Plant growth promotion in cereal and leguminous agricultural important plants: from microorganism capacities to crop production.

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.

The art of isolating nitrogen-fixing bacteria from non-leguminous plants using N-free semi-solid media: a practical guide for microbiologists

Although diazotrophs are important in the nitrogen (N)-cycle and contribute to the pool of plant available N, the population response to long-term inorganic fertilization is largely unknown. Here, we investigated the diazotrophic populations in both the bulk and rhizosphere soils of maize grown in an acidic farmland soil that experienced 25 years of inorganic fertilization. The fertilization regimes included unfertilized control, N fertilizer alone, N fertilizer with quicklime, phosphorus (P) and potassium (K) fertilizers, N + P + K fertilizers, and N + P + K fertilizers with quicklime. Quantitative PCR and high-throughput pyrosequencing of the nifH gene were used to analyze diazotrophic abundance and community composition. All of the fertilizer treatments improved soil nutrient availability, but those without quicklime caused soil acidification. Maize biomasses and nifH copy numbers were significantly lower under N and N + P + K treatments but increased under P + K fertilization. Quicklime applications effectively alleviated the inhibitory effect of N input. Fertilization led to decreases in operational taxonomic unit richness and shifts in diazotrophic community composition. Soil pH and nutrient availability had a cooperative effect on diazotrophic abundance, while soil nutrient availability appeared to be the main factor shaping diazotrophic community structure. Rhizosphere effects increased the nifH gene copy number but did not obviously change the diazotrophic community composition on the current research scale. Overall, the long-term inorganic fertilization affected both diazotrophic abundance and community composition, and the fertilizer treatment had a greater influence than quicklime remediation or crop cultivation on community composition.

Impact of 25 years of inorganic fertilization on diazotrophic abundance and community structure in an acidic soil in southern China

Plant growth-promoting bacteria, mainly diazotrophs and phosphate solubilizers, can reduce the use of chemical fertilizers for rice crops. Here, diazotrophic bacteria isolated from rice were screened for their ability to solubilize inorganic P (Pi) in vitro and in association with rice plants cultivated in pots. Forty-nine isolates were tested for the ability to solubilize Pi on NBRIP and GL agar plate media and seven selected strains were further evaluated in NBRIP liquid medium. Three of these strains were inoculated in rice plants grown in soil pots containing 15N-labeled fertilizer and two sources of P: tricalcium phosphate (TCP) or simple superphosphate (SSP). The dry matter, yield, N, P, and the 15N content accumulated in plant tissues were measured at 135 days after planting. Seven strains belonging to the genera Herbaspirillum and Burkholderia formed a halo of solubilized Pi on agar plates. The Burkholderia strains showed peak soluble P (around 200 mg P L−1) on the fifth day when grown in NBRIP liquid medium for 14 days. Inoculation of Herbaspirillum strains (H18, ZA15) and a Burkholderia vietaminensis strain (AR114) increased rice grain yield from 33 to 47 % with TCP and 18 to 44 % with TSS, respectively. The bacterial inoculation led to enhanced N-use efficiency of the 15N-labeled fertilizer. These results suggest that the selection and use of P-solubilizing diazotrophic bacteria are a good strategy to promote P solubilization and/or N use efficiency in rice plants.

Selection of phosphate-solubilizing diazotrophic Herbaspirillum and Burkholderia strains and their effect on rice crop yield and nutrient uptake

Phosphorus (P) is one of the most important minerals required for plant growth occupying a strong position among soil macro nutrients. Soil P deficiency is often fulfilled by phosphate fertilizers....

Phosphorus and phosphate solubilizing bacteria: Keys for sustainable agriculture

http://www.alice.cnptia.embrapa.br/bitstream/doc/955171/1/Almeida2.pdf

Response of traditional upland rice varieties to inoculation with selected diazotrophic bacteria isolated from rice cropped at the Northeast region of Brazil

Aims Previous studieshaveshownthatelephantgrassis colonized by nitrogen-fixing bacterial species; however, these results were based on culture-dependent methods, an approach that introduces bias due to an incomplete assessment of the microbial community. In this study, we used culture-independent methods to survey the diversity of endophytes and plant-associated bacterial communities in five elephant grass genotypes used in bioenergy production. Methods The plants of five genotypes of elephant grass were harvested from the experimental area of Embrapa Agrobiologia and divided into stem and root tissues. Total DNA and RNAwere extracted from plant tissues and the bacterial communities were analyzed by DGGE and clone library of the 16S rRNA and nifH genes at both the cDNA and DNA levels. Results Overall, the patterns based on DNA- and RNAderivedDGGE-profilesdiffered,especiallywithintissue samples. DNA-based DGGE indicated that both total bacterial and diazotrophic communities associated with roots (rhizoplane+endophytes) differed clearly from those obtained from stems (endophytes). These results were confirmed by the phylogenetic analyses of RNAderived sequences of 16S rRNA (total bacteria; 586 sequences), but not for nifH (186). In fact, rarefaction analyses showed a higher diversity of diazotrophic organisms associated with stems than roots. Based on 16S rRNA sequences, the clone libraries were dominated by sequences affiliated to members of Leptotrix (12.8 %) followed by Burkholderia (9 %) and Bradyrhizobium (6.5 %), while most of the nifH clones were closely related to the genus Bradyrhizobium (26 %). Conclusions Our results revealed an unexpectedly large diversity of metabolically active bacteria, providing new insights into the bacterial species predominantly found in

Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum—a bioenergy crop

The objective of this study was to evaluate the viability of real-time polymerase chain reaction (PCR) to detect Gluconacetobacter diazotrophicus in sugarcane inoculated and non-inoculated with diazotrophs which are grown under field conditions. The primer pair PAL5F and PAL5R yielded a specific band of 189 bp using real time PCR with SYBER Green I. This primer pair was the most sensitive one to detect endophytic bacteria in sugarcane plants grown under field conditions and inoculated or not with bacterium. The lower limit of detection was 5 fg of template DNA, which corresponds to 12 bacterial cells. In contrast, a cultivation-dependent approach was not capable of detecting the bacteria in the same sample. The quantification of G. diazotrophicus from field grown plants using real-time PCR and a set of specific primers can be used to determine the number of bacterial cells that colonize endophytically the plant after inoculation. A highly sensitive and specific assay was developed to quantify G. diazotrophicus in sugarcane plants grown under field conditions.  This assay can be used to evaluate the occurrence of the bacterium in different sample types. 

	Key words: Inoculant, diazotrophs, endophyte, colonization, quantitative polymerase chain reaction (qPCR).

Development of a real-time PCR assay for the detection and quantification of Gluconacetobacter diazotrophicus in sugarcane grown under field conditions

Péricles de Souza Galisa

Papers

Response of traditional upland rice varieties to inoculation with selected diazotrophic bacteria isolated from rice cropped at the Northeast region of Brazil

Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum—a bioenergy crop

Development of a real-time PCR assay for the detection and quantification of Gluconacetobacter diazotrophicus in sugarcane grown under field conditions