HUR 137 and VL 63) and two Rhizobium spp. strains (ND 1 and ND 2) produced maximum nodulation, nitrogenase activity, plant N contents, and grain yields in saline-sodic soil, with 12.5 mg N kg^–1, compared with the other strains. However, interactions between strains (USDA 2689, USDA 2674, and ND 5) and genotypes (PDR 14, HUR 15, and HUR 138) were significant and resulted in more nodulation, and greater plant N contents, nitrogenase activity, and grain yields in normal soils with 12.5 mg N kg^–1 compared with salt-tolerant strains. Higher levels of N inhibited nodulation and nitrogenase activity without affecting grain yields. To achieve high crop yields from saline-sodic and normal soils in the plains area, simultaneous selection of favourably interacting symbionts is necessary for N economy, so that bean yields can be increased by the application of an active symbiotic system.     

Synergistic effects of vesicular-arbuscular mycorrhizal fungi and diazotrophic bacteria on nutrition and growth of sweet potato (Ipomoea batatas)

Summary Sweet potatoes were micropropagated and then transplanted from axnic conditions to fumigated soil in pots in the greenhouse. Spores of Glomus clarum were obtained from Brachiaria decumbens or from sweet potatoes grown in soil infected with this fungus and with an enrichment culture of Acetobacter diazotrophicus. Three experiments were carried out to measure the beneficial effects of vesicular-arbuscular mycorrhizal (VAM) fungi-diazotroph interactions on growth, nutrition, and infection of sweet potato by A. diazotrophicus and other diazotrophs obtained from sweet potato roots. In two of these experiments the soils had been mixed with ¹⁵N-containing organic matter. The greatest effects of mycorrhizal inoculation were observed with co-inoculation of A. diazotrophicus and/or mixed cultures of diazotrophs containing A. diazotrophicus and Klebsiella sp. The tuber production was dependent on mycorrhization, and total N and P accumulation were increased when diazotrophs and G. clarum were applied together with VAM fungal spores. A. diazotrophicus infected aerial plant parts only when inoculated together with VAM fungi or when present within G. clarum spores. More pronounced effects on root colonization and intraradical sporulation of G. clarum were observed when A. diazotrophicus was co-inoculated. In non-fumigated soil, dual inoculation effects, however, were of lower magnitude. ¹⁵N analysis of the aerial parts and roots and tubers at the early growth stage (70 days) showed no statistical differences between treatments except for the VAM+Klebsiella sp. treatment. This indicates that the effects of A. diazotrophicus and other diazotrophs on sweet potato growth were caused by enhanced mycorrhization and, consequently, a more efficient assimilation of nutrients from the soil than by N₂ fixation. The possible interactions between these effects are discussed.     

Response of the pigeonpea-Rhizobium symbiosis to salinity stress: variation among Rhizobium strains in symbiotic ability

Summary There were significant differences among pigeonpea [Cajanus cajan (L.) Millsp] Rhizobium sp. strains (IC 3506, IC 3484, IC 3195, and IC 3087) in their ability to nodulate and fix N₂ under saline conditions. Pigeonpea plants inoculated with IC 3087 and IC 3506 were less affected in growth by salinity levels of 6 and 8 dS m^-1 than plants inoculated with the other strains. For IC 3506, IC 3484, and IC 3195, there was a decrease in the number of nodules with increasing salinity, while the average nodule dry weight and the specific nitrogenase activity remained unaffected. However, in IC 3087, the number of nodules increased slightly with increasing salinity. Leaf-P concentrations increased with salinity in the inoculated plants irrespective of the Rhizobium sp. strain, and leaf-N concentrations decreased with increasing salinity in IC 3484 and IC 3195 only. Shoot-Na and-Cl levels were further increased in these salt-sensitive strains only at 8 dS m^-1. Therefore there may be scope for selecting pigeonpea Rhizobium sp. symbioses better adapted to saline conditions. The Rhizobium sp. strains best able to form effective symbioses at high salinity levels are not necessarily derived from saline soils.Submitted as JA No. 919 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Nitrogen fixation by Azospirillium brasilense in soil and the rhizosphere under controlled environmental conditions

Summary Acetylene reduction activity by Azospirillum brasilense, either free-living in soils or associated with wheat roots, was determined in a sterilised root environment at controlled levels of O₂ tension and with different concentrations of mineral N. In an unplanted, inoculated soil nitrogenase activity remained low, at approximately 40 nmol C₂H₄ h^-1 per 2kg fresh soil, increasing to 300 nmol C₂H₄ h^-1 when malic acid was added as a C source via a dialyse tubing system. The N₂ fixation by A. brasilense in the rhizosphere of an actively growing plant was much less sensitive to the repressing influence of free O₂ than the free-living bacteria were. An optimum nitrogenase activity was observed at 10 kPa O₂, with a relatively high level of activity remaining even at an O₂ concentration of 20 kPa. Both NO _inf3 ^sup- and NH _inf4 ^sup+ repressed nitrogenase activity, which was less pronounced in the presence than in the absence of plants. The highest survival rates of inoculated A. brasilense and the highest rates of acetylene reduction were found in plants treated with azospirilli immediately after seedling emergence. Plants inoculated at a later stage of growth showed a lower bacterial density in the rhizosphere and, as a consequence, a lower N₂-fixing potential. Subsequent inoculations with A. brasilense during plant development did not increase root colonisation and did not stimulate the associated acetylene reduction. By using the ¹⁵N dilution method, the affect of inoculation with A. brasilense in terms of plant N was calculated as 0.067 mg N₂ fixed per plant, i.e., 3.3% of the N in the root and 1.6% in the plant shoot were of atmospheric origin. This ¹⁵N dilution was comparable to that seen in plants inoculated with non-N₂-fixing Psudomonas fluorescens.     

Does Biochar Improve Establishment of Tree Seedlings in Saline Sodic Soils?

Reforestation of saline sodic soil is increasingly undertaken as a means of reclaiming otherwise unproductive agricultural land. Currently, restoration of degraded land is limited to species with high tolerances of salinity. Biochar application has the potential to improve physical, biological and chemical properties of these soils to allow establishment of a wider range of plants. In a glasshouse trial, we applied biochar made from Acacia pycnantha (5 Mg ha⁻¹) or no biochar to either a low (EC_e 4·75 dS m⁻¹, ESP 6·9), a moderate (EC_e 27·6 dS m⁻¹, ESP 29·3) or a high (EC_e 49·4 dS m⁻¹, ESP 45·1) saline sodic soil. The regional common reforestation species Eucalyptus viminalis and Acacia mearnsii were planted as tubestock in to the soils. Early establishment indicators, including growth, plant condition and nutrition, were assessed at the end of a simulated growing season, 108 days after biochar application. Application of biochar increased height, and decreased root : shoot and the concentration of Mn, N and S in plants of E. viminalis when grown in the highly saline sodic soil. Biochar application increased the concentration of B in leaves of E. viminalis and increased the concentration of P, K and S in leaves of A. mearnsii when grown in the low saline sodic soil. The results confirm that there is potential for biochar to assist in reforestation of saline sodic soils. Copyright © 2015 John Wiley & Sons, Ltd.     

Soil surface disturbances in cold deserts: effects on nitrogenase activity in cyanobacterial-lichen soil crusts

Cyanobacterial-lichen soil crusts can be a dominant source of nitrogen for cold-desert ecosystems. Effects of surface disturbance from footprints, bike and vehicle tracks on the nitrogenase activity in these crusts was investigated. Surface disturbances reduced nitrogenase activity by 30–100%. Crusts dominated by the cyanobacterium Microcoleus vaginatus on sandy soils were the most susceptible to disruption; crusts on gypsiferous soils were the least susceptible. Crusts where the soil lichen Collema tenax was present showed less immediate effects; however, nitrogenase activity still declined over time. Levels of nitrogenase activity reduction were affected by the degree of soil disruption and whether sites were dominated by cyanobacteria with or without heterocysts. Consequently, anthropogenic surface disturbances may have serious implications for nitrogen budgets in these ecosystems.     

Infection potential of farm soils as mycorrhizal inocula for Leucaena leucocephala

A greenhouse experiment was conducted to assess the arbuscular mycorrhizal (AM) fungi inoculum potential for Leucaena leucocephala in a range of nutrient-depleted farm soils in western Kenya. Leucaena was grown in 12 uninoculated farm soils with pH 5.0–6.7, with or without rock P and farmyard manure. Root infection, nodulation, and shoot and root weight were determined 25, 46, and 69 days after planting. Spore concentrations in the farm soils at the start of the experiment ranged from 44 to 126 live spores per 100 g dry soil with 6–10 species per soil, principally Scutellospora spp. and Acaulospora spp. Nodulation was absent or poor in all soils, indicating the need for rhizobial inoculation of species belonging to the leucaena crossinoculation group in this agrosystem. Rock-P alone increased final shoot dry weight by a factor of 1.4, manure alone by 1.8, and rock-P plus manure by 1.9, compared with no ameliorant. Root infection with AM fungi was detected in all soils 25 days after planting and increased linearly in the different farm soils to values of 33–65% 69 days after planting. Soil pH and root infection 25 days after planting accounted for much of the variation in final shoot weight among soils with no amelioront (87%). As early root infection increased from 20 to 40% at soil pH 5.0, the predicted final shoot dry weight was doubled, and the response to ameliorants was reduced by two-thirds. The growth responses to increased infection became smaller as pH increased from 5.0 to 6.5.     

Diversity of diazotroph populations in the rhizosphere of maize (Zea mays L.) growing on different French soils

Summary We studied the dominant diazotrophs associated with maize roots and rhizosphere soil originating from three different locations in France. An aseptically grown maize plantlet, the spermosphere model, was used to isolate N₂-fixing (acetylene-reducing) bacteria. Bacillus circulans was the dominant N₂-fixing bacterium in the rhizosphere of maize-growing soils from Ramonville and Trogny, but was not found in maize-growing sandy soil from Pissos. In the latter soil, Enterobacter cloacae, Klebsiella terrigena, and Pseudomonas sp. were the most abundant diazotrophs. Azospirillum sp., which has been frequently reported as an important diazotroph accociated with the maize rhizosphere, was not isolated from any of these soils. The strains were compared for their acetylene-reducing activity in the spermosphere model. The Bacillus circulans strains, which were more frequently isolated, also exhibited significantly greater acetylene-reducing activity (3100 nmol ethylene day^-1 plant^-1) than the Enterobacteriaceae strains (180 nmol ethylene day^-1 plant^-1). This work indicates for the first time that Bacillus circulans is an important maizerhizosphere-associated bacterium and a potential plant growth-promoting rhizobacterium.     

Biological nitrogen fixation at elevated temperature in different Azospirillum species and strains

Nitrogenase activity, nitrogenase synthesis, and the growth of four species of Azospirillum were examined at 30°C and 42°C. Azospirillum brasilense Sp 7 did not synthesize nitrogenase at 42°C nor was the enzyme stable at this temperature. In A. lipoferum 708 and A. brasilense 12S the nitrogenase activity was stable at 42°C but the synthesis of nitrogenase was reduced. In A. brasilense Sp 9 the nitrogenase activity was stable and showed the highest C₂H₂ reduction activity at 42°C.     

Effect of inoculum rate on the performance of chickpea (Cicer arietinum L.) Rhizobium strains in the field

Summary The influence of three inoculum rates on the performance of three chickpea (Cicer arietinum L.) Rhizobium strains was examined in the field on a Mollisol soil. Increasing amounts of inoculum improved the performance of the strains. A normal dose (10⁴ cells per seed) applied at different intervals gave non-significant increases in nodulation, nitrogenase activity (acetylene reduction assay), nitrogen uptake and grain yield. A ten-fold increase in inoculum increased nodule number, shoot dry weight, nitrogenase activity (ARA) and grain yield, but increases over the control were significant only for nodule dry weight and nitrogen uptake by shoot and grain. The highest level of inoculum (100 × normal) significantly increased nodule dry weight, grain yield, total nitrogenase activity (ARA) and nitrogen uptake by shoot and grain. Strain TAL 620 was more effective than the other two. Combined nitrogen (60 kg N ha^–1) suppressed nodulation and nitrogenase activity (ARA).Research paper No. 4345 from the Experiment Station, G. B. P. U. A. & T., Pantnagar, Nainital, U. P.     

Effects of root-associated N2-fixing cyanobacteria on the growth and nitrogen content of wheat (Triticum vulgare L.) seedlings

Summary Following co-cultivation of wheat with N₂-fixing cyanobacterial isolates capable of forming associations, Nostoc 2S6B, 2S9B or Anabaena C5, for 15 days in the presence or absence of combined N a large stimulation of root length was observed without any increase in root dry weight. Increases in the N concentrations of both roots and shoots occurred following co-cultivation with most cyanobacteria tested. The increase in plant N concentrations appeared to be dependent on the wheat cultivar and the cyanobacterial isolate used. Nostoc isolates had similar nitrogenase activities when associated with roots and when grown in shake-flask cultures. The nitrogenase activity of roots colonized by Anabaena C5 or Nostoc 2S6B was higher following removal of loosely associated cyanobacteria.     

<Emphasis Type="Italic"> Gluconacetobacter diazotrophicus</Emphasis>: a natural endophytic diazotroph of Nile Delta sugarcane capable of establishing an endophytic association with wheat

Gluconacetobacter- like diazotrophs were encountered as dense populations inside the root and stem tissues of sugarcane cultivated in ancient agricultural fields of the Nile Delta. Counts of >10⁵ cells g^-1 were recorded in root and stem samples. The leaves contained a smaller population (<10³ g^-1). The typical dark-orange colonies which developed on LGIP agar plates were purified. Identification was performed with the API microtube systems: API 20E for Enterobacteriaceae and API 20NE for non-Enterobacteriaceae. API profiles of the local isolates were closely related to those of the type culture Gluconacetobacter diazotrophicus (ATTC 49037). The isolates successfully reduced C₂H₂ and produced appreciable amounts of ethylene in the presence of cane juice. This suggested that the local isolates are closely related to the type strain G. diazotrophicus. Wheat seedlings were inoculated with a number of isolates under gnotobiotic conditions. Both optical and scanning electron microscopy showed that endophytic Gluconacetobacter spp. were present in all the samples tested. They were observed in apparently intact and enlarged epidermal root cells, and also in stem tissues, indicating that the bacterium was able to migrate upward into the shoot tissues. Although Gluconacetobacter inoculation did not stimulate the growth of the cereal plant, the results obtained are particularly interesting because this bacterial species was capable of colonizing the internal tissues of wheat, not considered a natural host until now.     

The effect of earthworms (Lumbricidae) on nitrogenase activity in soil

Summary Nitrogenase activity associated with earthworms, their faeces and activity in soil was measured by the acetylene reduction technique. A clear increase in nitrogenase activity was found in field-deposited casts of Aporrectodea caliginosa in comparison with surrounding soil, although potential nitrogenase activity was significantly higher in soil than in casts. Nitrogenase activity associated directly with earthworms (Lumbricus rubellus ) was detected, indicating the presence of active N₂-fixing bacteria on the body surface and/or in the gut. Laboratory experiments showed that nitrogenase activity in the casts of L. rubellus was higher than in unmodified soil, and that nitrogenase activity in soil was significantly increased by the burrowing and feeding activity of these worms. This paper discusses the possible causes of these earthworm effects on soil nitrogenase activity and some methodological problems of determining the nitrogenase activity.     

Effects of inoculation with N2-fixing cyanobacteria on the nitrogenase activity in soil and rhizosphere of wetland rice (Oryza sativa L.)

Summary A greenhouse experiment was conducted with wetland rice (Oryza sativa cv. IR-50) in a clay-loam soil (Fluventic Eutrochrept) to study the effect of cyanobacterial inoculation a mixed culture of Aulosira fertilissima, Nostoc muscorum, Nostoc spp., and Anabaena spp., applied at the rate of 0.15 g (dry weight pot^-1 or 43 kg ha^-1) on acetylene reduction activity in soil and the root system (excised root), and the grain and straw yield. The effects of applying P (40 kg ha^-1), N (60 kg ha^-1), and P+N to the soil were also evaluated. Cyanbacterial inoculation significantly increased (more than 200% on average) photo-dependent acetylene reduction activity in soils, particularly where the indigenous activity was considerably low, i.e. under unfertilized and N-fertilized conditions. The effect of inoculation was prominent at the maximum tillering and grain formation stages of the crop. This inoculation benefit was, however, marginal in P-applied soils (P and P+N), where the indigenous activity was stimulated more than threefold. The inoculation led to a remarkable increase in root-associative acetylene reduction activity after the maximum tillering stage of the crop, particularly with applied N but for other treatments this inoculation effect was not significant. Cyanobacterial inoculation also increased the grain and straw yield of the crop when N was not applied. The grain and straw yield was significantly correlated with the acetylene reduction activity in flooded soils and in the root system during the tillering and maximum tillering stages of rice growth, respectively.