Nod factor-independent 'crack-entry' symbiosis in dalbergoid legume Arachis hypogaea.

Dalbergoids are typified by crack-entry symbiosis which is evidenced to be Nod Factor (NF)-independent in several Aeschynomene legumes. Natural symbionts of the dalbergoid legume Arachis hypogaea are always NF-producing, prompting us to check whether symbiosis in this legume could also be NF-independent. For this, we followed the symbiosis with two NF-containing bradyrhizobial strains - SEMIA6144, a natural symbiont of Arachis and ORS285, a versatile nodulator of Aeschynomene legumes, along with their corresponding nodulation (nod) mutants. Additionally, we investigated NF-deficient bradyrhizobia like BTAi1, a natural symbiont of Aeschynomene indica and the WBOS strains that were natural endophytes of Oryza sativa, collected from an Arachis-Oryza intercropped field. While SEMIA6144ΔnodC was non-nodulating, both ORS285 and ORS285ΔnodB could induce functional nodulation, although with lower efficiency than SEMIA6144. On the other hand, all the NF-deficient strains - BTAi1, WBOS2 and WBOS4 showed comparable nodulation with ORS285 indicating Arachis to harbour an NF-independent mechanism of symbiosis. Intriguingly, symbiosis in Arachis, irrespective of whether it was NF-dependent or independent, was always associated with the curling or branching of the rosette root hairs at the lateral root bases. Thus, despite being predominantly described as an NF-dependent legume, Arachis does retain a vestigial, less-efficient form of NF-independent symbiosis.

Identification of miRNAs linked to peanut nodule functional processes.

microRNAs (miRNAs) are non-coding small RNAs that regulate gene expression at post-transcriptional level. Thousands of miRNAs have been identified in legumes, but studies about miRNAs linked to peanut nodule functionality are scarce. In this work we analyzed transcriptional changes in peanut nodules to identify miRNAs involved in functional processes of these organs. We found 32 miRNAs precursors differentially expressed in nodules compared with roots, and predicted the potential targets of their corresponding mature miRNAs. Among them, 20 belong to 14 conserved miRNAs families and 12 are Arachis hypogaea-specific miRNAs. Expression levels of 3 miRNAs (ahy-miR399, ahy-miR159 and ahy-miR3508) were confirmed experimentally by qPCR. We also demonstrated that the expression of these miRNAs was not affected by inoculation of a biocontrol bacterium or a fungal pathogen. The catalogue of differentially expressed miRNA precursors and the expression of the corresponding mature miRNA potential targets in the nodules of A. hypogaea obtained in this work is a database of strong candidates, including A. hypogaea-specific miRNAs, for the regulation of the nodule functionality. The analysis of their role in this process will certainly lead to the characterization of essential regulators in these particular aeschynomenoid nodules.

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.

Induced systemic resistance and symbiotic performance of peanut plants challenged with fungal pathogens and co-inoculated with the biocontrol agent Bacillus sp. CHEP5 and Bradyrhizobium sp. SEMIA6144.

Synergism between beneficial rhizobacteria and fungal pathogens is poorly understood. Therefore, evaluation of co-inoculation of bacteria that promote plant growth by different mechanisms in pathogen challenged plants would contribute to increase the knowledge about how plants manage interactions with different microorganisms. The goals of this work were a) to elucidate, in greenhouse experiments, the effect of co-inoculation of peanut with Bradyrhizobium sp. SEMIA6144 and the biocontrol agent Bacillus sp. CHEP5 on growth and symbiotic performance of Sclerotium rolfsii challenged plants, and b) to evaluate field performance of these bacteria in co-inoculated peanut plants. The capacity of Bacillus sp. CHEP5 to induce systemic resistance against S. rolfsii was not affected by the inoculation of Bradyrhizobium sp. SEMIA6144. This microsymbiont, protected peanut plants from the S. rolfsii detrimental effect, reducing the stem wilt incidence. However, disease incidence in plants inoculated with the isogenic mutant Bradyrhizobium sp. SEMIA6144 V2 (unable to produce Nod factors) was as high as in pathogen challenged plants. Therefore, Bradyrhizobium sp. SEMIA6144 Nod factors play a role in the systemic resistance against S. rolfsii. Bacillus sp. CHEP5 enhanced Bradyrhizobium sp. SEMIA6144 root surface colonization and improved its symbiotic behavior, even in S. rolfsii challenged plants. Results of field trials confirmed the Bacillus sp. CHEP5 ability to protect against fungal pathogens and to improve the yield of extra-large peanut seeds from 2.15% (in Río Cuarto) to 16.69% (in Las Vertientes), indicating that co-inoculation of beneficial rhizobacteria could be a useful strategy for the peanut production under sustainable agriculture system.

Starting points in plant-bacteria nitrogen-fixing symbioses: intercellular invasion of the roots.

Agricultural practices contribute to climate change by releasing greenhouse gases such as nitrous oxide that are mainly derived from nitrogen fertilizers. Therefore, understanding biological nitrogen fixation in farming systems is beneficial to agriculture and environmental preservation. In this context, a better grasp of nitrogen-fixing systems and nitrogen-fixing bacteria-plant associations will contribute to the optimization of these biological processes. Legumes and actinorhizal plants can engage in a symbiotic interaction with nitrogen-fixing rhizobia or actinomycetes, resulting in the formation of specialized root nodules. The legume-rhizobia interaction is mediated by a complex molecular signal exchange, where recognition of different bacterial determinants activates the nodulation program in the plant. To invade plants roots, bacteria follow different routes, which are determined by the host plant. Entrance via root hairs is probably the best understood. Alternatively, entry via intercellular invasion has been observed in many legumes. Although there are common features shared by intercellular infection mechanisms, differences are observed in the site of root invasion and bacterial spread on the cortex reaching and infecting a susceptible cell to form a nodule. This review focuses on intercellular bacterial invasion of roots observed in the Fabaceae and considers, within an evolutionary context, the different variants, distribution and molecular determinants involved. Intercellular invasion of actinorhizal plants and Parasponia is also discussed.

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.

Influence of cadmium on the symbiotic interaction established between peanut (Arachis hypogaea L.) and sensitive or tolerant bradyrhizobial strains.

Heavy metals in soil are known to affect rhizobia-legume interaction reducing not only rhizobia viability, but also nitrogen fixation. In this work, we have compared the response of the symbiotic interaction established between the peanut (Arachis hypogaea L.) and a sensitive (Bradyrhizobium sp. SEMIA6144) or a tolerant (Bradyrhizobium sp. NLH25) strain to Cd under exposure to this metal. The addition of 10 µM Cd reduced nodulation and nitrogen content in both symbiotic associations, being the interaction established with the sensitive strain more affected than that with the tolerant one. Plants inoculated with the sensitive strain accumulated more Cd than those inoculated with the tolerant strain. Nodules showed an increase in reactive oxygen species (ROS) production when exposed to Cd. The histological structure of the nodules exposed to Cd revealed a deposit of unknown material on the cortex and a significant reduction in the infection zone diameter in both strains, and a greater number of uninfected cells in those nodules occupied by the sensitive strain. In conclusion, Cd negatively impacts on peanut-bradyrhizobia interaction, irrespective of the tolerance of the strains to this metal. However, the inoculation of peanut with Bradyrhizobium sp. NLH25 results in a better symbiotic interaction suggesting that the tolerance observed in this strain could limit Cd accumulation by the plant.

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.

Contribution of phytochelatins to cadmium tolerance in peanut plants.

Cadmium (Cd) is a well known heavy metal considered as one of the most toxic metals on Earth, affecting all viable cells that are exposed even at low concentration. It is introduced to agricultural soils mainly by phosphate fertilizers and causes many toxic symptoms in cells. Phytochelatins (PCs) are non-protein thiols which are involved in oxidative stress protection and are strongly induced by Cd. In this work, we analyzed metal toxicity as well as PCs implication on protection of peanut plants exposed to Cd. Results showed that Cd exposure induced a reduction of peanut growth and produced changes in the histological structure with a deposit of unknown material on the epidermal and endodermal cells. When plants were exposed to 10 µM Cd, no modification of chlorophyll, lipid peroxides, carbonyl groups, or hydrogen peroxide (H2O2) content was observed. At this concentration, peanut leaves and roots glutathione (GSH) content decreased. However, peanut roots were able to synthesize different types of PCs (PC2, PC3, PC4). In conclusion, PC synthesis could prevent metal disturbance on cellular redox balance, avoiding oxidative damage to macromolecules.

Involvement of glutathione and enzymatic defense system against cadmium toxicity in Bradyrhizobium sp. strains (peanut symbionts).

In this study, the effects of cadmium (Cd) on cell morphology and antioxidant enzyme activities as well as the distribution of the metal in different cell compartments in Bradyrhizobium sp. strains were investigated. These strains were previously classified as sensitive (Bradyrhizobium sp. SEMIA 6144) and tolerant (Bradyrhizobium sp. NLH25) to Cd. Transmission electron micrographs showed large electron-translucent inclusions in the sensitive strain and electron-dense bodies in the tolerant strain, when exposed to Cd. Analysis of Cd distribution revealed that it was mainly bounded to cell wall in both strains. Antioxidant enzyme activities were significantly different in each strain. Only the tolerant strain was able to maintain a glutathione/oxidized glutathione (GSH/GSSG) ratio by an increase of GSH reductase (GR) and GSH peroxidase (GPX) enzyme activities. GSH S-transferase (GST) and catalase (CAT) activities were drastically inhibited in both strains while superoxide dismutase (SOD) showed a significant decrease only in the sensitive strain. In conclusion, our findings suggest that GSH content and its related enzymes are involved in the Bradyrhizobium sp. tolerance to Cd contributing to the cellular redox balance.

Phenotypic and phylogenetic characterization of native peanut Bradyrhizobium isolates obtained from Córdoba, Argentina.

Peanut is an economically important legume nodulated by slow-growing bacteria of the genus Bradyrhizobium. In this study, a collection of native slow-growing peanut rhizobial isolates from Argentina was obtained and characterized. The phenotypical characterization included the determination of the symbiotic properties, whereas the genetic and phylogenetic diversity was assessed through ERIC-PCR and sequencing of the internal transcribed spacer (ITS) region, as well as the dnaK and nodA genes. The results obtained indicated that peanut nodulating bradyrhizobia were phenotypically and genotypically diverse, and included locally adapted variants of B. yuanmingense and B. iriomotense carrying novel nodA alleles.

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.

Cadmium accumulation and tolerance in Bradyrhizobium spp. (peanut microsymbionts).

In this study, the effect of cadmium (Cd) on cell viability and its accumulation in Bradyrhizobium spp. (peanut microsymbionts) as well as the role of glutathione (GSH) in the tolerance to this metal were investigated. A reference strain recommended as peanut inoculant (Bradyrhizobium sp. SEMIA6144) grew up to 10 µM Cd meanwhile a GSH-deficient mutant strain (Bradyrhizobium sp. SEMIA6144-S7Z) was unable to grow at this concentration. Two native peanut isolates obtained from Córdoba soils (Bradyrhizobium sp. NLH25 and Bradyrhizobium sp. NOD31) tolerated up to 30 µM Cd. The analysis of Cd content showed that Bradyrhizobium sp. SEMIA6144 accumulated a high amount of this metal, but a considerable inhibition of growth was observed compared to tolerant strains at 10 µM Cd. At this concentration, the intracellular GSH content of all the Bradyrhizobium sp. strains was not modified in comparison to control conditions. However, at 30 µM Cd, the intracellular GSH content significantly increased in Bradyrhizobium sp. strains NLH25 and NOD31. Thus, the distinct response of each Bradyrhizobium sp. strain to Cd reveals that, even in closely related lineages, there are strain-specific variations influencing the levels of tolerance to this metal. Indeed, the native peanut isolates tolerated higher Cd concentration than the reference strain, possibly due to an increase in GSH levels which could act as a detoxifying agent.

Experimental evidences of pSym transfer in a native peanut-associated rhizobia.

In previous works we have characterized the native bacterial diversity associated with root nodules of peanut grown in Córdoba province, Argentina. Studies performed in the isolate Rhizobium sp. NET30 revealed a phylogenetic incongruence between housekeeping and nodulation genes. This discrepancy could be explained by the horizontal transfer of nodulation genes from the native peanut isolate Rhizobium sp. NCHA22, a bacterium that showed high identity percentages with Rhizobium tropici IIB strains in the basic and symbiotic genes analyzed. In this work, we demonstrate that, in R. sp. NCHA22, genes required for nodule formation in common bean (Phaseolus vulgaris L.) are plasmid-borne. A symbiotic plasmid capable of conjugal transfer to different genetic backgrounds was identified in this isolate. The mechanism involved in the plasmidic transfer differs from that described for R. tropici CIAT899 (R. tropici IIB type strain). The transfer of a symbiotic plasmid, and the subsequent homologous recombination of nodulation genes in R. sp. NET30 genetic background could account for the phylogenetic incongruence determined in this isolate. Results are also indicating that the transfer of the R. tropici NCHA22 pSym may be a frequent event in native conditions.

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.

Role of glutathione in the growth of Bradyrhizobium sp. (peanut microsymbiont) under different environmental stresses and in symbiosis with the host plant.

Glutathione (GSH) plays an important role in the defence of microorganisms and plants against different environmental stresses. To determine the role of GSH under different stresses, such as acid pH, saline shock, and oxidative shock, a GSH-deficient mutant (Bradyrhizobium sp. 6144-S7Z) was obtained by disruption of the gshA gene, which encodes the enzyme gamma-glutamylcysteine synthetase. Growth of the mutant strain was significantly reduced in liquid minimal saline medium, and the GSH content was very low, about 4% of the wild-type level. The defect, caused by disruption of the gshA gene in the growth of mutant strain, cannot be reversed by the addition of GSH (up to 100 micromol/L) to the liquid minimal saline medium, and the endogenous GSH level was approximately the same as that observed without the addition of GSH. In contrast, the wild-type strain increased the GSH content under these conditions. However, the growth of the mutant strain in a rich medium (yeast extract--mannitol) increased, suggesting that at least some but not all of the functions of GSH could be provided by peptides and (or) amino acids. The symbiotic properties of the mutant were similar to those found in the wild-type strain, indicating that the mutation does not affect the ability of the mutant to form effective nodules.