Effect of inoculation ofAzospirillum spp. on nitrogen accumulation by field-grown wheat

Summary Two experiments were performed to examine the effects of inoculation of field grown wheat with various Azospirillum strains. In the first experiment the soil was sterilized with methyl bromide to reduce the Azospirillum population and¹⁵N labelled fertilizer was added to all treatments. Two strains ofAzospirillum brasilense isolated from surface sterilized wheat roots and theA. brasilense type strain Sp7 all produced similar increases in grain yield and N content. From the¹⁵N and acetylene reduction data it was apparent that these increases were not due to N₂ fixation. In the second experiment performed in the same (unsterilized) soil, twoA. brasilense strains (Sp245, Sp246) and oneA. amazonense strain (Am YTr), all isolated from wheat roots, produced responses of dry matter and N content while the response to the strain Sp7 was much smaller. These data confirm earlier results which indicate that if natural Azospirillum populations in the soil are high (the normal situation under Brazilian conditions), strains which are isolated from wheat roots are better able to produce inoculation responses than strains isolated from other sources. The inoculation of a nitrate reductase negative mutant of the strain Sp245 produced only a very small inoculation response in wheat. This suggests that the much greater inoculation response of the original strain was not due to N₂ fixation but to an increased nitrate assimilation due to the nitrate reductase activity of the bacteria in the roots. Consultant Inter-American Institute for Cooperation in Agriculture IICA/EMBRAPA World Bank Project.     

Field inoculation of sorghum and rice withAzospirillum spp. andHerbaspirillum seropedicae

Two field experiments were carried out at the UAPNPBS experimental station, Seropédica, with two sorghum and one rice cultivars. The establishment, and inoculation effects, ofAzospirillum spp. andHerbaspirillum strains marked with antibiotic resistance were investigated. One grain sorghum (BR 300) and one sugar sorghum (Br 505) cultivar were used.Azospirillum lipoferum strain S82 (isolated from surface sterilized roots of sorghum) established in both cultivars and comprised 40 to 80% of theAzospirillum spp. population in roots and stems 60 days after plant emergence (DAE).Azospirillum amazonense strain AmS91 (isolated from surface-sterilized roots of sorghum) reached only 50%. At 90 DAE, S82 almost disappeared (less than 30% of establishment) while the establishment of AmS91 remained constant in roots and stems. No establishment ofH. seropedicae strain H25 (isolated from surface-sterilized roots of sorghum) orA. lipoferum strain S65 (isolated from the root surface of sorghum) could be observed on inoculated roots. Inoculation with S82, AmS91 or S65 but not withH. seropedicae H25, increased plant dry weight of both cultivars and total N in grain of the grain sorghum. In rice,A. lipoferum Al 121 andA. brasilense Sp 245 (isolated from surface sterilized rice and wheat roots respectively) established in the roots but there was no increase inAzospirillum spp. numbers due to inoculation. None of the strains affected plant growth or rice grain yield.Azospirillum amazonense, A82 andH. seropedicae Z95, which did not establish in roots, significantly enhanced seed germination.     

Associative effect ofAzospirilium brasilense withRhizobium japonicum on nodulation and yield of soybean (Glycine max)

Summary Azospirillum was associated with nodules of soybean. In general, seed inoculation with a broth culture ofAzospirillum brasilense alone significantly increased nodulation and grain yield of soybean grown in pots in unsterilized soil with different levels of urea ranging from 0 to 80 kg N/ha. This trend was significantly reproducible in a second experiment when a carrier based inoculant of the bacterium was used for seed inoculation.Inoculation withRhizobium japonicum andA. brasilense in combination generally increased grain yield in both the experiments, although the data were not significant.     

Yield response of a common spring wheat cultivar to inoculation withAzospirillum brasilense at various levels of nitrogen fertilization

Summary The yield response of a common spring wheat cultivar,Triticum aestivum, to inoculation withAzospirillum brasilense was studied at four levels of N fertilization. Plant yield increased due to the inoculation treatment only at medium and high levels of N fertilization, with a maximum yield increase of about 8.0 per cent at the highest level (approximately 1.0 g of pure N per plant). Yield increase was mostly due to an increase in the number of grains per spike, and at the highest level of fertilization, also due to a higher number of spikes per plant. At all N levels, the inoculation caused an increase of 0.5–1.4 per cent in the number of fertile spikelets per main spike.Grain protein percentage was unaffected by the inoculation, though significantly increase due to the fertilization treatments.The occurrence of maximum yield response at the highest N level, the response by early-determined yield components, i.e. spikelet number, and the unaffected grain protein content are in accord with the suggestion that the contribution ofAzospirillum brasilense to wheat yield is not through N₂-fixation.     

Yield response of various wheat genotypes to inoculation withAzospirillum brasilense

Summary Twenty different wheat genotypes representing a wide genetic variability, were tested for their yield and yield components response to inoculation withAzospirillum brasilense, at two levels of N fertilization. Only two cultivars responded by a significant yield increase of 7.4 and 8.0 per cent — both at the higher N level. The response reflected an increase in the number of grains per plant added as additional spikes. The importance of the host plant genotype for a successful wheat-bacteria association is discussed.     

Selection and evaluation ofAzospirillum brasilense strains growing at a sub-optimum temperature in rhizocoenosis with wheat

FourteenAzospirillum brasilense strains growing at a sub-optimum temperature were selected based on their ability to grow and carry out plant growth promoting activities at 22°C. The strains were tested for their response to inoculation in wheat (two popular cultivars, HD2285 and WH547, under sterile conditions) crop using sterile and nonsterile rooting medium. Significant increase in plant growth parameters was observed: the overall response to inoculation was better in cultivar HD 2285. Based on their performance under sterile conditions, 4 strains were selected and compared under nonsterile conditions with strain sensitive to a sub-optimum temperature in pots using wheat variety HD2285. The strains capable of growing at the sub-optimum temperature can colonize the wheat endorhizosphere efficiently and improve the plant growth and yield as compared to sensitive strain; a 25–27% increase in grain yield was found on inoculating two selected strains compared to NO ₃ ^- control.     

Soil and litter respiration rates in different microhabitats of a mixed oak-conifer forest and their control by edaphic conditions and substrate quality

Summary Seven isolates belonged toA. brasilense and 3 belonged toA. lipoferum. Isolates having more denitrifying capacity fixed less nitrogen in nitrogen free semi-solid malate medium. One strain ofA. lipoferum having high nitrogen fixing capacity with negative test for denitrification was tested as inoculant to supplement the nitrogen need of a wheat crop in field condition with different doses of N with and without the inoculant. While control without nitrogen yielded 1260 kg/ha the yield in inoculated treatment was 2070 kg/ha resulting in significant increase. In a treatment receiving 40 kg N/ha the grain yield was 2370 kg/ha as against yield of 3110 kg/ha in a similar treatment receiving fertiliser plus inoculant. Thus increase in yield was about 30%. Further the treatment receiving 80 kg N/ha yielded 2970 kg/ha as against yield of 4150 kg/ha in a treatment receiving inoculant alongwith the above dose of the fertiliser. Thus increase in yield due to application of inoculant was about 36%. Similarly, the uptake of N in different treatments was augmented due to inoculation of seeds with the culture.     

Growth promoting effect of Azospirillum brasilense on Casuarina cunninghamiana Miq. seedlings

The growth of Casuarina cunninghamiana seedlings was stimulated when inoculated with Azospirillum brasilense. This resulted in a higher biomass production than in uninoculated controls in the presence or absence of a non-nodulating strain of Frankia.Increase in whole plant dry weight was due to a significant increase in both shoot and root biomass, which corresponded with a higher total N content of the plants inoculated with Azospirillum. No such effects were observed under inoculation with a non-nodulating Frankia strain. These results suggest that the growth-promoting substances provided by A. brasilense may have enhanced the growth of Casuarina seedlings.     

The participation of the cell wall hydrolytic enzymes in the initial colonization of Azospirillum brasilense on wheat roots

The effect of cellulase and pectinase on bacterial colonization of wheat was studied by three different experiments. In the first experiment, the root colonization of 3 wheat cultivars (Ghods, Roshan and Omid) by two A. brasilense strains (Sp7 and Dol) was compared using pre-treated roots with cellulase and pectinase, and non-treated with these enzymes (control). Although the root colonization varied greatly among strain-plant combinations in controls, the pre-treatment of roots with polysaccharide degrading enzymes significantly increased the bacterial count in roots, regardless of the strain-plant combination. This might be an indication that cell wall may act as an important factor in plant-Azospirillum interaction. In the second experiment, the root cellulase activity of the same wheat cultivars treated with and without the two Azospirillum brasilense, strains (Sp7 and Dol) was compared. The pre-treatment of wheat roots with Azospirillum enhanced the cellulase activity of wheat root extracts. Thus, the cellulase activity might participate in the initial colonization of wheat roots by Azospirillum. The comparison of the cellulase activity of root extracts within inoculated and non-inoculated seedlings showed that the inoculation had enhanced the cellulase activity in root extracts, but this effect was directly dependent on the strain-plant combination. Strain Sp7 stimulated the highest cellulase activity in cv. Roshan, but strain Dol induced the highest enzyme activity in cv. Ghods. In the third experiment, several growth parameters of those 3 wheat cultivars treated with and without those two bacterial strains (Sp7 and Dol) were compared. The highest magnitude of growth responses caused by Sp7 strain was in the cv Roshan, but Dol strain stimulated the highest growth in cv Ghods. Therefore, effective colonization may contribute to more growth responses.     

Seed inoculation withAzospirillum brasilense andAzotobacter chroococcum and the root biomass of rice (Oryza sativa L.)

Summary The root biomass of rice seedlings increased due to inoculation withAzospirillum brasilense andAzotobacter chroococcum singly or in combination. In general, the increase in the biomass of roots was better in unsterilized soil than in sterilized one with or without inorganic nitrogen in the form of urea.     

Nutrition of sorghum plants fertilized with nitrogen or inoculated withAzospirillum brasilense

Summary Sorghum plants were inoculated withAzospirillum brasilense or received an N-amended nutrient solution. Azospirillum inoculation increased plant dry weight and nitrogen assimilation by 25%. Most plant growth responses to Azospirillum were comparable to application of 2.0 mM N. Increased scavenging of nutrients, altered root permeability or nitrogen fixation are possible explanations for these effects.This work was supported by the United States Department of Agriculture, Agricultural Research Service (CRIS No. 5102-20170-001) in collaboration with the University of California, Berkeley. Requests for reqrints to G. J. Bethlenfalvay.     

The effect of aluminium on phosphate uptake by three isolated ectomycorrhizal fungi

Three wheat cultivars with different tolerances against free aluminium were grown monoxenically in association with Azospirillum brasilense. In situ nitrogen fixation, measured with the acetylene reduction assay, was higher by the aluminium-tolerant cultivars than by the sensitive cultivar. The transfer of fixed nitrogen to the host plant, determined by the ¹⁵N dilution technique, was also significantly higher in the aluminium-resistant wheat plants. The total accumulation of fixed nitrogen in the host plants due to an A. brasilense inoculation varied from approximately 13% to 17% of the total nitrogen in the root and 2.9% to 3.9% of the nitrogen in the shoot.The quantity and quality of exudates released in liquid nutrient solution were analysed separately for two of the wheat cultivars, one aluminium-tolerant and one aluminium-sensitive. After 29 days of growth the aluminium-tolerant plants exudated significantly higher total amounts of carbon than aluminium-sensitive plants. No differences between the two cultivars existed in the carbon exudation rate per gram dry root.Much higher concentrations of low molecular dicarboxylic acids i.e. succinic, malic and oxalic acid, were found in the exudates of aluminium-tolerant plants. Dicarboxylic acids are potential chelating compounds for positively charged metals such as aluminium and they may play an important role in protecting the plant against aluminium incorporation. They are also very suitable substrates for Azospirillum spp. It is therefore suggested that these factors may be causing the higher associative nitrogen fixation rates which was found in the aluminium-tolerant wheat cultivars.     

Fatty acid analysis of four Azospirillum species reveals three groups

Cellular fatty acid composition of 14 strains from the four species of Azospirillum was determined by gas chromatographic analysis. All strains of Azospirillum lipoferum and Azospirillum brasilense were similar in fatty acid data, thus not revealing an expected distinction between the two long established species. Strains of both Azospirillum halopraeferens and Azospirillum amazonense, however, differed significantly from this first group of strains.     

Field release of genetically marked Azospirillum brasilense in association with Sorghum bicolor L.

The agronomic impact of genetically tagged azospirilla (Azospirillum brasilense)was assessed in open field and their fluctuation were monitored in the soil/rhizosphere. Strain performance, upon inoculation of sorghum, was evaluated over a two-years period; agronomic treatments included nitrogen application (0, 80, 160 kg ha^–1), and types of inoculant (Sp245 lacZ, Sp6 gusA, Sp6 IAA⁺⁺ gusA). Grain yield was higher for inoculated seed plots than in non-inoculated ones, whereas nitrogen content, biomass of plant residues and nitrogen in plant residues gave values that were not statistically different. Root length density (RLD) of sorghum at the end of the stem elongation stage was affected only by the indole-3-acetic acid (IAA) overproducer Azospirillum strain (A. brasilense Sp6 IAA⁺⁺ gusA) with respect to the normal IAA producer (A. brasilense Sp6 gusA), being higher in the first 40 cm of depth, notwithstanding the level of nitrogen fertilization. The traceability of the released genetically modified strains enabled to monitor their ability to colonise soil and roots. Moreover, the genetic modification per se vs. the non-modified counterpart, did not affect the culturable aerobic population in soil, microfungi, streptomycetes, fluorescent pseudomonads, soil microbial biomass, or some microbial activities, all selected as important indicators.     

Effect of Azospirillum inoculation on some growth parameters and n-content of wheat,sorghum and panicum

The potential of the nitrogen fixing bacteriumAzospirillum brasilense to enhance development and increase growth of several gramineae was investigated. In both sterilized and non-sterilized systems heading and flowering occurred earlier in the inoculated plants as compared to the noninoculated ones. Total shoot and root weights, total-N content, plant height and leaf length were significantly increased by inoculation.From the Department of Agricultural Botany.     

Association between N2-fixing bacteria and pearl millet plants: Responses,mechanisms and persistence

Responses to inoculation with N₂-fixing bacteria were studied in relation to genotypic differences in pearl millet, effect of nitrogen levels, and FYM additions in India. In some experiments, inoculation increased mean grain yield up to 33% over the uninoculated control, whereas in the remaining 11 experiments there was no significant increase. Increased grain yields, >10% over the uninoculated controls were observed in 46% of the experiments withAzospirillum lipoferum (18.7% average increase) and withAzotobacter chroococcum (13.6% average increase). Yield increases were nil or reduced in three experiments withAzos. lipoferum and four experiments withAztb. chroococcum. In two experiments continued inoculation for two or three years resulted in increased grain, plant biomass yield, and N uptake. Interactions of bacterial cultures with cultivars or years were not observed. The counts of the inoculated strains increased two to three-fold when inoculation was continued for three years. Repeated inoculations increased the mean cumulative N uptake from season 1 to season 3 by 19 kg ha^–1. Repeated inoculations withAztb. chroococcum andAzos. lipoferum increased mean grain yield of a succeeding crop by 14.4% and 9.8%, respectively, over the uninoculated control. Inoculation increased the efficiency of N-assimilation by pearl millet. Marginal increase in nitrogenase activity, associated with the inoculated plants was observed during later stages of plant growth. Increased leaf nitrate reductase activity (NRA) was observed after inoculation with these bacteria. The responses to inoculation are mainly attributable to increased plant N assimilation which could be the effect of growth promoting substances secreated by the bacteria; and thus the contribution from BNF may be small.CRISAT, journal article 732.     

Interaction between barley and mixed cultures of nitrogen fixing and phosphate-solubilizing bacteria

Pot experiments were carried out to investigate the effect of inoculation with pure and mixed cultures of nitrogen fixers Azospirillum lipoferum 137, Arthrobacter mysorens 7 and the phosphate-solubilizing strain Agrobacterium radiobacter 10 on growth and mineral nutrition of two barley cultivars. A significant positive effect on grain yield both of the studied barley cultivars was obtained after inoculation with mixtures of A. lipoferum 137 + A. radiobacter 10 and A. lipoferum 137 + A. mysorens 7 only. The acetylene reduction activity on roots or in batch culture was significantly higher when A. lipoferum 137 and A. radiobacter 10 were combined. Using ¹⁵N isotope dilution technique it was established that these mixed cultures significantly increased the accumulation of nitrogen fertilizer in the plants. The strain A. radiobacter 10 promoted a better accumulation of phosphorus fertilizer by plants and A. mysorens 7 increased the total phosphorus content in plant tissues. The maximum positive effect of joint inoculation on plant development was observed when the combined nitrogen in soil was in short supply. It was concluded that inoculation with bacterial mixtures provided a more balanced nutrition for the plants and the improvement in root uptake of nitrogen and phosphorus was the major mechanism of interaction between plants and bacteria. The introduced bacteria were able to colonize actively the rhizoplane of barley. No interspecific competition or antagonism were established between components of the bacterial mixtures in the rhizoplane. The strains A. mysorens 7 and A. radiobacter 10 improved viability of A. lipoferum 137 when the plants were grown in acid soil. Field experiments carried out on 3 barley cultivars confirmed the assertion that inoculation with mixed cultures significantly increases the grain yield and nitrogenous nutrition of plants as compared with single cultures.     

Strain-specific salt tolerance and chemotaxis ofAzospirillum brasilense and their associative N-fixation with finger millet in saline calcareous soil

Three salt-tolerantAzospirillum brasilense strains were isolated from the roots of finger millet grown in saline calcareous soil and characterized. The effect of various salts on growth and N₂ase activity of these strains was tested and strain STR1 was found more tolerant at higher concentrations of Cl^-, SO₄ ² and HCO₃ ^-. Bicarbonate was found to be the most toxic. The content and concentrations of root exudates of finger millet genotypes were different and chemotaxis to sugars, amino acids, organic acids and root exudates was strain specific. Under salt stress, significant interactions between strains and genotypes of finger millet resulted in different responses of N₂ase activity, endo- and exorhizospheric population, dry weight of root, shoot and grain yield.     

Effect of Rhizobium andAzospirillum lipoferum inoculation on the nodulation,yield and nitrogen uptake of peanut cultivars

Peanut (Arachis hypogaea Linn.) Cvs. Robut 33-1 and JL 24 were inoculated with Rhizobium strain NC 92 and a strain ofAzospirillum lipoferum singly and as mixed inoculum. Seed inoculation with these bacteria enhanced nodulation, N content and yield of these cultivars under field conditions. While a mix inoculation of these two diazotrophic cultures had an adverse effect on these parameters as compare to single inoculation.     

The response of maize (Zea mays) to Azospirillum inoculation in various types of soils in the field

Application of a peat-based powder inoculant of Azospirillum brasilense, as well as a granular inoculant (each containing 0.5–1.0×10⁷ Azospirillum/g moist peat), in the seed furrows of Zea mays resulted in significantly increased yields (11 to 14%) in light soils at low rates of N fertilization. In general, there was no effect of inoculation on plant yields in heavier soils nor when N fertilization was high. Pre-emergence application of granular inoculant and inoculation associated with irrigation were more efficient in increasing yield than inoculation post-emergence or seed coating.E. Fallik is with ARO-The Volcani Centre, Department of Postharvest Science of Fresh Produce, Bet-Dagan 50250, Israel. Y. Okon is with The Hebrew University of Jerusalem, Faculty of Agriculture, Department of Plant Pathology and Microbiology, Rehovot 76100, Israel