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Rhizobia are common members of plant microbiomes. This study aimed to evaluate if rhizobia can colonize sugarcane mini-setts and if and how they affect sprouting and morphological traits of plant development. Bradyrhizobium sacchari strain BR 10280T and Rhizobium sp. strain BR 10268 were inoculated on sugarcane mini-setts and sprouting and plant development were evaluated. Bacterial production of hormones and their effect on plant growth were studied using HPLC and GC-MS / SIM and sugarcane sprouting assays. Colonization was investigated by colony counting and CLSM. Sequence analysis of recA, gyrB and rpoB genes was applied to refine the phylogenetic classification of strain BR 10268. BR 10268 had a positive effect on sugarcane mini-sett sprouting and shoot and root growth at 30 and 75 days after transplantation (DAT) and colonized sprouting mini-sett tissues. Few significant effects were observed for B. sacchari BR 10280T. Cell-free BR 10268 supernatant was found to contain gibberellins (GAs) and low concentrations of indole-3-acetic acid (IAA) and trans-zeatin; treatment of mini-setts with GA3 (0.15 or 1.5 μg mL−1) affected sprouting in a similar manner as BR 10268 inoculation. BR 10268 was found to be phylogenetically close to R. freirei. Rhizobium sp. BR 10268 positively affects sprouting and growth of sugarcane, probably via production of plant hormonal substances, notably gibberellins, thus naturally occurring rhizobia may have potential as sugarcane inoculants.

We examined the influence of inoculation with five species/strains of diazotrophic bacteria on the modulation of two enzymes involved in the assimilation of N and on the soluble N fractions in the sugarcane varieties RB867515 (adapted for low fertility soils) and IACSP95-5000 (adapted for medium to high fertility soils) under high- (3 mM) and low (0.3 mM)-N conditions in hydroponic cultivation for 59 days. The sugarcane plants were produced in three steps to obtain the hydroponic cultivation: the supply of 3 mM N for 30 days (first harvest), N depletion for 72 h (second harvest), and cultivation in high- and low-N conditions over 26 days (final harvest). Inoculation was performed by immersion of the minisetts in a diluted solution of five diazotrophic bacteria. After the final harvest, plants were divided into roots and shoots to assess their dry weight and N, P, and K accumulation. The variety played an important role in the interaction with diazotrophs, each showing distinct behavior in the activity of their N-assimilation enzymes. The nitrate reductase activity (NRa—EC 1.7.1.1) was increased in var. RB867515 by 26% in the shoots and by 48% in the roots after 72 h under N depletion, while var. IACSP95-5000 showed a reduced enzymatic activity in the roots (by 62%) but not in the shoots. Under high-N conditions, the inoculated IACSP95-5000 plants showed 31% higher glutamine synthetase activity (GSa—EC 6.31.2) compared with 19% in RB867515. Under low-N conditions, the GSas were 21% and 16% higher in the inoculated RB867515 and IACSP95-5000 plants, respectively, compared with that of the control. The content of nitrogen in the form of nitrate (N-nitrate) confirmed these varietal differences, but the soluble sugar content did not. The varieties utilized N sources differently, and inoculation modified the activity of two N-assimilation enzymes as well as the biomass accumulation, with the highest improvement seen in the low fertility-adapted variety RB867515; it showed a greater response to inoculation compared with that of the high fertility-adapted variety IACSP95-5000, with an increase in biomass and nutrient accumulation (N, P, K), especially when cultivated under low-N conditions. This suggests that the best response to inoculation with diazotrophs will be achieved using low fertility-adapted sugarcane varieties under low-N conditions.

The inoculation of cereal crops with plant growth-promoting bacteria (PGPB) is a potential strategy to improve fertilizer-N acquisition by crops in soils with low capacity to supply N. A study was conducted to assess the impact of three inoculants on grain yield, protein content, and urea-15 N recovery in maize (Zea mays L.) under Cerrado soil and climate conditions. The main treatments included inoculants containing (i) Azospirillum brasilense strain Sp245, (ii) A. brasilense strains AbV5 + AbV6, (iii) Herbaspirillum seropedicae strain ZAE94, and (iv) a non-inoculated control. The subtreatments were (i) urea-N fertilization (100 kg N ha−1) at 30 days after sowing and (ii) no N addition at the stage. To determine fertilizer-N recovery, 15N–labelled urea was applied in microplots. Inoculants carrying A. brasilense improved urea-15 N acquisition efficiency in maize and also improved grain yield compared to the non-inoculated control, while urea-N fertilization enhanced grain quality by providing higher protein content. Our results suggest that the inoculation of maize grains with PGPB represents a strategy to improve fertilizer-N recovery and maize yield in Cerrado soil with a low capacity to supply N.

Culture media compositions and bioprocess conditions were studied to improve the production of cell biomass and indolic phytohormones by Herbaspirillum seropedicae BR11471, a plant growth promoting bacterium, and different inoculant formulations were also produced and tested for their stability and shelf life. Response surface methodology (RSM) based on central composite rotation designs (CCRD) was used to find bioprocess variables that lead to an increase in bacterial biomass and yield of indolic compounds. The major components of DYGS medium were optimized in small-scale shaken cultivations, in two sets of CCRD. High performance liquid chromatography was used to determine nutrient consumption and to correlate it with cell biomass production, and the Salkowski method was used to quantify indoles. Hydrolytic activity in the formulations was quantified with the fluorescein diacetate assay. Glycerol (5.5 g L−1) and yeast extract (2.8 g L−1), as the main carbon and nitrogen sources, respectively, increased biomass production by 87.5% when compared to original DYGS medium, reaching 3.0 g L−1 of dry cell weight (DCW). In a 2.0 L bioreactor, the optimized medium was used to enhance process conditions for DCW and indole-3-acetic acid (IAA). Biomass production reached 3.4 g L−1 and was restrained at highest air flow levels. The conditions of 34-36 °C, 150 rpm and 4.0 L min−1 of air flow rate resulted in 11.97 mg L−1 of IAA, an increase of 370% over original DYGS at 30 °C. Peat can still be regarded as a good cell carrier for solid state inoculants, whilst the additives tested for liquid formulations are individually more efficient than the mixture. The production of inoculants containing H. seropedicae strain BR11471 can be efficiently improved with the use of the RSM approach i.e. it maximizes the production of biomass and indolic compounds, and reduces culture media components, both key factors for large-scale industrial production.

To assess the impacts of soil microbes and plant genotype on the composition of maize associated bacterial communities. Two genotypes of Brazilian maize were planted indoors on sterile sand, a deep underground subsoil, and a nutrient-rich topsoil from the Amazon jungle (terra preta). DNA was extracted from rhizospheres, phyllospheres, and surface sterilized roots for 16S rDNA fingerprinting and next generation sequencing. Neither plant genotype nor soil type appeared to influence bacterial diversity in phyllospheres or endospheres. Rhizospheres showed strikingly similar 16S rDNA ordination of both fingerprinting and sequencing data, with soil type driving grouping patterns and genotype having a significant impact only on sterile sand. Rhizospheres grown in non-sterile soils contained greater bacterial diversity than sterile-sand grown ones, however the dominant OTUs (species of Proteobacteria and Bacteroidetes) were found in all rhizospheres suggesting seeds as a common source of inoculum. Rhizospheres of the commercial hybrid appeared to contain less bacterial diversity than the landrace. Maize rhizospheres receive diverse bacteria from soil, are influenced by the genotype or treatment of the seed, and are dominated by species of Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. As many dominant 16S rDNA sequences were observed in rhizospheres grown in both sterile and non-sterile substrate, we conclude that the most common bacterial cells in juvenile maize rhizospheres are seed transmitted.

The genus Burkholderia is highly diverse and may mediate plant growth via several mechanisms. The current study evaluates the role of strains from four Burkholderia species in maize growth promotion. Twenty three strains of diazotrophic Burkholderia species were applied to two maize genotypes (SHS5050 and BRS4157) under greenhouse and field conditions. Strains were tested for growth promotion in greenhouse during 40 days. Strain 41 from B. silvatlantica was used under field conditions. Parameters evaluated: biomass accumulation, N and root measurements in pots and grain yield, N % and N in the grains under field conditions. Eleven strains were evaluated under soil substrate. The inoculation of the hybrid SHS5050 showed root biomass improvement using three strains, namely M130, M209 and PPe7. The highest acetylene reduction activity was observed in strain 41 from B. silvatlantica. Strain 41 from B. silvatlantica improved plant dry mass on both genotypes. This strain produced 31 % more aerial tissue and 7 % more root dry mass in the BRS4157 variety than it did in the control. Both genotypes planted at Seropedica, RJ, produced 7 % more grains under field conditions and the variety produced 10.9 % more grains in comparison to the non-inoculated control. Strain 41 from B. silvatlantica showed beneficial plant-growth promotion of maize under greenhouse and field experiments specially in a low fertility soil.

Herbaspirillum spp. has been described as bacteria that colonizes not only maize roots but also stems and leaves. This study aimed to access the growth-promoting effect of 21 strains of Herbaspirillum on maize plants. This study is composed by 12 strains of the H. seropedicae, 5 strains of the H. rubrisubalbicans and 4 strains of the H. frisingense. Bacterial selection was performed in two phases, consisting on planting the BRS4157 maize variety and hybrid SHS5050 in the greenhouse, with sterile and non-sterile substrate and in the field. After 40 days, the greenhouse plants were harvested and the dry matter accumulation of the aerial part and roots were measured. The best-performing strain was tested in field trials treated with 40 and 80 kg ha−1 of N fertilizer, parallel to the same treatments without inoculation. In this trial, the grain yield was evaluated as well as the contribution of biological nitrogen fixation (BNF), using the technique of natural abundance of 15 N. The results showed that H. seropedicae ZAE94 was the best strain under controlled conditions and its application as a field inoculant increased maize yield up to 34 %, depending on the plant genotype. The BNF quantification revealed that 37 % BNF-derived plant nitrogen in the hybrid SHS5050 inoculated with the H. seropedicae strain ZAE94. The difference in response to nitrogen as well as to inoculation depends on the selected plant genotype and the bacterial inoculant. H. seropedicae ZAE94 can promote plant growth and contribute biologically with fixed nitrogen in the maize hybrid SHS5050.

Nitrogen-fixing bacteria or diazotrophs have been isolated for many years using different formulations of N-free semi-solid media. However, the strategies used to isolate them, and the recipes of these media, are scattered through the published literature and in other sources that are more difficult to access and which are not always retrievable. Therefore, the aim of this work was to collate the various methods and recipes, and to provide a comprehensive methodological guide and their use by the scientific community working in the field of biological nitrogen fixation (BNF), particularly with non-leguminous plants. Procedures used for bacterial counting and identification either from rhizosphere soil or on the surface of, or within, plant tissues (to access “endophytic” bacteria) are presented in detail, including colony and cell morphologies. More importantly, appropriate recipes available for each N-free semi-solid culture medium that are used to count and isolate various diazotrophs are presented. It is recognized by those working in the field of BNF with non-legumes that the development of the N-free semi-solid medium has allowed a tremendous accumulation of knowledge on the ecology and physiology of their associated diazotrophs. At least 20 nitrogen-fixing species have been isolated and identified based on the enrichment method originally developed by Dobereiner, Day and collaborators in the 70’s. In spite of all the advances in molecular techniques used to detect bacteria, in most cases the initial isolation and identification of these diazotrophs still requires semi-solid media. The introduction of the N-free semi-solid medium opened new opportunities for those working in the area of BNF with non-legumes not only for elucidating the important role played by their associated microorganisms, but also because some of these bacteria that were isolated using semi-solid media are now being recommended as plant growth-promoting inoculants for sugarcane (Saccharum sp.), maize (Zea mays) and wheat (Triticum aestivum) in Brazil and other countries. Further progress in the field could be made by using a combination of culture-independent molecular community analyses, in situ activity assessments with probe-directed enrichment, and isolation of target strains using modified or standard semi-solid media.

Studies have been conducted to evaluate maintenance of cell viability and stability, as well as to select cheap carriers to extend the shelf life of plant beneficial bacterial inoculants for agricultural crops. The purpose of this study was to evaluate the shelf life and the colonization efficiency of novel liquid and gel-based inoculant formulations for sugarcane. The different inoculant formulations were all composed of a mixture of five strains of diazotrophic bacteria (Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicans, Azospirillum amazonense and Burkholderia tropica), which are recognized as sugarcane growth promoters. Different inoculant formulations containing as carrier the polymers carboxymethylcellulose (CMC) and corn starch (60/40 ratio) at five different concentrations (named PIC, for Polymeric Inoculant Carrier) were supplemented, or not, with 2 % MgO, an interfacial stabilizing agent. Bacterial survival in the different formulations during storage was evaluated under controlled conditions, and two experiments with mini-cuttings of sugarcane variety RB72454 were carried out under greenhouse conditions. Laboratory tests showed that in the formulation composed of 0.8 g of the polymeric mixture per 100 g of the final product (PIC 0.8), survival of G. diazotrophicus and A. amazonense was around 109 CFU∙mL−1 after 120 days of storage, regardless of the supplementation with MgO. The other formulation (2.2 g of polymeric mixture, PIC 2.2) presented survival levels of 108 CFU∙mL−1 for up to 60 days of storage for all the individual strains. In the greenhouse, sugarcane seedlings showed a positive growth response 50 days after inoculation when inoculated with the mixture of five bacteria, with and without PIC 2.2. The polymer carriers described here allowed for the long-term survival of the five different bacterial strains tested. In addition, short-term experiments in the greenhouse showed that their application as part of an inoculant on sugarcane cuttings was at least as effective in terms of bacterial colonization and the promotion of plant growth as that of the bacterial mixture without carriers.

Because of its high dry matter (DM) productivity, elephant grass (Pennisetum purpureum) is an ideal candidate for biomass production for biofuel production if low N fertilizer rates are used to avoid high fossil fuel inputs. The objective of this study was to investigate the potential of different elephant grass genotypes to obtain contributions of plant-associated biological N2 fixation (BNF). Three field experiments with 4 or 5 different genotypes were conducted on low-fertility Acrisols, two in Rio de Janeiro State and one in Espirito Santo for the evaluation of DM and N accumulation and 15N abundance. DM and N accumulation rates of four genotypes in the two experiments in Rio State stabilized at high levels after 2 years of growth. In all experiments the spontaneously-occurring weeds in the plots were significantly higher in 15N abundance than the elephant grass genotypes. The lower 15N abundance of the elephant grass was shown not to be due to lower δ15N abundance at depth in the soil. Four of the grass genotypes obtained between 18 and 70% of their N from BNF amounting to inputs of between 36 and 132 kg N ha−1 yr−1.

To identify active diazotrophs in sugarcane, 16S rRNA and nifH transcript analyses were applied. This should help to better understand the basis of the biological nitrogen fixation (BNF) activity of a high nitrogen fixing sugarcane variety. A field experiment using the sugarcane variety RB 867515 was conducted in Seropedica, RJ, Brazil, receiving the following treatments: unfertilised and fertilised controls without inoculation, unfertilised with inoculation. The five-strain mixture developed by EMBRAPA-CNPAB was used as inoculum. Root and leaf sheath samples were harvested in the third year of cultivation to analyse the 16S rRNA and nifH transcript diversity. In addition to nifH expression from Gluconacetobacter spp. and Burkholderia spp., a wide diversity of nifH sequences from previously uncharacterised Ideonella/Herbaspirillum related phylotypes in sugarcane shoots as well as Bradyrhizobium sp. and Rhizobium sp. in roots was found. These results were confirmed using 16S cDNA analysis. From the inoculated bacteria, only nifH transcripts from G. diazotrophicus and B. tropica were detected in leaf sheaths and roots. Known as well as yet uncultivated diazotrophs were found active in sugarcane roots and stems using molecular analyses. Two strains of the inoculum mix were identified at the late summer harvest.

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.

It is well described that the beneficial interactions between plants and bacteria are genotype and site specific. Brazilian sugarcane varieties can obtain up to 70% of their nitrogen requirement from biological nitrogen fixation (BNF), and this contribution is related to the Brazilian breeding and selection processes, by example of the variety SP70-1143. In this study the effect of two inoculation mixtures containing diazotrophic bacteria in our earlier pot experiment was evaluated with two sugarcane varieties, a known responder, SP70-1143, and a newly selected variety, SP81-3250, to investigate the sugarcane genotype effect and the role of the mixtures. The sugarcane varieties SP70-1143 and SP81-3250 were grown under commercial field conditions at three sites with contrasting soil types: an Alfisol, an Oxisol and an Ultisol that means a low, medium and high natural fertility respectively. The stem yield and BNF contribution in response to bacterial inoculation were influenced by the strain combinations in the inoculum, the plant genotype, and the soil type and nitrogen fertilization, confirming the genetic and environmental influence in PGP-bacteria interactions. Inoculation effects on the BNF contribution and stem yield increased in the variety SP70-1143 grown in the Alfisol without nitrogen fertilization for three consecutive crops, and it was equivalent to the annual nitrogen fertilization. The plants grown in the Oxisol showed small increases in the productivity of the variety SP70-1143, and in the Ultisol the sugarcane plants presented even decreases in the stem productivity due to inoculation with diazotrophic bacteria mixtures. The results demonstrate the feasibility of the inoculation technology using diazotrophic bacteria in micropropagated sugarcane varieties grown in soils with low to medium levels of fertility. In addition, the results also indicated that specific plant – bacteria – environment combinations are needed to harness the full benefits of BNF.

Biological nitrogen fixation (BNF) in sugarcane is considered one of the principal reasons for the success of the Brazilian Ethanol Program (PRO-ALCOOL) for motor car fuel. The BNF influences positively the energy balance of sugarcane crops for alcohol production. Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium associated with sugarcane, and is particularly abundant and active in the early stages after germination. The objective of this work was to evaluate the effect of the addition of increasing amounts of two sources of mineral nitrogen (ammonium sulphate and calcium nitrate) on the population of G. diazotrophicus and also to evaluate its effect on nitrogenase (acetylene reduction) activity and accumulation of N by two sugarcane hybrids, SP 701143 and SP 792312. The results showed that both varieties differed in the form of nitrogen they prefer to uptake from the soil. The variety SP 701143 preferred ammonium sulphate, whilst the variety SP 792312 preferred N from calcium nitrate. In both varieties, the addition of increased doses of ammonium and nitrate inhibited the population of G. diazotrophicus but in the variety SP 701143 the inhibition was more pronounced in the presence of calcium nitrate. The acetylene reduction activity was inhibited in both varieties, especially in variety SP 792312 in the presence of either of the two nitrogen sources.

In Brazil the long-term continuous cultivation of sugarcane with low N fertiliser inputs, without apparent depletion of soil-N reserves, led to the suggestion that N2-fixing bacteria associated with the plants may be the source of agronomically significant N inputs to this crop. From the 1950s to 1970s, considerable numbers of N2-fixing bacteria were found to be associated with the crop, but it was not until the late 1980s that evidence from N balance and 15N dilution experiments showed that some Brazilian varieties of sugarcane were able to obtain significant contributions from this source. The results of these studies renewed the efforts to search for N2-fixing bacteria, but this time the emphasis was on those diazotrophs that infected the interior of the plants. Within a few years several species of such `endophytic diazotrophs' were discovered including Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, H. rubrisubalbicansand Burkholderia sp. Work has continued on these endophytes within sugarcane plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed BNF and in what site, or sites, within the cane plants the N2 fixation mainly occurs. Until such important questions are answered further developments or extension of this novel N2-fixing system to other economically important non-legumes (e.g. cereals) will be seriously hindered. As far as application of present knowledge to maximise BNF with sugarcane is concerned, molybdenum is an essential micronutrient. An abundant water supply favours high BNF inputs, and the best medium term strategy to increase BNF would appear to be based on cultivar selection on irrigated N deficient soils fertilised with Mo.

Micropropagated plantlets of sugar cane were inoculated with the N2-fixing bacterium Acetobacter diazotrophicus. Various modifications on the basic plant culture medium MS were made for the plant/bacteria association. The protocol required the inoculation of the bacteria at the end of the rooting period in a medium without hormones or vitamins, and with the concentration of sugar and mineral nutrients reduced by a factor of 10. Individual plants were inoculated with A. diazotrophicus and maintained under the appropriate light and temperature condition used for micropropagation up to 7 days. The system favored the infection and the establishment of the bacteria within the plant tissue. Bacteria colonized the plant tissue and accumulated in inter-cellular cavities and the region of lateral root emergence and also colonizes the xylem vessels. The inoculated plantlets were subsequently transferred to the acclimatization phase and after 30 days it was possible to isolate the bacteria from plant tissue. This protocol permitted studies of infection and comparison among strains.

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.