Wheat represents a fundamental component of the diet that contributes to ensuring global food security. However, its cultivation faces various abiotic and biotic constraints that result in fluctuating yields. The use of plant growth promoting rhizobacteria (PGPR) with the ability to solubilize phosphate presents itself as an environmentally friendly solution aimed to at enhancing sustainable agriculture practices. The objective of the present study is to assess the potential of PGPR in augmenting both yield and phosphorus (P) levels in wheat plants (Triticum durum L). under semi-controlled environmental conditions. Initially, the strains under investigation were screened for their capacity to dissolve different forms of mineral complex P, along with other plant growth promoting attributes such as iron chelation, Indole acetic acid (IAA) production, nitrogen fixation, and production of exopolysaccharides. Plant response to inoculation with PGPR was studied through an experiment conducted in a greenhouse involving four distinct treatments: (1) Control plants without any inoculation, (2) Inoculation with the PGP27 strain (Rahnella aquatilis), (3) Inoculation with the PGP25 strain (Erwinia persicina), and (4) Inoculation with the PGP374 strain (Raoultella terrigena). Results demonstrated that the studied strains exhibited significant promoting activities, including the solubilization of complex phosphate, the production of exopolysaccharides, IAA, siderophores, and atmospheric nitrogen fixation. Furthermore, the inoculation of plants with PGPR led to an increase in both biomass and plant phosphate concentrations, surpassing those observed in the control group. In conclusion, the findings propose that species like Raoultella and Erwinia have the potential to offer agronomic advantages as rhizobacteria that promote plant growth.

Bradyrhizobium diazoefficiens, a bacterial symbiont of soybean and other leguminous plants, enters a nodulation-promoting genetic programme in the presence of host-produced flavonoids and related signalling compounds. Here, we describe the crystal structure of an isoflavonoid-responsive regulator (FrrA) from Bradyrhizobium, as well as cocrystal structures with inducing and noninducing ligands (genistein and naringenin, respectively). The structures reveal a TetR-like fold whose DNA-binding domain is capable of adopting a range of orientations. A single molecule of either genistein or naringenin is asymmetrically bound in a central cavity of the FrrA homodimer, mainly via C-H contacts to the pi-system of the ligands. Strikingly, however, the interaction does not provoke any conformational changes in the repressor. Both the flexible positioning of the DNA-binding domain and the absence of structural change upon ligand binding are corroborated by small-angle X-ray scattering (SAXS) experiments in solution. Together with a model of the promoter-bound state of FrrA our results suggest that inducers act as a wedge, preventing the DNA-binding domains from moving close enough together to interact with successive positions of the major groove of the palindromic operator.

Among abiotic stresses, drought is considered the most important growth-limiting factor, particularly in arid and semiarid regions. Therefore, new management strategies are needed to resolve and mitigate these negative consequences, improve soil quality and plant growth, and rationalize water use. In this context, we investigated the role of beneficial plant growth–promoting rhizobacteria (PGPR), arbuscular mycorrhizal fungi (AMF) consortium, and compost (Comp) in improving tomato growth and yield, and drought tolerance. A completely randomized design was used in this experiment with the water stress as the main factor consisting of two treatments: (1) control well-watered (WW) plants (75% field capacity (FC)) and (2) water-stressed (WS) plants (35% FC), and the fertilization as a subfactor consisting of eight treatments. Growth parameters (shoot and root dry weight, leaf number, and area), productivity (fruit number and weight), mineral content (Ca 2+ , Na + , K + , and P), biochemical parameters (sugar, protein, and polyphenols), and antioxidant enzyme activities (polyphenoloxidase, peroxidase, catalase, and superoxide dismutase) were evaluated to investigate the effect of both factor. Soil physicochemical and microbial properties were examined after the experiment to assess the impact of water stress and applied biofertilizers on these parameters. Water stress affected negatively plant growth traits and yield and unbalanced the antioxidant enzymes. However, application of biofertilizers attenuated the negative effect of drought stress. For instance, a significant increase in shoot biomass of 160%, 120%, and 156% was obtained by Comp, PGPR + Comp, and AMF + Comp treatments compared to the control, respectively. Indeed, all treatments (except AMF under both conditions and PGPR + AMF + Comp under WS conditions) significantly increased the fruit number per plant. Concerning fruit yield, Comp, PGPR + Comp, AMF + Comp, PGPR, and PGPR + AMF treatments were the most effective treatments resulting in 179%, 149%, 111%, 203%, and 181% of the increment, respectively. Concerning the fruit quality, our finding showed a positive effect on sugar content and a significant decrease in the amount of polyphenol content was recorded by the different applied treatments compared to control plants under WW conditions. Under WS conditions, the PGPR significantly enhanced sugar and protein by 24% and 177%, respectively. However, they significantly decreased the polyphenol content under WS conditions by 42%. According to the antioxidant enzymes, a significant decrease in polyphenoloxidase, peroxidase, catalase, and superoxide dismutase activities in roots was recorded by the different applied treatment plants than WS control plants. In the shoot part, treatments with PGPR increased the catalase activity under WS conditions. PGPR + Comp, AMF + Comp, PGPR, and PGPR + AMF treatments significantly decreased the polyphenoloxidase activity and increased the peroxidase activity under WS conditions. In light of these findings, the use of compost alone or in combinations with the beneficial microorganisms (PGPR and AMF) enhanced the water stress tolerance of tomato plants by improving plant growth, osmolyte accumulation, and mineral accumulation and by decreasing the amount of antioxidant enzyme activity. This strategy could be vital to resolve and mitigate the negative consequences of drought stress and rationalize water use.

Host-specific legume-rhizobium symbiosis is strictly controlled by rhizobial type III effectors (T3Es) in some cases. Here, we demonstrated that the symbiosis of Vigna radiata (mung bean) with Bradyrhizobium diazoefficiens USDA110 is determined by NopE, and this symbiosis is highly dependent on host genotype. NopE specifically triggered incompatibility with V. radiata cv. KPS2, but it promoted nodulation in other varieties of V. radiata , including KPS1. Interestingly, NopE1 and its paralogue NopE2, which exhibits calcium-dependent autocleavage, yield similar results in modulating KPS1 nodulation. Furthermore, NopE is required for early infection and nodule organogenesis in compatible plants. Evolutionary analysis revealed that NopE is highly conserved among bradyrhizobia and plant-associated endophytic and pathogenic bacteria. Our findings suggest that V. radiata and B. diazoefficiens USDA110 may use NopE to optimize their symbiotic interactions by reducing phytohormone-mediated ETI-type (PmETI) responses via salicylic acid (SA) biosynthesis suppression.

Plant growth promoting rhizobacteria (PGPR) may improve crop yield and reduce or optimize the use of chemical fertilizers.We investigated the effect of co-inoculation on growth, phosphorus nutrition, and phosphatase-phytase activities of Vicia faba under different phosphorus availability conditions.The studied strains possess several PGPR traits such as nitrogen fixation ability, solubilization of phosphate and potassium, and production of exopolysaccharides, auxins, and siderophores.Based on their 16S rDNA sequences, the strains were identified as Rahnella aquatilis (PGP30), Pseudomonas brassicacearum (PGP291) and Rhizobium sp.(RhOF57A).In a greenhouse experiment, plants were inoculated with an individual strain or co-inoculated with two strains in addition to uninoculated controls.Each setup was supplemented either with tricalcium phosphate Ca 3 (PO 4 ) 2 (TCP treatment) or irrigated with potassium phosphate KH 2 PO 4 (PO 4 treatment).Regardless of the applied phosphorus source, co-inoculation significantly increased biomass and phosphorus concentrations in plants as well as in bean pods.Moreover, especially co-inoculation increased phosphatase-phytase activities in roots supplemented with tricalcium phosphate.This study suggests that co-inoculation of plants is a means to prevent phosphorus limitation and for judicious use of chemical fertilizers.

In recent years, more attention has been paid to plant growth promoting (PGP) rhizobacteria use as a biofertilizer alternative to chemical fertilizers, which might cause damage to the environment. The main objective of this work was to evaluate the field application of PGP bacteria and rhizobial strains on the productivity of two food crops extensively used in Morocco; Vicia faba L. and Triticum durum L. A field experiment with four treatments was designed: (1) control without inoculation, (2) PGP bacteria alone (P), (3) rhizobia alone (R) and (4) a mixture of PGP-rhizobia (PR). Furthermore, the PGP strains were tested for their ability to solubilize complex mineral phosphorus and potassium and for their production of indole acetic acid and exopolysaccharides. The strains showed several plant growth promoting traits. Field inoculation by these rhizobacteria improved phosphorus uptake and the agronomic parameters of faba bean and wheat plants, such as biomass of shoots and roots, as well as the weight of bean pods and wheat spikes. The most pronounced effect was displayed by rhizobial strains or the combination of PGP-rhizobia. The rhizobacterial inoculation significantly stimulated the growth of both crops and could be used as potential biofertilizers to optimize growth and phosphorus retention capacity.

Abstract This study supports the idea that the evolution of type III secretion system (T3SS) is one of the factors that controls Vigna radiata–bradyrhizobia symbiosis. Based on phylogenetic tree data and gene arrangements, it seems that the T3SSs of the Thai bradyrhizobial strains SUTN9‐2, DOA1, and DOA9 and the Senegalese strain ORS3257 may share the same origin. Therefore, strains SUTN9‐2, DOA1, DOA9, and ORS3257 may have evolved their T3SSs independently from other bradyrhizobia, depending on biological and/or geological events. For functional analyses, the rhcJ genes of ORS3257, SUTN9‐2, DOA9, and USDA110 were disrupted. These mutations had cultivar‐specific effects on nodulation properties. The T3SSs of ORS3257 and DOA9 showed negative effects on V. radiata nodulation, while the T3SS of SUTN9‐2 showed no effect on V. radiata symbiosis. In the roots of V. radiata CN72, the expression levels of the PR1 gene after inoculation with ORS3257 and DOA9 were significantly higher than those after inoculation with ORS3257 ΩT3SS, DOA9 ΩT3SS, and SUTN9‐2. The T3Es from ORS3257 and DOA9 could trigger PR1 expression, which ultimately leads to abort nodulation. In contrast, the T3E from SUTN9‐2 reduced PR1 expression. It seems that the mutualistic relationship between SUTN9‐2 and V. radiata may have led to the selection of the most well‐adapted combination of T3SS and symbiotic bradyrhizobial genotype.

AimsThe symbiosis between Sinorhizobium fredii HH103 and its host legumes is influenced by the type 3 secretion system (T3SS), which delivers proteins (effectors) directly into the host cells to promote infection. GunA, one of the predicted HH103 effectors, potentially codes for a cellulase. In this work we tried to characterise GunA and elucidate its role in symbosis with soybean and cowpea.MethodsA GunA::HA fusion protein was constructed to study T3SS-dependent secretion. Cellulase activity of GunA was measured and gunA::uidA-GFP and gunA::cyA fusions were constructed to monitor gunA expression in nodules and to study translocation to the host cells, respectively. Finally, the symbiotic performance of a gunA mutant was studied in soybean and cowpea.ResultsGunA from S. fredii HH103 shows cellulase activity and is secreted through the T3SS in response to the inducer flavonoid genistein. Interestingly, at the beggining of the symbiotic process, GunA was partially responsible for the induction of the expression of the soybean GmPR1 gene, a gene used as a marker for plant defense responses. However, GunA was also detected in soybean and cowpea developed nodules. Finally, nodulation assays indicate that GunA is beneficial for symbiosis with soybean but detrimental with cowpea.ConclusionSecretion of GunA through the S. fredii HH103 T3SS clearly and differentially impacts the symbiotic performance of this strain with soybean and cowpea. GunA, or its cellulase activity, is recognised by soybean root cells very early in the symbiotic process but, curiously, its secretion can also be detected in mature nodules. This suggests different symbiotic roles at different symbiotic stages that need to be further elucidated.

Strains of the Gram-negative bacterium Vibrio coralliilyticus cause the bleaching of corals due to decomposition of symbiotic microalgae. The V. coralliilyticus strain ATCC BAA-450 (Vc450) encodes a type III secretion system (T3SS). The gene cluster also encodes a protein (locus tag VIC_001052) with sequence homology to the T3SS-secreted nodulation proteins NopE1 and NopE2 of Bradyrhizobium japonicum (USDA110). VIC_001052 has been shown to undergo auto-cleavage in the presence of Ca2+ similar to the NopE proteins. We have studied the hitherto unknown secondary structure, Ca2+-binding affinity and stoichiometry of the “metal ion-inducible autocleavage” (MIIA) domain of VIC_001052 which does not possess a classical Ca2+-binding motif. CD and fluorescence spectroscopy revealed that the MIIA domain is largely intrinsically disordered. Binding of Ca2+ and other di- and trivalent cations induced secondary structure and hydrophobic packing after partial neutralization of the highly negatively charged MIIA domain. Mass spectrometry and isothermal titration calorimetry showed two Ca2+-binding sites which promote structure formation with a total binding enthalpy of −110 kJ mol−1 at a low micromolar Kd. Putative binding motifs were identified by sequence similarity to EF-hand domains and their structure analyzed by molecular dynamics simulations. The stoichiometric Ca2+-dependent induction of structure correlated with catalytic activity and may provide a “host-sensing” mechanism that is shared among pathogens that use a T3SS for efficient secretion of disordered proteins.

Several genes coding for proteins with metal ion-inducible autocleavage (MIIA) domains were identified in type III secretion system tts gene clusters from draft genomes of recently isolated Bradyrhizobium spp. MIIA domains have been first described in the effectors NopE1 and NopE2 of Bradyrhizobium diazoefficiens USDA 110. All identified genes are preceded by tts box promoter motifs. The identified proteins contain one or two MIIA domains. A phylogenetic analysis of 35 MIIA domain sequences from 16 Bradyrhizobium strains revealed four groups. The protein from Bradyrhizobium sp. LmjC strain contains a single MIIA domain and was designated MdcE (MdcELmjC). It was expressed as a fusion to maltose-binding protein (MalE) in Escherichia coli and subsequently purified by affinity chromatography. Recombinant MalE-MdcELmjC-Strep protein exhibited autocleavage in the presence of Ca2+, Cu2+, Cd2+ and Mn2+, but not in the presence of Mg2+, Ni2+ or Co2+. Site-directed mutagenesis at the predicted cleavage site abolished autocleavage activity of MdcELmjC. An LmjC mdcE- mutant was impaired in the ability to nodulate Lupinus angustifolius and Macroptilium atropurpureum.

Bradyrhizobium elkanii USDA61 is incompatible with mung bean (Vigna radiata cv. KPS1) and soybean (Glycine max cv. BARC2) and unable to nodulate either plant. This incompatibility is due to the presence of a functional type III secretion system (T3SS) that translocates effector protein into host cells. We previously identified five genes in B. elkanii that are responsible for its incompatibility with KPS1 plants. Among them, a novel gene designated as innB exhibited some characteristics associated with the T3SS and was found to be responsible for the restriction of nodulation on KPS1. In the present study, we further characterized innB by analysis of gene expression, protein secretion, and symbiotic phenotypes. The innB gene was found to encode a hypothetical protein that is highly conserved among T3SS-harboring rhizobia. Similar to other rhizobial T3SS-associated genes, the expression of innB was dependent on plant flavonoids and a transcriptional regulator TtsI. The InnB protein was secreted via the T3SS and was not essential for secretion of other nodulation outer proteins. In addition, T3SS-dependent translocation of InnB into nodule cells was confirmed by an adenylate cyclase assay. According to inoculation tests using several Vigna species, InnB promoted nodulation of at least one V. mungo cultivar. These results indicate that innB encodes a novel type III effector controlling symbiosis with Vigna species.

Rhizobia are symbiotic nitrogen-fixing bacteria in root nodules of legumes. In Morocco, faba bean (Vicia faba L.), which is the main legume crop cultivated in the country, is often grown in marginal soils of arid and semi-arid regions. This study examines the phenotypic diversity of rhizobia nodulating V. faba isolated from different regions in Morocco for tolerance to some abiotic stresses. A total of 106 rhizobia strains isolated from nodules were identified at the species level by analysing 16S rDNA. Additionally, for selected strains recA, otsA, kup and nodA fragments were sequenced. 102 isolates are likely to belong to Rhizobium leguminosarum or R. laguerreae and 4 isolates to Ensifer meliloti. All strains tolerating salt concentrations of 428 or 342mM NaCl as well as 127 or 99mM Na2SO4 were highly resistant to alkaline conditions (pH 10) and high temperature (44°C). Three strains: RhOF4 and RhOF53 (both are salt-tolerant) and RhOF6 (salt-sensitive) were selected to compare the influence of different levels of salt stress induced by NaCl on growth and on trehalose and potassium accumulation. We find a direct correlation between the trehalose contents of the rhizobial strains and their osmotolerance.

Irrigation of crops with microcystins (MCs)-containing waters—due to cyanobacterial blooms—affects plant productivity and could be a way for these potent toxins entering the food chain. This study was performed to establish whether MC-tolerant rhizobia could benefit growth, nodulation, and nitrogen metabolism of faba bean plants irrigated with MC-containing waters. For that, three different rhizobial strains—with different sensitivity toward MCs—were used: RhOF96 (most MC-sensitive strain), RhOF125 (most MC-tolerant strain), or Vicz1.1 (reference strain). As a control, plants grown without rhizobia and fertilized by NH 4 NO 3 were included in the study. MC exposure decreased roots (30–37 %) and shoots (up to 15 %) dry weights in un-inoculated plants, whereas inoculation with rhizobia protects plants toward the toxic effects of MCs. Nodulation and nitrogen content were significantly impaired by MCs, with the exception of plants inoculated with the most tolerant strain RhOF125. In order to deep into the effect of inoculation on nitrogen metabolism, the nitrogen assimilatory enzymes (glutamine synthetase (GS) and glutamate synthase (GOGAT)) were investigated: Fertilized plants showed decreased levels (15–30 %) of these enzymes, both in shoots and roots. By contrast, inoculated plants retained the levels of these enzymes in shoots and roots, as well as the levels of NADH-GOGAT activity in nodules. We conclude that the microcystin-tolerant Rhizobium protects faba bean plants and improves nitrogen assimilation when grown in the presence of MCs.

Sinorhizobium fredii HH103 is a rhizobial strain showing a broad host range of nodulation. In addition to the induction of bacterial nodulation genes, transition from a free-living to a symbiotic state requires complex genetic expression changes with the participation of global regulators. We have analyzed the role of the zinc-finger transcriptional regulator MucR1 from S. fredii HH103 under both free-living conditions and symbiosis with two HH103 host plants, Glycine max and Lotus burttii. Inactivation of HH103 mucR1 led to a severe decrease in exopolysaccharide (EPS) biosynthesis but enhanced production of external cyclic glucans (CG). This mutant also showed increased cell aggregation capacity as well as a drastic reduction in nitrogen-fixation capacity with G. max and L. burttii. However, in these two legumes, the number of nodules induced by the mucR1 mutant was significantly increased and decreased, respectively, with respect to the wild-type strain, indicating that MucR1 can differently affect nodulation depending on the host plant. RNA-Seq analysis carried out in the absence and the presence of flavonoids showed that MucR1 controls the expression of hundreds of genes (including some related to EPS production and CG transport), some of them being related to the nod regulon.

After nitrogen, phosphorus is the main element for plant growth. Most agricultural soils worldwide are deficient in phosphorus and therefore require a contribution of phosphorus for the plant needs. There is a continuing need to improve soil fertility, to increase yields and agricultural productivity. During the application of phosphate fertilizers, soluble phosphorus assimilated by plants is rare because of its precipitation and then become unavailable to the plant. Rhizospheric bacteria including the plant growth promoting rhizobacteria (PGPR) are of growing interest for their potential role in improving soil fertility and enhancing an increase of crop yields and their nutrients contents. These bacteria make the insoluble phosphorus in soluble forms during the application of phosphate fertilizers and make the phosphorus available to the plant. This work gives a review of methodology and techniques used for the research of phosphate solubilization bacteria (PSB), their molecular characterization and the biochemical mechanisms and genes tools involved in solubilization of phosphate and their relationships with symbiotic plants.

Knöllchenbakterium – the “microbe of the year” In 2015, the VAAM selected Knöllchenbakterium as “microbe of the year”. Knöllchenbakterium ist a collective term for a number of different bacterial species that are able to establish a root nodule symbiosis with legumes. During nodule development the bacteria differentiate into bacteroids that are confined by an additional membrane. These organelle-like structures are now called symbiosomes, whose task is to fix molecular nitrogen for the benefit of the plant. In return, the plant has to supply all nutrients. The symbiotic interaction is initiated by a specific signal exchange. The first signals are flavonoids secreted by the plant. This leads to the activation of the bacterial nod genes. The Nod proteins synthesize and secrete Nod factors: modified and fatty acid-carrying oligosaccharide. They serve as a specific signal to the plant and induce nodule formation. Besides this core signaling, a number of extracellular components, e.g. exopolysaccharides, lipopolysaccharides and secreted proteins influence the symbiotic interaction very specific for each individual system.

In this study three bacteria were isolated from groundnut root nodules grown in three regions in the Sudan, El-Obied, El-Gezira and El-Gadarif, DNA was extracted, nifH genes were amplified and sequenced and the sequences were compared with reference strain. The analysis of the sequences of nifH genes revealed that all the isolates and the reference strain are Klebsiella with identity of 99% and ensured the presence of nifH gene in all isolates and the reference strain and there is no difference in the sequences except the sequence of El-Gezira strain was found shorter than the others which led to difference of the nifH gene translated amino acids of this strain compared to the others.

Sinorhizobium fredii HH103 is a fast-growing rhizobial strain infecting a broad range of legumes including both American and Asiatic soybeans. In this work, we present the sequencing and annotation of the HH103 genome (7.25 Mb), consisting of one chromosome and six plasmids and representing the structurally most complex sinorhizobial genome sequenced so far. Comparative genomic analyses of S. fredii HH103 with strains USDA257 and NGR234 showed that the core genome of these three strains contains 4,212 genes (61.7% of the HH103 genes). Synteny plot analysis revealed that the much larger chromosome of USDA257 (6.48 Mb) is colinear to the HH103 (4.3 Mb) and NGR324 chromosomes (3.9 Mb). An additional region of the USDA257 chromosome of about 2 Mb displays similarity to plasmid pSfHH103e. Remarkable differences exist between HH103 and NGR234 concerning nod genes, flavonoid effect on surface polysaccharide production, and quorum-sensing systems. Furthermore a number of protein secretion systems have been found. Two genes coding for putative type III–secreted effectors not previously described in S. fredii, nopI and gunA, have been located on the HH103 genome. These differences could be important to understand the different symbiotic behavior of S. fredii strains HH103, USDA257, and NGR234 with soybean.

The divergently oriented Sinorhizobium meliloti emrAB (SMc03168 and SMc03167) and emrR (SMc03169) genes are predicted to encode an efflux system of the major facilitator superfamily and a TetR-like transcriptional regulator, respectively. The transcription of the emrA gene was found to be inducible by flavonoids, including luteolin and apigenin, which are known inducers of the nodulation genes in S. meliloti. Interestingly, quercetin, which does not induce nodulation genes, was also a potent inducer of emrA, indicating that NodD is not directly involved in regulation of emrA. The likely regulator of emrAB is EmrR, which binds to palindrome-like sequences in the intergenic region. Several modifications of the palindromes, including an increase of the spacing between the two half sites, prevented binding of EmrR. Binding was also impaired by the presence of luteolin. Mutations in emrA had no obvious effect on symbiosis. This was in contrast to the emrR mutant, which exhibited a symbiotic deficiency with Medicago sativa. Conserved binding sites for TetR-like regulators within the intergenic regions between the emrAB and emrR genes were identified in many symbiotic and pathogenic members of the order Rhizobiales.