Azospirillum amazonense inoculation: effects on growth, yield and N2 fixation of rice (Oryza sativa L.)

Bacteria of the genus Azospirillum are considered to be plant-growth promoting bacteria (PGPR) and stimulate plant growth directly either by synthesising phyto-hormones or by promoting nutrition by the process of biological nitrogen fixation (BNF). Although this genus extensively studied, the effects of inoculation and the possible BNF contribution of the Azospirillum amazonense specie are very scarce. The aim of this study was to isolate, characterise and evaluate auxin production and nitrogenase activity of this species and to select, by inoculation of rice plants, promising isolates based on their ability to improve plant growth, yield and the BNF contribution. One hundred and ten isolates obtained from rice were characterised and grouped according to colony features. Forty-two isolates, confirmed as A. amazonense by the fluorescent in situ hybridization (FISH) technique, were tested for auxin production and nitrogenase activity in vitro. Subsequently plant growth promotion related to plant nutrition effect was evaluated, in vitro and in greenhouse experiments. The BNF contribution to plant growth was evaluated using the ¹⁵N isotope dilution technique. All A. amazonense strains tested produced indoles, but only 10% of them showed high production, above 1.33 μM mg protein⁻¹. The nitrogenase activity also was variable and only 9% of isolates showed high nitrogenase activity and the majority (54%) exhibited a low potential. The inoculation of selected strains in rice under gnotobiotic conditions reduced the growth of root and aerial part when compared to the control, showing the negative effects of excess of phytohormone accumulation in the medium. However, in the greenhouse experiment, inoculation of strains of A. amazonense increased grain dry matter accumulation (7 to 11.6%), the number of panicles (3 to 18.6%) and nitrogen accumulation at grain maturation (3.5 to 18.5%). BNF contributions up to 27% were observed for A. amazonense Y2 (wild type strain). The data presented here is the first report that the PGPR effect of A. amazonense for rice plants grown under greenhouse conditions is mainly due the BNF contribution as measured by ¹⁵N isotope dilution technique, in contrast to the hormonal effect observed with other Azospirillum species studied.     

Nitrogen fixation associated with grasses and cereals: Recent results and perspectives for future research

Over the last few years research in the area of biological nitrogen fixation (BNF) associated with cereals and grasses has become divided into two areas. On the one hand there have been a large number of reports of responses of field-grown plants to inoculation with N₂-fixing bacteria, principallyAzospirillum spp. On the other hand there have been several reports of significant contributions of associated BNF to the nutrition of several crops, including wetland rice, sugar cane and some forage grasses. However, where BNF contributions have definitely been established no certain information is available as to the diazotrophic organisms responsible. Furthermore, certain recent reports indicate that, at least in some cases, responses of plants to inoculation withAzospirillum spp. have been shown not to be due to BNF contributions. In this paper we review some recent progress in this field, particularly at our institute in Rio de Janeiro, concerning specificity of selected Azospirillum strains in the infection of cereal roots and the promotion of responses in the host plants. The possible mechanisms of plant response are discussed including the possibility that plant growth substances or bacterial nitrate reductase are involved. The application of¹⁵N and N balance techniques to the quantification of plant associated BNF are considered and the possible strategies that may be adopted to further the understanding of true N₂-fixing plant/diazotroph associations. The recent discovery of many more plant-associated N₂-fixing bacteria suggests that further research in this area may eventually lead to the development of such associations with applications for agricultural productivity.     

Application of 15N natural abundance technique for evaluating biological nitrogen fixation in oil palm ecotypes at nursery stage in pot experiments and at mature plantation sites

A range of different species of diazotrophic bacteria has been found in tissues and the rhizosphere of oil palm plants, suggesting a potential to benefit from biological nitrogen fixation (BNF). A few studies have confirmed that plantlets at nursery stage can benefit significantly from BNF after inoculation with Azospirillum spp. but no data are available regarding the benefit from naturally-occurring diazotrophic bacteria in oil palm. The results described here were derived from two pot trials laid out under controlled conditions with plantlets from two important regions for palm oil production in Brazil, as well as from different field sites of mature oil palm plantations. The ¹⁵N natural abundance technique was employed to estimate plant dependence on BNF (%Ndfa) by the different ecotypes grown in soil and previously characterized as hosting diazotrophic bacteria. From both pot trials it was possible to identify some ecotypes of high potential for N₂-fixation that reached in some cases approximately 50%Ndfa. However, the accuracy of measurement still needs to be improved using more suitable reference plants for pot experiments. Values of δ ¹⁵N signals from oil palm and reference plants in the field were inconclusive concerning any benefit from BNF to oil palm, owing to apparently high temporal and spatial variability of δ ¹⁵N of the plant-available N in the heterogeneous soil matrix for the different palm and reference plant tested.     

<Emphasis Type="Italic">Azospirillum</Emphasis> spp. from native forage grasses in Brazilian Pantanal floodplain: biodiversity and plant growth promotion potential

A sustainable alternative to improve yield and the nutritive value of forage is the use of plant growth-promoting bacteria (PGPB) that release nutrients, synthesize plant hormones and protect against phytopathogens (among other mechanisms). Azospirillum genus is considered an important PGPB, due to the beneficial effects observed when inoculated in several plants. The aim of this study was to evaluate the diversity of new Azospirillum isolates and select bacteria according to the plant growth promotion ability in three forage species from the Brazilian Pantanal floodplain: Axonopus purpusii, Hymenachne amplexicaulis and Mesosetum chaseae. The identification of bacterial isolates was performed using specific primers for Azospirillum in PCR reactions and partial sequencing of the 16S rRNA and nifH genes. The isolates were evaluated in vitro considering biological nitrogen fixation (BNF) and indole-3-acetic acid (IAA) production. Based on the results of BNF and IAA, selected isolates and two reference strains were tested by inoculation. At 31 days after planting the plant height, shoot dry matter, shoot protein content and root volume were evaluated. All isolates were able to fix nitrogen and produce IAA, with values ranging from 25.86 to 51.26 mg N mL^?1 and 107–1038 µmol L^?1, respectively. The inoculation of H. amplexicaulis and A. purpusii increased root volume and shoot dry matter. There were positive effects of Azospirillum inoculation on Mesosetum chaseae regarding plant height, shoot dry matter and root volume. Isolates MAY1, MAY3 and MAY12 were considered promising for subsequent inoculation studies in field conditions.     

Biological nitrogen fixation associated with sugar cane and rice: Contributions and prospects for improvement

¹⁵N isotope and N balance studies performed over the last few years have shown that several Brazilian varieties of sugarcane are capable of obtaining over 60% of their nitrogen (<150 kg N ha^-1 year^-1) from biological nitrogen fixation (BNF). This may be due to the fact that this crop in Brazil has been systematically bred for high yields with low fertilizer N inputs. In the case of wetland rice, N balance experiments performed both in the field and in pots suggest that 30 to 60 N ha^-1 crop^-1 may be obtained from plant-associated BNF and that different varieties have different capacities to obtain N from this source. ¹⁵N₂ incorporation studies have proved that wetland rice can obtain at least some N from BNF and acetylene reduction (AR) assays also indicate differences in N₂-fixing ability between different rice varieties. However in situ AR field estimates suggest plant-associated BNF inputs to be less than 8 kg N ha^-1 crop^-1. The problems associated with the use of the ¹⁵N dilution technique for BNF quantification are discussed and illustrated with data from a recent study performed at EMBRAPA-CNPAB. Although many species of diazotrophs have been isolated from the rhizosphere of both sugarcane and wetland rice, the recent discovery of endophytic N₂-fixing bacteria within roots, shoots and leaves of both crops suggests, at least in the case of sugarcane, that these bacteria may be the most important contributors to the observed BNF contributions. In sugarcane both Acetobacter diazotrophicus and Herbaspirillum spp. have been found within roots and aerial tissues and these microorganisms, unlike Azospirillum spp. and other rhizospheric diazotrophs, have been shown to survive poorly in soil. Herbaspirillum spp. are found in many graminaceous crops, including rice (in roots and aerial tissue), and are able to survive and pass from crop to crop in the seeds. The physiology, ecology and infection of plants by these endophytes are fully discussed in this paper. The sugarcane/endophytic diazotroph association is the first efficient N₂-fixing system to be discovered associated with any member of the gramineae. As yet the individual roles of the different diazotrophs in this system have not been elucidated and far more work on the physiology and anatomy of this system is required. However, the understanding gained in these studies should serve as a foundation for the improvement/development of similar N₂-fixing systems in wetland rice and other cereal crops.     

Effect of seed inoculation,mycorrhizal infection and organic amendment on wheat growth

Summary The effect of seed inoculation withAzotobacter spp. orAzospirillum spp., and garbage amendment (0.5%), on the growth of wheat was studied in a field experiment under sub-tropical conditions. Two levels of N fertilizer were applied, the usual field rate (150 kg N ha^–1) and half this amount. Tillering of plants, dry matter contents and nitrogenase activity were determined 30, 60 and 90 days after sowing. At the end of the experimental period, spore numbers and percentage of mycorrhizal infection were observed in the rhizosphere and root systems of plants. Straw and grain yields were also determined. The results of this study showed that seed inoculation and/or organic amendment stimulated plant growth, nitrogenase activity and mycorrhizal infection. This was more noticeable withAzotobacter than withAzospirillum. Inoculation withAzotobacter together with 1/2 N dose and organic amendment was the most effective application (19.75 and 10.70 t ha^–1 were recorded for straw and grain yield, respectively).     

Influence of Azospirillum Strains on the Nodulation of Clovers by Rhizobium Strains

Mixed cultures of several Azospirillum and Rhizobium trifolii strains caused either an inhibition or stimulation of nodule formation on plant hosts as compared with nodulation of plants inoculated with R. trifolii alone. Azospirillum strains affected the nodulation process at a precise cell ratio (R. trifolii/Azospirillum cells) and time of inoculation. All Azospirillum strains used showed a variation in their ability to inhibit or enhance nodulation by R. trifolii strains. When nonviable cell preparations of Azospirillum strains were used for mixing experiments, no effect on nodulation was observed. A decrease in the effectiveness of normally Nod⁺ Fix⁺R. trifolii strains was observed when an Azospirillum strain caused an increase in nodule number.     

Activation of a calcium-dependent protein kinase involved in the <Emphasis Type="Italic">Azospirillum</Emphasis> growth promotion in rice

Rice seedlings (Oryza sativa) inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense FT326 showed an enhanced development of the root system 3 days after inoculation. Later on, a remarkable enlargement of shoots was also evident. An increase in the Ca²⁺-dependent histone kinase activity was also detected as a result of inoculation. The biochemical characterization and Western-blot analysis of the kinase strongly supports the hypothesis that it belongs to a member of the rice CDPK family. The fact that the amount of the protein did not change upon inoculation seems to indicate that a posttranslational activation is responsible for the change in the enzymatic activity. An in-gel kinase experiment identified a 46 kDa CDPK like protein kinase as a putative component of the signal transduction pathway triggered by Azospirillum inoculation. To our knowledge, this is the first report on the possible involvement of a Ca²⁺-dependent protein kinase in promotion of rice plants growth by A. brasilense.     

The effect of inoculation with Azospirillum brasilense on growth and nitrogen utilization by wheat plants

Azospirillium brasilense is a rhizosphere bacteria that has been reported to improve yield when inoculated on wheat plants. However, the mechanisms through which this effect is induced is still unclear. In the present work, we have studied the effects of inoculating a highly efficient A. brasilense strain on wheat plant grown in 5 kg pots with soil in a greenhouse, under three N regimes (0, 3 or 16 mM NO₃ ^–, 50 ml/pot once or twice-a -week), and in disinfected or non-disinfected soil. At the booting stage, the inoculated roots in both soils showed a similar colonization by Azospirillum sp. that was not affected by N addition. The plants grown in the disinfected soil showed a higher biomass, N content and N concentration than those in the non-disinfected soil, and in both soils the inoculation stimulated plant growth, N accumulation, and N and NO₃ ^– concentration in the tissues.At maturity, the inoculated plants showed a higher biomass, grain yield and N content than the uninoculated ones in both soils, and a higher grain protein concentration than the uninoculated. It is concluded that in the present experiments, A. brasilenseincreased plant growth by stimulating nitrogen uptake by the roots.     

Influence of Soil Type,Cultivar and Verticillium dahliae on the Structure of the Root and Rhizosphere Soil Fungal Microbiome of Strawberry

Sustainable management of crop productivity and health necessitates improved understanding of the ways in which rhizosphere microbial populations interact with each other, with plant roots and their abiotic environment. In this study we examined the effects of different soils and cultivars, and the presence of a soil-borne fungal pathogen, Verticillium dahliae, on the fungal microbiome of the rhizosphere soil and roots of strawberry plants, using high-throughput pyrosequencing. Fungal communities of the roots of two cultivars, Honeoye and Florence, were statistically distinct from those in the rhizosphere soil of the same plants, with little overlap. Roots of plants growing in two contrasting field soils had high relative abundance of Leptodontidium sp. C2 BESC 319 g whereas rhizosphere soil was characterised by high relative abundance of Trichosporon dulcitum or Cryptococcus terreus, depending upon the soil type. Differences between different cultivars were not as clear. Inoculation with the pathogen V. dahliae had a significant influence on community structure, generally decreasing the number of rhizosphere soil- and root-inhabiting fungi. Leptodontidium sp. C2 BESC 319 g was the dominant fungus responding positively to inoculation with V. dahliae. The results suggest that 1) plant roots select microorganisms from the wider rhizosphere pool, 2) that both rhizosphere soil and root inhabiting fungal communities are influenced by V. dahliae and 3) that soil type has a stronger influence on both of these communities than cultivar.     

Stable cesium uptake and accumulation capacities of five plant species as influenced by bacterial inoculation and cesium distribution in the soil

The effects of inoculation with Bacillus and Azospirillum strains on growth and cesium accumulation of five plant species, Komatsuna, Amaranth, sorghum, common millet and buckwheat, grown on cesium-spiked soil were assessed for potential use in cesium remediation. Pot experiments were performed using “artificially” Cs-contaminated soil. Three treatments were applied based on Cs location in the soil. For a soil height of 15 cm in the pots, Cs was added as follows: in the top five cm to imitate no ploughing condition; in the bottom five cm simulating inverted ploughing; and uniformly distributed Cs reproducing normal plowing. Generally, inoculation of Cs-exposed plants significantly enhanced growth and tolerance to this element. Transfer factor (ratio of Cs concentration in the plant tissues to that in surrounding soil) was strongly influenced by Cs distribution, with higher values in the top-Cs treatment. Within this treatment, inoculation of Komatsuna with Bacillus and Azospirillum strains resulted in the greatest transfer factors of 6.55 and 6.68, respectively. Cesium content in the shoots was high in the Azospirillum-inoculated Komatsuna, Amaranth, and buckwheat, i.e., 1,830, 1,220, and 1,030 µg per pot, respectively (five plants were grown in each pot). Therefore, inoculation of Komatsuna and Amaranth with the strains tested here could be effective in enhancing Cs accumulation. The decrease of Cs transfer under uniform- and bottom-Cs treatments would suggest that countermeasures aiming at decreasing the transfer of Cs could rely on ploughing practices.     

L-Tryptophan-dependent biosynthesis of indole-3-acetic acid (IAA) improves plant growth and colonization of maize by <Emphasis Type="BoldItalic">Burkholderia phytofirmans</Emphasis> PsJN

Burkholderia phytofirmans PsJN is a well-known plant growth-promoting bacterium that establishes rhizospheric and endophytic colonization in different plants. PsJN inoculation promotes growth of different horticultural crops. L-Tryptophan (L-TRP) application may further improve its effectiveness, due to substrate (L-TRP)-dependent inoculum (PsJN)-derived auxins in the rhizosphere. In the present study, the substrate (L-TRP)-dependent response of PsJN inoculation to maize growth and auxin biosynthesis was evaluated under pot conditions. In vitro auxin biosynthesis by PsJN was determined in the absence and presence of L-TRP, a physiological precursor of auxins. Surface-disinfected seeds were treated with peat-based inoculum and L-TRP solutions (10^?4 and 10^?5 M). Results revealed that L-TRP application and PsJN inoculation, when applied separately, significantly increased the growth parameters of maize compared to untreated control. However, PsJN inoculation supplemented with L-TRP (10^?5 M) gave the most promising results and significantly increased plant height, photosynthesis, chlorophyll content, root biomass and shoot biomass up to 18, 16, 45, 62 and 55 %, respectively, compared to the uninoculated control. Similarly, higher values of N, P and IAA content were observed with precursor (L-TRP)–inoculum (PsJN) interaction. The inoculant strain efficiently colonized maize seedlings and was recovered from the rhizosphere, root and shoot of plants. The results imply that substrate (L-TRP)-derived IAA biosynthesis in the rhizosphere by PsJN inoculation could be a useful approach for improving the growth, photosynthesis and nutrient content of maize plants.     

Trends in biological nitrogen fixation research and application

In the world each year 17.2×10⁷ tons of N are biologically fixed. Biological nitrogen fixation (BNF) contributes to plant production in arable lands and in natural ecosystems. Research to improve BNF is progressing through the breeding of efficient N-fixing organisms and host plants, selection of the best combinations of host plant and microsymbiont, and by the improvement of inoculation techniques and field management. Biotechnology is useful for the creation of promising N₂-fixing organisms. However, to increase plant production through enhanced BNF the constraints in establishing effective N₂-fixing systems in the field should be understood and eliminated.     

Azospirillum lipoferum strain AZm5 containing 1-aminocyclopropane-1-carboxylic acid deaminase improves early growth of tomato seedlings under nitrogen deficiency

In this study we evaluated the ability of two wild strains of Azospirillum, A. lipoferum AZm5 and A. brasilense VS9, to produce ACC deaminase. We tested the effects of a deficiency and medium doses of nitrogenous fertilizers on the growth and physiology of tomato plants (Lycopersicon esculentum Mill cv. ACE VF55) inoculated with both Azospirillum strains independently. Tomato plants were evaluated by root elongation assay and grown in pot soil culture with different nitrogen levels (0 kg N ha^–1 and 170 kg N ha^–1). The root:shoot ratio (R:S) and some ecophysiological traits were determined after 42 days of plant growth. Results showed very different physiological characteristics in both strains. We found three relevant aspects related to the AZm5 strain: it produces high amounts of cytokinins, it contains the gene acdS, which encodes ACC deaminase, and it promotes plant growth. We conclude that AZm5 maybe useful to increase N uptake in N-deficient soil by production of cytokinins and the promotion of ACC deaminase activity, which favored leaf expansion and higher leaf N investment. Therefore, for tomato culture, a simultaneous biofertilization with AZm5 and a relatively low fertilization with N (170 kg N ha^–1) to promote AZm5 activity could be advantageous.     

Nitrate Reductase in Wheat Plants Grown Under Water Stress and Inoculated with Azospirillum spp.

The present investigation has been performed to evaluate nitrate reductase (NR) and nitrogenase activities as well as growth and mineral nutrition of wheat plants grown under drought stress and inoculated with different Azospirillum strains (NR⁻ and NR⁺). Fresh, dry mass and water content decreased with decreasing soil moisture content, which was accompanied with low soluble sugars and soluble protein content and increase in the total amino acids content. Azospirillum inoculation with either bacterial strain (NR⁻ and NR⁺) significantly increased the above characteristics even at 40 % moisture content. NR activity decreased in both the shoots and roots by decreasing soil moisture content. NR⁺ strain exhibited increased root NR activity compared with uninoculated plants or inoculated with NR⁻ strain. However, plants inoculated with NR⁻strain increased NR activity in the shoot more than in the root of the same plant and in the shoot of control plants. Inoculation with either NR⁻ and NR⁺ Azospirillum strains gave higher nitrogenase activity than uninoculated control plants. The low N supply (0.5 mM) did not affect nitrogenase activity. NR⁻strain was less effective than NR⁺strain in promoting total N-yield, spike numbers and their mass per pot. Azospirillum inoculation exhibited no significant changes in wheat Mg²⁺ content. However, K⁺ and Ca²⁺ have shown significantly increased values. Azospirillum beneficial effect on plant N balance and growth are most probably composed of multiple mechanisms and beneficial NR is one of them. The importance of Azospirillum NR⁺strains for increasing wheat resistance to water stress is also supported by the obtained data. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of co-inoculation of methylotrophic Methylobacterium oryzae with Azospirillum brasilense and Burkholderia pyrrocinia on the growth and nutrient uptake of tomato, red pepper and rice

The present greenhouse study was undertaken to evaluate the effects of co-inoculating methylotrophic Methylobacterium oryzae CBMB20 along with nitrogen-fixing Azospirillum brasilense CW903 or a phosphate solubilizing bacterium Burkholderia pyrrocinia CBPB-HOD on the growth and nutrient uptake of tomato, red pepper and rice. Seed inoculation and soil/foliar application of the bacterial strains alone or under dual inoculation increased the plant growth in terms of shoot or root length and increased the nutrient uptake in the plants studied compared to uninoculated control plants. Co-inoculation of M. oryzae CBMB20 with A. brasilense CW903 or B. pyrrocinia CBPB-HOD improved the N and P concentration of plants, while the results varied among the plant species tested. Also, co-inoculation of the bacterial strains increased the activity of nitrogenase, urease and phosphatase enzymes in soil when compared to uninoculated control or individual inoculations. Though the inoculation effects were analyzed at an early stage of plant growth, the results conclusively suggest that M. oryzae being compatible with other microorganisms in the rhizosphere can potentially be used as individual inoculant or co-inoculated with other plant growth promoting bacteria to increase the production in sustainable agricultural systems.     

Bacterial inoculation of maize affects carbon allocation to roots and carbon turnover in the rhizosphere

To assess the influence of bacteria inoculation on carbon flow through maize plant and rhizosphere,¹⁴C allocation after¹⁴CO₂ application to shoots over a 5-day period was determined. Plants were grown on C- and N-free quartz sand in two-compartment pots, separating root and shoot space. While one treatment remained uninoculated, treatments two and three were inoculated withPantoea agglomerans (D5/23) andPseudomonas fluorescens (Ps I A12), respectively, five days after planting. Bacterial inoculation had profound impacts on carbon distribution within the system. Root/rhizosphere respiration was increased and more carbon was allocated to roots of plants being inoculated. After five days of¹⁴CO₂ application, more ethanol-soluble substances were found in roots of inoculated treatments and lower rhizodeposition indicated intensive C turnover in the rhizosphere. In both inoculated treatments the intensity of photosynthesis measured as net-CO₂-assimilation rates were increased when compared to the uninoculated plants. However, high C turnover in the rhizosphere reduced shoot growth of D5/23 inoculated plants, with no effect on shoot growth of Ps I A12 inoculated plants. A separation of labeled compounds in roots and rhizodeposition revealed that neutral substances (sugars) constituted the largest fraction. The relative fractions of sugars, amino acids and organic acids in roots and rhizodeposition suggest that amino acid exudation was particularly stimulated by bacterial inoculation and that turnover of this substance group is high in the rhizosphere.     

Interaction of Rhizosphere Bacteria, Fertilizer, and Vesicular-Arbuscular Mycorrhizal Fungi with Sea Oats

Plants must be established quickly on replenished beaches in order to stabilize the sand and begin the dune-building process. The objective of this research was to determine whether inoculation of sea oats (Uniola paniculata L.) with bacteria (indigenous rhizosphere bacteria and N₂ fixers) alone or in combination with vesicular-arbuscular mycorrhizal fungi would enhance plant growth in beach sand. At two fertilizer-N levels, Klebsiella pneumoniae and two Azospirillum spp. did not provide the plants with fixed atmospheric N; however, K. pneumoniae increased root and shoot growth. When a sparingly soluble P source (CaHPO₄) was added to two sands, K. pneumoniae increased plant growth in sand with a high P content. The phosphorus content of shoots was not affected by bacterial inoculation, indicating that a mechanism other than bacterially enhanced P availability to plants was responsible for the growth increases. When sea oats were inoculated with either K. pneumoniae or Acaligenes denitrificans and a mixed Glomus inoculum, there was no consistent evidence of a synergistic effect on plant growth. Nonetheless, bacterial inoculation increased root colonization by vesicular-arbuscular mycorrhizal fungi when the fungal inoculum consisted of colonized roots but had no effect on colonization when the inoculum consisted of spores alone. K. pneumoniae was found to increase spore germination and hyphal growth of Glomus deserticola compared with the control. The use of bacterial inoculants to enhance establishment of pioneer dune plants warrants further study.