Enterobacter sp. J49: A Native Plant Growth-Promoting Bacteria as Alternative to the Application of Chemical Fertilizers on Peanut and Maize Crops.

In agricultural soils the productivity is determined by several factors and among them are the metabolic activities of the microorganisms that reside in it. The inoculation of plants with these bacteria is an alternative to the use of agrochemicals in crops. In particular, in those soils in which P levels are low, phosphate-solubilizing bacteria became an important group of soil microorganisms. In order to propose a potential P-biofertilizer to replace chemical fertilizers, the objective of this study was to evaluate the response of peanut and maize plants to the inoculation with the phosphate solubilizer Enterobacter sp. J49 individually or in combination with chemical fertilizers on growth, yield, and nutrient contents on peanut and maize plants in field trials. Two field assays in the peanut growing region of Córdoba Province (Argentina) were carried out. The inoculation of peanut with Enterobacter sp. J49 showed an increase in the yield with respect to the other treatments. Maize plants inoculated with this strain, alone or combined with half dose of chemical fertilizer, presented the highest yields. The results indicated that Enterobacter sp. J49 has a growth-promoting effect on the yield of peanut and maize mainly under drought stress. In conclusion, the inoculation with this strain would be a more sustainable agricultural practice for improving yield of peanut and maize crops in Argentinian agricultural area.

Genome sequence of the endophytic strain Enterobacter sp. J49, a potential biofertilizer for peanut and maize.

Enterobacter sp. J49 is a plant growth promoting endophytic strain that promotes the growth of peanut and maize crops. This strain promotes plant growth by different mechanisms with the supply of soluble phosphorus being one of the most important. Enterobacter sp. J49 not only increases the phosphorus content in the plant but also in the soil favoring the nutrition of other plants usually used in rotation with these crops. The aims of this study were to analyze the genome sequence of Enterobacter sp. J49 in order to deepen our knowledge regarding its plant growth promoting traits and to establish its phylogenetic relationship with other species of Enterobacter genus. Genome sequence of Enterobacter sp. J49 is a valuable source of information to continuing the research of its potential industrial production as a biofertilizer of peanut, maize and other economically important crops.

Effects of P limitation and molecules from peanut root exudates on pqqE gene expression and pqq promoter activity in the phosphate-solubilizing strain Serratia sp. S119.

The mineral phosphate-solubilizing phenotype in bacteria is attributed predominantly to secretion of gluconic acid produced by oxidation of glucose by the glucose dehydrogenase enzyme and its cofactor, pyrroloquinoline quinone. This study analyzes pqqE gene expression and pqq promoter activity in the native phosphate-solubilizing bacterium Serratia sp S119 growing under P-limitation, and in the presence of root exudates obtained from peanut plants, also growing under P-limitation. Results indicated that Serratia sp. S119 contains a pqq operon composed of six genes (pqqA,B,C,D,E,F) and two promoters, one upstream of pqqA and other between pqqA and pqqB. PqqE gene expression and pqq promoter activity increased under P-limiting growth conditions and not under N-deficient conditions. In the plant-bacteria interaction assay, the activity of the bacterial pqq promoter region varied depending on the concentration and type of root exudates and on the bacterial growth phase. Root exudates from peanut plants growing under P-available and P-limiting conditions showed differences in their composition. It is concluded from this study that the response of Serratia sp. S119 to phosphorus limitation involves an increase in expression of pqq genes, and that molecules exuded by peanut roots modify expression of these phosphate-solubilizing bacterial genes during plant-bacteria interactions.

Growth promotion of peanut (Arachis hypogaea L.) and maize (Zea mays L.) plants by single and mixed cultures of efficient phosphate solubilizing bacteria that are tolerant to abiotic stress and pesticides.

The aims of this study were, to analyze in vitro phosphate solubilization activity of six native peanut bacteria and to determine the effect of single and mixed inoculation of these bacteria on peanut and maize plants. Ability to produce organic acids and cofactor PQQ, to solubilize FePO4 and AlPO4 and phosphatase activity were analyzed. Also, the ability to solubilize phosphate under abiotic stress and in the presence of pesticides of the selected bacteria was determined. The effect of single and mixed bacterial inocula was analyzed on seed germination, maize plant growth and in a crop rotation plant assay with peanut and maize. The six strains produced gluconic acid and five released cofactor PQQ into the medium. All bacteria showed ability to solubilize phosphate from FePO4 and AlPO4 and phosphatase activity. The ability of the bacteria to solubilize tricalcium phosphate under abiotic stress and in presence of pesticides indicated encouraging results. Bacterial inoculation on peanut and maize increased seed germination, plant́s growth and P content. Phosphate solubilizing bacteria used in this study showed efficient phosphate mineralizing and solubilization ability and would be potential P-biofertilizers for peanut and maize.

Sequence and expression analysis of putative Arachis hypogaea (peanut) Nod factor perception proteins.

Peanut, like most legumes, develops a symbiotic relationship with rhizobia to overcome nitrogen limitation. Rhizobial infection of peanut roots occurs through a primitive and poorly characterized intercellular mechanism. Knowledge of the molecular determinants of this symbiotic interaction is scarce, and little is known about the molecules implicated in the recognition of the symbionts. Here, we identify the LysM extracellular domain sequences of two putative peanut Nod factor receptors, named AhNFR1 and AhNFP. Phylogenetic analyses indicated that they correspond to LjNFR1 and LjNFR5 homologs, respectively. Transcriptional analysis revealed that, unlike LjNFR5, AhNFP expression was not induced at 8 h post bradyrhizobial inoculation. Further examination of AhNFP showed that the predicted protein sequence is identical to GmNFR5 in two positions that are crucial for Nod factor perception in other legumes. Analysis of the AhNFP LysM2 tridimensional model revealed that these two amino acids are very close, delimiting a zone of the molecule essential for Nod factor recognition. These data, together with the analysis of the molecular structure of Nod factors of native peanut symbionts previously reported, suggest that peanut and soybean could share some of the determinants involved in the signalling cascade that allows symbiosis establishment.

Interrelationships between Bacillus sp. CHEP5 and Bradyrhizobium sp. SEMIA6144 in the induced systemic resistance against Sclerotium rolfsii and symbiosis on peanut plants.

Plant-growth-promoting bacteria are often used to enhance crop yield and for biological control of phytopathogens. Bacillus sp. CHEP5 is a biocontrol agent that induces systemic resistance (ISR) in Arachis hypogaea L. (peanut) against Sclerotium rolfsii, the causal agent of root and stem wilt. In this work, the effect of the co-inoculation of Bacillus sp. CHEP5 and the peanut nodulating strain Bradyrhizobium sp. SEMIA 6144 was studied on induction of both systemic resistance and nodulation processes. Bradyrhizobium sp. SEMIA 6144 did not affect the ability of Bacillus sp. CHEP5 to protect peanut plants from S. rolfsii by ISR and the priming in challenged-plants, as evidenced by an increment in phenylalanine ammonia-lyase enzyme activity. Additionally, the capacity of Bradyrhizobium sp. SEMIA 6144 to induce nodule formation in pathogen-challenged plants was improved by the presence of Bacillus sp. CHEP5.

The effects of pesticides on bacterial nitrogen fixers in peanut-growing area.

In the peanut production, the applications of herbicides and fungicides are a common practice. In this work, studies done under field conditions demonstrated that pesticides affected negatively the number and nitrogenase activity of diazotrophic populations of soil. Agrochemical effects were not transient, since these parameters were not recovered to pre-treatment levels even 1 year after pesticides application. Results obtained from greenhouse experiments revealed that the addition of herbicide or fungicides diminished the free-living diazotrophs number reaching levels found in soil amended with the pesticides and that the number of symbiotic diazotrophs was not affected by the insecticide assayed. The soil nitrogenase activity was not affected by fungicides and glyphosate. The effect of pesticides on the nitrogen-fixing bacteria diversity was evaluated both in field and greenhouse experiments. Analysis of clone libraries generated from the amplification of soil nifH gene showed a diminution in the genetic diversity of this bacterial community.

Diversity and symbiotic effectiveness of indigenous rhizobia-nodulating Adesmia bicolor in soils of Central Argentina.

Native perennial legume Adesmia bicolor reveals characteristics that are key to securing persistence under grazing. Literature on the diversity and symbiotic effectiveness of indigenous rhizobia-nodulating A. bicolor in central Argentina is limited. The purpose of this study was therefore to determine phenotypic and genotypic variability as well as biological N-fixation effectiveness in rhizobia isolated from A. bicolor nodules. To this end, repetitive genomic regions were analyzed using ERIC primers. In the greenhouse, plants were grown under a (i) N-fertilized treatment, (ii) N-free control treatment, and (iii) rhizobia inoculation treatment. Dry weight and N-content were analyzed. All isolates belonged to Rhizobium genus and showed high symbiotic effectiveness. The N-content/subterranean N-content ratio in aerial and subterranean parts of inoculated plants was higher than that observed in N-fertilized plants during the vegetative stage. Results from this study demonstrate that symbiosis between native rhizobial strains and A. bicolor is very effective.

A study on the prevalence of bacteria that occupy nodules within single peanut plants.

In this study, bacteria hosted in root nodules of single plants of legume Arachis hypogaea L. (peanut) cv Tegua Runner growing at field were isolated. The collection of nodule isolates included both fast and slow growing strains. Their genetic diversity was assessed in order to identify the more frequently rhizobial strain associated to nodules from single plants. Molecular fingerprinting of 213 nodular isolates indicated heterogeneity, absence of a dominant genotype and, therefore, of a unique strains highly competitive. Efficient nitrogen-fixing isolates were identified as Bradyrhizobium sp. by phylogenetic analysis of the sequences of their 16S rRNA genes. The genetic diversity of 68 peanut nodulating isolates from all the collected plants was also analyzed. Considering their ERIC-PCR profiles, they were grouped in eighteen different OTUs for 60% similarity cut-off. Results obtained in this study indicate that the genetic diversity of rhizobia occupying nodules from single plant is very high, without the presence of a dominant strain. Therefore, the identification of useful peanut rhizobia for agricultural purposes requires strongly the selection, among the diverse population, of a very competitive genotype in combination with a high-symbiotic performance.

Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria.

Several bacterial isolates were recovered from surface-sterilized root nodules of Arachis hypogaea L. (peanut) plants growing in soils from Córdoba, Argentina. The 16S rDNA sequences of seven fast-growing strains were obtained and the phylogenetic analysis showed that these isolates belonged to the Phylum Proteobacteria, Class Gammaproteobacteria, and included Pseudomonas spp., Enterobacter spp., and Klebsiella spp. After storage, these strains became unable to induce nodule formation in Arachis hypogaea L. plants, but they enhanced plant yield. When the isolates were co-inoculated with an infective Bradyrhizobium strain, they were even found colonizing pre-formed nodules. Analysis of symbiotic genes showed that the nifH gene was only detected for the Klebsiella-like isolates and the nodC gene could not be amplified by PCR or be detected by Southern blotting in any of the isolates. The results obtained support the idea that these isolates are opportunistic bacteria able to colonize nodules induced by rhizobia.

Signal molecules in the peanut-bradyrhizobia interaction.

Main nodulation signal molecules in the peanut-bradyrhizobia interaction were examined. Flavonoids exuded by Arachis hypogaea L. cultivar Tegua were genistein, daidzein and chrysin, the latest being released in lower quantities. Thin layer chromatography analysis from genistein-induced bacterial cultures of three peanut bradyrhizobia resulted in an identical Nod factor pattern, suggesting low variability in genes involved in the synthesis of these molecules. Structural study of Nod factor by mass spectrometry and NMR analysis revealed that it shares a variety of substituents with the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium spp. Nodulation assays in legumes nodulated by these rhizobia demonstrated differences between them and the three peanut bradyrhizobia. The three isolates were classified as Bradyrhizobium sp. Their fixation gene nifD and the common nodulation genes nodD and nodA were also analyzed.

Peanut nodulation kinetics in response to low pH.

In this work, the peanut nodulation kinetics by acid-sensitive and tolerant isolates under acid stress condition was analyzed. The results demonstrated that the acid pH produced a decrease in the number of nodules formed only when peanut plants were inoculated with acid-sensitive isolates but increased steadily by the addition of 10 mM Ca2+, reaching higher values than those obtained at pH 7.0. On the contrary, the peanut nodulation by acid-tolerant isolates was not affected by this stressing condition. These data suggest that acid-tolerant isolates could be used as a potential source of strains for preparing highly effective inoculants for peanut plants growing in acid soils.

A calcium-dependent bacterial surface protein is involved in the attachment of rhizobia to peanut roots.

As part of a project to characterize molecules involved in the crack-entry infection process leading to nodule development, a microscopic assay was used to visualize the attachment of cells of Bradyrhizobium sp. strains SEMIA 6144 and TAL 1000 (labelled by introducing a plasmid expressing constitutively the green fluorescent protein GFP-S65T) to Arachis hypogaea L. (peanut). Qualitative and quantitative results revealed that attachment was strongly dependent on the growth phase of the bacteria. Optimal attachment occurred when bacteria were at the late log or early stationary phase. Cell surface proteins from the Bradyrhizobium sp. strains inhibited the attachment when supplied prior to the attachment assay. Root incubation with a 14-kDa protein (eluted from sodium dodecyl sulphate - gel electrophoresis of the cell surface fraction) prior to the attachment assay resulted in a strong decrease of attachment. The adhesin appeared to be a calcium-binding protein, since cells treated with EDTA were found to be able to bind to adhesin-treated peanut roots. Since this protein has properties identical to those reported for rhicadhesin, we propose that this adhesin is also involved in the attachment process of rhizobia to root legumes that are infected by the crack-entry process.