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.

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.

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.