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Drought is the most detrimental abiotic stress in agriculture, limiting crop growth and yield and, currently, its risk is increasing due to climate change. Thereby, ensuring food security will be one of the greatest challenges of the agriculture in the nearest future, accordingly it is essential to look for sustainable strategies to cope the negative impact of drought on crops. Inoculation of pulses with biostimulants such as rhizobium strains with high nitrogen fixation efficiency and drought-tolerance, has emerged as a promising and sustainable production strategy. However, some commercial inoculums are not effective under field conditions due to its lower effectiveness against indigenous rhizobium strains in the establishment of the symbiosis. Thus, in the present study, we evaluated the ability to improve drought tolerance in common bean plants of different indigenous rhizobia strains isolated from nearby crop fields in the Basque Country either affected by drought or salinity. The plants in this trial were grown in a climatic chamber under controlled conditions and exposed to values of 30% relative soil water content at the time of harvest, which is considered a severe drought. From the nine bacteria strains evaluated, three were found to be highly efficient under drought (namely 353, A12 and A13). These strains sustained high infectiveness (nodulation capacity) and effectiveness (shoot biomass production) under drought, even surpassing the plants inoculated with the CIAT899 reference strain, as well as the chemically N-fertilized plants. The tolerance mechanisms developed by plants inoculated with 353, A12 and A13 strains were a better adjustment of the cell wall elasticity that prevents mechanical damages in the plasma membrane, a higher WUE and an avoidance of the phenological delay caused by drought, developing a greater number of flowers. These results provide the basis for the development of efficient common bean inoculants able to increase the yield of this crop under drought conditions in the Northern Spain and, thus, to be used as biostimulants. In addition, the use of these efficient nitrogen fixation bacteria strains is a sustainable alternative to chemical fertilization, reducing cost and minimizing its negative impact on environment. This work was founded by Basque Government: Grupo de Investigación del Sistema Universitario Vasco IT1022-16 and T1682-22; and proyects from the Dirección de Calidad e Industrias Alimentarias. Dpto. de Desarrollo Económico e Infraestructuras (32-2016-00043; 37-2017-00047; 00049-IDA-2019-38) and Dpto. De Desarrollo Económico Sostenibilidad y Medio Ambiente (00039-IDA2021-45). A. Del-Canto was the recipient of a predoctoral fellowship granted by the Education Department of the Basque Country Government, Spain.

Bacterial surface motility is a complex microbial trait that contributes to host colonization. However, the knowledge about regulatory mechanisms that control surface translocation in rhizobia and their role in the establishment of symbiosis with legumes is still limited. Recently, 2-tridecanone (2-TDC) was identified as an infochemical in bacteria that hampers microbial colonization of plants. In the alfalfa symbiont Sinorhizobium meliloti, 2-TDC promotes a mode of surface motility that is mostly independent of flagella. To understand the mechanism of action of 2-TDC in S. meliloti and unveil genes putatively involved in plant colonization, Tn5 transposants derived from a flagellaless strain that were impaired in 2-TDC-induced surface spreading were isolated and genetically characterized. In one of the mutants, the gene coding for the chaperone DnaJ was inactivated. Characterization of this transposant and newly obtained flagella-minus and flagella-plus dnaJ deletion mutants revealed that DnaJ is essential for surface translocation, while it plays a minor role in swimming motility. DnaJ loss-of-function reduces salt and oxidative stress tolerance in S. meliloti and hinders the establishment of efficient symbiosis by affecting nodule formation efficiency, cellular infection, and nitrogen fixation. Intriguingly, the lack of DnaJ causes more severe defects in a flagellaless background. This work highlights the role of DnaJ in the free-living and symbiotic lifestyles of S. meliloti.

Soybean (Glycine max [L.] Merr.) is a food and oil crop whose growth and yield are influenced by root and nodule development. In the present study, GmNMHC5 was found to promote the formation of nodules in overexpressing mutants. In contrast, the number of nodules in Gmnmhc5 edited with CRISPR/Cas9 decreased sharply. In 35S:GmNMHC5 mutants, expression levels of genes involved in nodulation were significantly up-regulated. Both in vitro and in vivo biochemical analyses showed that GmNMHC5 directly interacted with GmGAI (a DELLA protein), and the content of gibberellin 3 (GA3) in overexpressing mutants was lower than that in the wild type. These results revealed that GmNMHC5 participates in the classical GA signaling pathway, and may regulate the content of GA3 to match the optimal concentration required for nodule formation, thereby promoting nodulation by directly interacting with GmGAI. A model illustrating the mechanism by which GmNMHC5 promotes soybean nodulation is presented.

To improve the nitrogen fixation, legume crops are often inoculated with selected effective rhizobia. However, there is large variation in how well the inoculant strains compete with the indigenous microflora in soil. To assess the success of the inoculant, it is necessary to distinguish it from other, closely related strains. Methods used until now have generally been based either on fingerprinting methods or on the use of reporter genes. Nevertheless, these methods have their shortcomings, either because they do not provide sufficiently specific information on the identity of the inoculant strain, or because they use genetically modified organisms that need prior authorization to be applied in the field or other uncontained environments. Another possibility is to target a gene that is naturally present in the bacterial genomes. Here we have developed a method that is based on amplicon sequencing of the bacterial housekeeping gene rpoB, encoding the beta-subunit of the RNA polymerase, which has been proposed as an alternative to the 16S rRNA gene to study the diversity of rhizobial populations in soils. We evaluated the method under laboratory and field conditions. Peanut seeds were inoculated with various Bradyrhizobium strains. After nodule development, DNA was extracted from selected nodules and the nodulating rhizobia were analysed by amplicon sequencing of the rpoB gene. The analyses of the sequence data showed that the method reliably identified bradyrhizobial strains in nodules, at least at the species level, and could be used to assess the competitiveness of the inoculant compared to other bradyrhizobia.

Summary Soybean is one of the most important oilseed and fodder crops. Benefiting from the efforts of soybean breeders and the development of breeding technology, large number of germplasm has been generated over the last 100 years. Nevertheless, soybean breeding needs to be accelerated to meet the needs of a growing world population, to promote sustainable agriculture and to address future environmental changes. The acceleration is highly reliant on the discoveries in gene functional studies. The release of the reference soybean genome in 2010 has significantly facilitated the advance in soybean functional genomics. Here, we review the research progress in soybean omics (genomics, transcriptomics, epigenomics and proteomics), germplasm development (germplasm resources and databases), gene discovery (genes that are responsible for important soybean traits including yield, flowering and maturity, seed quality, stress resistance, nodulation and domestication) and transformation technology during the past decade. At the end, we also briefly discuss current challenges and future directions.

Soybean cropping has been growing in recent years in environments with sandy soils and with climatic risk, but yield is low, especially in the early years. The objective of this study was to evaluate the effect of cover crops and nitrogen management in a sandy soil previously under degraded pastures on soybean yield. The study was conducted in Western São Paulo state, Brazil. The experimental design was randomized blocks, with four replicates, and the treatments were: black oats; black oats + 50 kg ha-1 of N in black oats; black oats + 50 kg ha-1 of N in soybean; black oats + lupine; black oats + lupine + 50 kg ha-1 of N in soybean; lupine; fallow; fallow + 50 kg ha-1 of N in soybean. Nitrogen concentration of the microbial biomass was higher with oats + N in soybean applied at the beginning of flowering (R1). The number of nodules in soybean roots increased by 2.3 times with oats and oats + N in soybean as compared to fallow. Soybean yield was higher in treatments with oats + N in oats (2,130 kg ha-1), oats (2,038 kg ha-1) and oats + N in soybean (1,872 kg ha-1). In the absence of cover crops, N fertilization in soybean increased yield by 19% (262 kg ha-1) compared to fallow. Black oats are the best option to increase soybean yield. However, in the absence of cover crops, nitrogen fertilization in soybean is necessary. RESUMO O cultivo de soja vem crescendo nos últimos anos em ambientes de solos arenosos e com risco climático, mas a produtividade é baixa, principalmente nos primeiros anos. Objetivou-se neste estudo avaliar o efeito do manejo de culturas de cobertura e nitrogênio em solo arenoso previamente ocupado por pastagem degradada na produtividade da soja. O estudo foi conduzido no Oeste de São Paulo. O delineamento experimental foi em blocos casualizados, com quatro repetições e os tratamentos foram: aveia preta; aveia preta + 50 kg ha-1 de N na aveia preta; aveia preta + 50 kg ha-1 de N na soja; aveia preta + tremoço; aveia preta + tremoço + 50 kg ha-1 de N na soja; tremoço; pousio; pousio + 50 kg ha-1 de N na soja. O teor de nitrogênio da biomassa microbiana foi maior com aveia preta + N na soja aplicado no início do florescimento (R1). O número de nódulos nas raízes da soja aumentou 2,3 vezes com aveia e aveia + N na soja em relação ao pousio. A produtividade da soja foi maior nos tratamentos com aveia + N na aveia (2.130 kg ha-1), aveia (2.038 kg ha-1) e aveia + N na soja (1.872 kg ha-1). Na ausência de culturas de cobertura, a adubação nitrogenada na soja aumentou a produtividade em 19% (262 kg ha-1) comparado ao pousio. A aveia preta é a melhor opção para aumentar a produtividade da soja. Porém na ausência de planta de cobertura é necessária a adubação nitrogenada na soja.

For sustainable agriculture nitrogen must be adequately supplied because it is the most limiting major nutrient required by all living plants. This experiment was conducted at the two ecological zones, Sudan savannah in Kano State and Guinea savannah in Bauchi state of Nigeria during the 2016 cropping season. The design of the experiment was Randomized Complete Block Design (RCBD) with seven treatments and four replications in each site. The treatments were; legume fix, alosca, 50 kg N ha-1, cattle manure, legume fix + cattle manure, alosca + cattle manure and control. The result showed that Legume fix had the highest grain N uptake (90.51 kg N ha-1) which differed significantly (P≤0.001) from the rest of the treatments. At Guinea savannah legume fix gave the highest N uptake value (95.53 kg N ha-1) which was significantly different from all treatments except legume fix + cattle manure. However, 50 kg N ha-1 gave the least (48.64 kg N ha-1) even lower than the control and differs significantly with the rest of treatments (P≤0.001). Legume fix gave the highest P uptake value (9.66 kg P ha-1) which significantly differed from all other treatments. At Sudan savannah, significant differences (P ≤ 0.001) occurred between some of the treatments. Alosca + cattle manure recorded the highest K uptake (49.99 kg K ha-1). Result for the fixed N in the Sudan and Guinea savannahs soybean field revealed a significant difference between the treatments and the control.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a leading cause of infectious disease mortality. Animal infection models have contributed substantially to our understanding of TB, yet their biological and non-biological limitations are a research bottleneck. There is a need for more ethically acceptable, economical, and reproducible TB infection models capable of mimicking key aspects of disease. Here, we demonstrate and present a basic description of how Galleria mellonella (the greater wax moth, Gm) larvae can be used as a low cost, rapid, and ethically more acceptable model for TB research. This is the first study to infect Gm with the fully virulent MTB H37Rv, the most widely used strain in research. Infection of Gm with MTB resulted in a symptomatic lethal infection, the virulence of which differed from both attenuated Mycobacterium bovis BCG and auxotrophic MTB strains. The Gm-MTB model can also be used for anti-TB drug screening, although CFU enumeration from Gm is necessary for confirmation of mycobacterial load reducing activity of the tested compound. Furthermore, comparative virulence of MTB isogenic mutants can be determined in Gm. However, comparison of mutant phenotypes in Gm against conventional models must consider the limitations of innate immunity. Our findings indicate that Gm will be a practical, valuable, and advantageous additional model to be used alongside existing models to advance tuberculosis research.

Legumes associate with Gram-negative soil bacteria called rhizobia, resulting in the formation of a nitrogen-fixing organ, the nodule. Nodules are an important sink for photosynthates for legumes, so these plants have developed a systemic regulation mechanism that controls their optimal number of nodules, the so-called autoregulation of nodulation (AON) pathway, to balance energy costs with the benefits of nitrogen fixation. In addition, soil nitrate inhibits nodulation in a dose-dependent manner, through systemic and local mechanisms. The CLE family of peptides and their receptors are key to tightly controlling these inhibitory responses. In the present study, a functional analysis revealed that PvFER1, PvRALF1, and PvRALF6 act as positive regulators of the nodule number in growth medium containing 0 mM of nitrate but as negative regulators in medium with 2 and 5 mM of nitrate. Furthermore, the effect on nodule number was found to be consistent with changes in the expression levels of genes associated with the AON pathway and with the nitrate-mediated regulation of nodulation (NRN). Collectively, these data suggest that PvFER1, PvRALF1, and PvRALF6 regulate the optimal number of nodules as a function of nitrate availability.

International audience; Legumes have the remarkable ability to develop root nodules with nitrogen-fixing rhizobial bacteria. This symbiosis enables them to overcome nitrogen limitation affecting their growth, and such a nutritional advantage is key to the ecological and agronomic success of legumes. Intriguingly, non-nodulating lines can be obtained through inbreeding work in cultivated peanut. Using elegant genetic approaches, Peng et al. (2021) have elucidated this 40-year-old mystery by identifying the underlying genes. They are the two homoeologs of the symbiotic signalling gene NSP2, pinpointing NSP2 as a gene of primary adaptative importance for nodulation in legumes.

In the symbiotic associations between rhizobia and legumes, the NodD regulators orchestrate the transcription of the specifc nodulation genes. This set of genes is involved in the synthesis of nodulation factors, which are responsible for initiating the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. Among the fve NodD regulators present in this rhizobium, only NodD1 and NodD2 seem to have a role in the symbiotic process. However, the individual role of each NodD in the absence of the other proteins has remained elusive. In this work, we show that the CIAT 899 NodD2 does not require activation by inducers to promote the synthesis of nodulation factors. A CIAT 899 strain overexpressing nodD2, but lacking all additional nodD genes, can nodulate three different legumes as effciently as the wild type. Interestingly, CIAT 899 NodD2- mediated gain of nodulation can be extended to another rhizobial species, since its overproduction in Sinorhizobium fredii HH103 not only increases the number of nitrogen-fxing nodules in two host legumes but also results in nodule development in incompatible legumes. These fndings potentially open exciting opportunities to develop rhizobial inoculants and increase legume crop production. Spanish Ministry of Science and Innovation funded by MCIN/AEI/10.13039/501100011033 AGL2016-77163-R and PID2019- 107634RB-I00 Ministerio de Economía y Competitividad FPU18/06248

International audience; Post-transcriptional control of gene expression allows plants to rapidly adapt to changes in their environment. Under low nitrogen conditions, legume plants engage into a symbiosis with soil bacteria that results in the formation of root nodules, where bacteria are allocated and fix atmospheric nitrogen for the plant's benefit. Recent studies highlighted the importance of small RNA-mediated mechanisms in the control of bacterial infection, nodule organogenesis, and the long-distance signaling that balances plant growth and nodulation. Examples of such mechanisms are shoot-to-root mobile microRNAs and small RNA fragments derived from degradation of bacterial transfer RNAs that repress complementary mRNAs in the host plant. Mechanisms of selective mRNA translation also contribute to rapidly modulate the expression of nodulation genes in a cell-specific manner during symbiosis. Here, the most recent advances made on the regulation of mRNA stability and translatability, and the emerging roles of long non-coding RNAs in symbiosis are summarized.

A field experiment was performed at Research Farm of the University of Agriculture Peshawar, Pakistan to evaluate the effect of biochar on soil and plant nitrogen and yielding parameters of mung bean. The experiment was performed in randomize complete block design with split plot arrangement with four replications. Area of each experimental unit was 10.5 m2 and was applied with biochar at the rate of 0, 20, and 40 t ha-1 along with full and half levels of P and K i-e., (90 kg P, 60 kg K ha-1 and 45 kg P, 30 kg K ha-1). Nitrogen was applied to all the experimental units uniformly @ 25 kg N ha-1. The data showed that all of the analyzed parameters were significantly influenced with various biochar levels except fresh and dry weight of nodules. With full and half dose of P and K application, soil total N and soil organic matter were found significant with an increase of 16% and 7% as compared to half dose. With application of various levels of biochar, a significant increase was recorded in most of the parameters. Data regarding Soil N and soil O.M were found significantly enhanced at 40 t ha-1 with values of 0.15% and 2.64% which were 200% and 94% higher as compared to 0 t ha-1 biochar applications. Other parameters like 1000 grain weight, biological yield (fresh and dry), plant N and number of nodules were significantly affected with 20 t ha-1 biochar application which were increased by 20 %, 33%, 21% and 21% as compared to control. Combined application of P&K and biochar significantly affected soil total nitrogen i-e., 0.16% at (full×40 t ha-1) and nodulation number i-e., 36 at (half×20 t ha-1). It was concluded that biochar application @ 20 t ha-1 along with half levels of P and K, proved the best treatments combination for most of the plant parameters and hence they are recommended.

Pinitol (3-O-methyl-D-chiro-inositol) is shown to be transported passively across the plant-derived symbiosome membrane in Lotus japonicus by the energy-independent polyol transporter LjPLT11. Accumulation of pinitol is crucial to maintenance of osmotic balance in nodules. RNAi lines reduced in LjPLT11 have disrupted symbiosome membranes, reduced N2 fixation by rhizobial bacteroids and reduced plant growth. There is also an increase in production of reactive oxygen species (ROS) in LjPLT11 RNAi lines, consistent with osmotic imbalance causing increased stress in nodules.

Out of 70 bacterial strains isolated from root nodules of Lupinus albus and L. angustifolius grown in the soils from the Maamora forest in Morocco, 56 isolates possessed the nodC symbiotic gene, as determined by nodC-PCR, and they were able to renodulate their original hosts. The phenotypic analysis showed that many strains had great potential for using different carbon compounds and amino acids as sole carbon and nitrogen sources. The majority of strains grew in media with pH values between 6 and 8. Only one strain isolated from L. angustifolius was able to grow at low pH values, whereas fourteen strains nodulating L. albus grew at pH 5. No strain developed at 40 °C, and eighteen strains grew at NaCl concentrations as high as 855 mM. A total of 17 strains solubilized phosphates, whereas 20 produced siderophores and seven produced IAA. Only three strains, Lalb41, Lang10 and Lang16, possessed all three plant growth promoting activities. The strains were grouped into eight genetic groups by rep-PCR. Analysis of the 16S rRNA sequences of eight strains representing the different groups showed that they were members of the genus Bradyrhizobium. The sequencing of the five housekeeping genes atpD, glnII, dnaK, gyrB and recA, from the eight representative strains, and the phylogenetic analysis of their concatenated sequences, showed that both plants were nodulated by different Bradyrhizobium species. Accordingly, two strains, Lalb41 and Lalb5.2, belonged to B. lupini, whereas two strains, Lalb2 and Lang17.2, were affiliated to B. cytisi, and one strain, Lang2, was close to B. canariense. The fourth group of strains, Lalb25, Lang14.3 and Lang8.3, which had similarity values of less than 96% with their closest named species, B. cytisi, may belong to two new genospecies in the genus Bradyrhizobium. All the strains nodulated Lupinus cosentinii, L. luteus, Retama sphaerocarpa, R. monosperma, Chamaecytisus albus, but not Vachellia gummifera, Phaseolus vulgaris or Glycine max. The nodA, nodC and nifH sequence analyses and their phylogeny confirmed that the strains isolated from the two lupines were members of the symbiovar genistearum., This work was supported by the Hassan II Academy of Sciences and Technology in Morocco. We are grateful to all the persons who contributed to the realization of this work.

This study aims to explore the possibility of a reduced application of inorganic nitrogen (N) fertiliser on the yield, yield qualities, and biological nitrogen fixation (BNF) of the hydroponic common bean (Phaseolus vulgaris L.), without compromising plant performance, by utilizing the inherent ability of this plant to symbiotically fix N2. Until the flowering stage, plants were supplied with a nutrient solution containing N-concentrations of either a, 100%, conventional standard-practice, 13.8 mM; b, 75% of the standard, 10.35 mM; or c, 50% of the standard, 6.9 mM. During the subsequent reproductive stage, inorganic-N treatments b and c were decreased to 25% of the standard, and the standard (100% level) N-application was not altered. The three different inorganic-N supply treatments were combined with two different rhizobia strains, and a control (no-inoculation) treatment, in a two-factorial experiment. The rhizobia strains applied were either the indigenous strain Rhizobium sophoriradicis PVTN21 or the commercially supplied Rhizobium tropici CIAT 899. Results showed that the 50–25% mineral-N application regime led to significant increases in nodulation, BNF, and fresh-pod yield, compared to the other treatment, with a reduced inorganic-N supply. On the other hand, the 75–25% mineral-N regime applied during the vegetative stage restricted nodulation and BNF, thus incurring significant yield losses. Both rhizobia strains stimulated nodulation and BNF. However, the BNF capacity they facilitated was suppressed as the inorganic-N input increased. In addition, strain PVTN21 was superior to CIAT 899—as 50–25% N-treated plants inoculated with the former showed a yield loss of 11%, compared to the 100%-N-treated plants. In conclusion, N-use efficiency optimises BNF, reduces mineral-N-input dependency, and therefore may reduce any consequential negative environmental consequences of mineral-N over-application.

The RWP-RK is a small family of plant-specific transcription factors that are mainly involved in nitrate starvation responses, gametogenesis, and root nodulation. To date, the molecular mechanisms underpinning nitrate-regulated gene expression in many plant species have been extensively studied. However, the regulation of nodulation-specific NIN proteins during nodulation and rhizobial infection under nitrogen starvation in soybean still remain unclear. Here, we investigated the genome-wide identification of RWP-RK transcription factors and their essential role in nitrate-inducible and stress-responsive gene expression in soybean. In total, 28 RWP-RK genes were identified from the soybean genome, which were unevenly distributed on 20 chromosomes from 5 distinct groups during phylogeny classification. The conserved topology of RWP-RK protein motifs, cis-acting elements, and functional annotation has led to their potential as key regulators during plant growth, development, and diverse stress responses. The RNA-seq data revealed that the up-regulation of GmRWP-RK genes in the nodules indicated that these genes might play crucial roles during root nodulation in soybean. Furthermore, qRT-PCR analysis revealed that most GmRWP-RK genes under Phytophthora sojae infection and diverse environmental conditions (such as heat, nitrogen, and salt) were significantly induced, thus opening a new window of possibilities into their regulatory roles in adaptation mechanisms that allow soybean to tolerate biotic and abiotic stress. In addition, the dual luciferase assay indicated that GmRWP-RK1 and GmRWP-RK2 efficiently bind to the promoters of GmYUC2, GmSPL9, and GmNIN, highlighting their possible involvement in nodule formation. Together, our findings provide novel insights into the functional role of the RWP-RK family during defense responses and root nodulation in soybean.

Resumen Introducción. Rhizobium ha sido utilizado como estimulador del crecimiento en leguminosas. Diferentes especies de este grupo se asocian al garbanzo (Cicer arietinum L.), por lo que resulta importante obtener aislados con capacidad de beneficiar su crecimiento y productividad. Objetivo. Seleccionar e identificar aislados de Rhizobium sp. en cuanto a sus atributos como rizobacterias promotoras del crecimiento vegetal con mayor efectividad en su asociación a cultivares de garbanzo. Materiales y métodos. Los estudios se realizaron entre los años 2017-2020, en el Instituto de Investigaciones Fundamentales en Agricultura Tropical “Alejandro de Humboldt”, La Habana, Cuba. Se identificaron por secuenciación del gen 16S ARN tres cepas de rizobios, obtenidas de nódulos de garbanzo y se caracterizaron por su potencial como bacterias promotoras del crecimiento vegetal. Se realizó un experimento en condiciones de campo en dos campañas para demostrar su efectividad en la interacción con las plantas. Para los experimentos en condiciones de laboratorio se utilizó un diseño completamente aleatorizado, mientras que los ensayos en campo se realizaron bajo un diseño de bloques al azar, en ambos casos se realizó un análisis de varianza. Resultados. Las tres cepas bacterianas asociadas al garbanzo e identificadas como Rhizobium sp., fijaron nitrógeno atmosférico, solubilizaron fosfato de calcio, liberaron fitohormonas y fueron capaces de inhibir el crecimiento de Fusarium oxysporum, Fusarium incarnatum y Fusarium moniliforme. La inoculación en el garbanzo, incrementó la nodulación y las variables de rendimiento del cultivo número de vainas por planta y masa fresca de los granos (g). Conclusión. Se identificaron cepas de Rhizobium sp. con atributos como bacterias promotoras del crecimiento vegetal asociadas a cultivares nacionales de garbanzo que incrementaron su nodulación y rendimiento. Abstract Introduction. Rhizobium has been used as a growth stimulator in leguminous. Different species of this group are associated with chickpea (Cicer arietium L.), so it is important to obtain isolated with capacity of benefitting their growth and productivity. Objective. To select and identify isolates of Rhizobium sp. in terms of their attributes as plant growth-promoting rhizobacteria with greater effectiveness in their association with chickpea cultivars. Materials and methods. The studies were carried out during the years 2017-2020, at the Instituto de Investigaciones Fundamentales en Agricultura Tropical “Alejandro of Humboldt”, Havana, Cuba. Three rhizobia strains, obtained from chickpea nodules were identified by 16S RNA gene sequencing and characterized for their potential as growth-promoting bacteria. An experiment was carried out in field conditions in two campaigns to demonstrate their effectiveness in the interaction with plants. For the experiments under laboratory conditions a completely randomized design was used, while the field trials were conducted under a randomized block design, in both cases an analysis of variance was performed. Results. The three bacterial strains associated with chickpea and identified as Rhizobium sp. fixed atmospheric nitrogen, solubilized calcium phosphate, released phytohormones and were able to inhibit the growth of Fusarium oxysporum, Fusarium incarnatum, and Fusarium moniliforme. Inoculation in chickpea increased the nodulation and the crop yield variables such as number of pods per plant and fresh grain mass (g). Conclusions. Rhizobium sp. strains were identified with attributes as plant growth-promoting bacteria associated with national chickpea cultivars, that increased their nodulation and yield.

Due to the ever-changing climate and deterioration of the earth���s ecosystem, environmental stresses like abiotic (drought, salinity) and biotic stresses (pathogen infection) gravely affected plant growth. Native plants are a great way of improving these effects on the urban landscape. They can be used as ornamental plants in landscaping as they are accustomed to their natural environment. The Center for Water-Efficient Landscaping at Utah State University has released a list of plants to be used for low water use landscaping. One such native plant is Ceanothus velutinus (snowbrush ceanothus). They are evergreen plants that can grow in dry and harsh conditions and are native to the Intermountain West region of North America. This study focused on the effect of rhizosphere and endosphere microbiome on the growth and development of snowbrush ceanothus plants. A comparative metagenomic study in the bulk soil, rhizosphere, and endosphere of snowbrush ceanothus revealed the microbial diversity and presence of several plant growth promoting rhizobacteria (PGPR). So next, the effect of this native soil was observed on the growth and development of snowbrush ceanothus under the greenhouse conditions. Inoculation of native soil to the propagation media enhanced the rooting and survival rate of snowbrush ceanothus cuttings. The inoculation of native soil in the snowbrush ceanothus plants developed from cutting propagation and seedlings in the greenhouse revealed an improved growth compared to control plants. The metagenomic study of the rhizosphere and endosphere of snowbrush ceanothus plants treated with native soil revealed the presence of several PGPR that were absent in the control plants. Nodulation was observed for the first time in snowbrush ceanothus plants grown in the greenhouse and inoculated with native soil. So finally, an attempt was made to isolate as many PGPR species as possible from the rhizosphere and endosphere of snowbrush ceanothus plants. Many of these isolates tested positive for one or more specific traits such as siderophore production, indole acetic acid production, catalase production, nitrogen fixation, and phosphate solubilization. The isolates were further tested for their plant growth-promoting properties in plants. We found many of these bacterial isolates could potentially be used as bio-fertilizers or bio-stimulants.

One of the limiting factors in organic farming is the scarcity of allowed fertilizers and chemicals for plant protection. Plant and compost extracts are a promising solution for fertilization because of their positive effect on plant growth and soil microbial activity. Nettle extract was already successfully applied to some vegetables. Not-aerated nettle extract, obtained from dry nettle leaves, was applied in experiments with green beans in a quantity of 1 L per pot at two-day intervals was studied. A three-factorial experimental design was applied with two soil types (brown—Calcic Gleysol and red—Eutric Cambisol), soil disinfection with dazomet or not, and irrigated with nettle extract or water. Nettle extract application increased all above-ground traits; plant height, leaf area, flower buds, shoot dry weight at flowering, pod length, pod diameter, and shoot dry weight at harvest by 49%, 66%, 43%, 36%, 11%, 9%, and 37%, respectively, the root length at harvest by 59%, total yield by 48%, soil respiration by 91% and 74% in two soil types, and alkaline phosphatase by 30%. Dehydrogenase activity was enhanced by nettle extract application on red soil, while nettle extract application had no effect on root nodulation. The nettle extract application benefits in green bean organic production were attributed to the nutrients and other components present in the extract and not to nitrogen fixation. The optimization of the dose of the extract and experiments in real conditions of green bean production would be the next step toward the implementation of nettle extract as an organic fertilizer.

Nodulation is a hallmark yet non-universal characteristic of legumes. It is unknown whether the mechanisms underlying nitrogen-fixing symbioses evolved within legumes and the broader nitrogen-fixing clade (NFC) repeatedly de novo or based on common ancestral pathways. Ten new transcriptomes representing members from the Cercidoideae and Caesalpinioideae subfamilies were supplemented with published omics data from 65 angiosperms, to investigate how gene content correlates with nodulation capacity within Fabaceae and the NFC. Orthogroup analysis categorized annotated genes into 64150 orthogroups, of which 19 were significantly differentially represented between nodulating versus non-nodulating NFC species and were most commonly absent in nodulating taxa. The distribution of six over-represented orthogroups within Viridiplantae representatives suggested that genomic evolution events causing gene family expansions, including whole-genome duplications (WGDs), were unlikely to have facilitated the development of stable symbioses within Fabaceae as a whole. Instead, an absence of representation of 13 orthogroups indicated that losses of genes involved in trichome development, defense and wounding responses were strongly associated with rhizobial symbiosis in legumes. This finding provides novel evidence of a lineage-specific predisposition for the evolution and/or stabilization of nodulation in Fabaceae, in which a loss of pathogen resistance genes may have allowed for stable mutualistic interactions with rhizobia.

MYB-CC transcription factors (TFs) are essential for plant growth and development. Members of the MYB-CC subfamily with long N terminal domains, such as phosphate starvation response 1 (PHR1) or PHR1-like TFs, have well documented functions, while those with short N terminal domains remain less understood. In this study, we identified a nodule specific MYB-CC transcription factor 1 (GmPHR1) in soybean that is different from other canonical PHR family genes in that GmPHR1 harbors a short N terminal ahead of its MYB-CC domain and was highly induced by rhizobium infection. The overexpression of GmPHR1 dramatically increased the ratio of deformed root hairs, enhanced subsequent soybean nodulation, and promoted soybean growth in pot experiments. The growth promotion effects of GmPHR1 overexpression were further demonstrated in field trails in which two GmPHR1-OE lines yielded 10.78% and 8.19% more than the wild type line. Transcriptome analysis suggested that GmPHR1 overexpression led to global reprogramming, with 749 genes upregulated and 279 genes downregulated, especially for genes involved in MYB transcription factor activities, root growth, and nutrient acquisition. Taken together, we conclude that GmPHR1 is a key gene involved in the global regulation of nodulation, root growth, and nutrient acquisition in soybeans, and is thus a promising candidate gene to target for soybean yield enhancement.

The response of soybean to biological nitrogen fixation (BNF) varies due to factors such as plant variety, soil, and environmental conditions, which can compromise the N2 fixation capacity. The objective of this study was to evaluate additional inoculation with B. japonicum at different soybean growth stages as a strategy to improve N2 fixation. This was determined by studying plant nodulation and BNF with the isotope dilution method. The experiment was carried out under greenhouse conditions in a soil without soybean cropping history. Inoculated soybean increased nodulation, plant biomass, BNF and yield components compared to un-inoculated soybean. Additional inoculation at the V3 growth stage showed promising results by maintaining BNF at later growth stages. A strong correlation was observed between nodule dry weight and N2 fixation, providing an easily available method to estimate differences in BNF activity between treatments. Moreover, efficient nodulation is crucial to sustain high yields with BNF, as shown by the correlation between nodule dry weight and pod dry weight.

Many Bradyrhizobium strains are able to establish a Nod factor-independent symbiosis with the leguminous plant Aeschynomene indica by the use of a type III secretion system (T3SS). Recently, an important advance in the understanding of the molecular factors supporting this symbiosis has been achieved by the in silico identification and functional characterization of 27 putative T3SS effectors (T3Es) of Bradyrhizobium vignae ORS3257. In the present study, we experimentally extend this catalog of T3Es by using a multi-omics approach. Transcriptome analysis under non-inducing and inducing conditions in the ORS3257 wild-type strain and the ttsI mutant revealed that the expression of 18 out of the 27 putative effectors previously identified, is under the control of TtsI, the global transcriptional regulator of T3SS and T3Es. Quantitative shotgun proteome analysis of culture supernatant in the wild type and T3SS mutant strains confirmed that 15 of the previously determined candidate T3Es are secreted by the T3SS. Moreover, the combined approaches identified nine additional putative T3Es and one of them was experimentally validated as a novel effector. Our study underscores the power of combined proteome and transcriptome analyses to complement in silico predictions and produce nearly complete effector catalogs. The establishment of the ORS3257 effectome will form the basis for a full appraisal of the symbiotic properties of this strain during its interaction with various host legumes via different processes.

The main objective of this study is to use bio-inoculants in relative to specific legume plant diversity for, enhanced nodulation and plant growth. Method involves organically based selection of 36 rhizobial strains, of which 6 strains were isolated to assess the efficiency of relative host-specific inoculation on nodulation and development in legumes viz. Vigna radiata. All promising combinations of the preferred rhizobial strain inoculants were tested under sterile conditions for improving nodulation and to screen the best isolate to be evaluated for its enhanced characteristics through inoculation by field trial in various soils. It was observed that the strains from Bhadrachalam forest BD1 are highly host specific for Vigna radiata plants and when inoculated, improved nodulation and enhanced plant growth. Because of the novel characters in BD1, further studies were carried out and was identified as Rhizobium sp. BD1 (NCBI Accession no. MT577595). The percentage of nitrogen content in Vigna radiata ranged between 1.2% to 2.9%. This Rhizobium sp. BD1 was tested for the unraveling and amelioration of crop production in barren, polluted and agricultural soils which showed enhanced characteristics in Vigna radiata plants. This method may be employed across the globe of same climatic conditions for the retrieval of plants in soils that carry agriculture unsuccessfully.

Roots have a fundamental role in plant growth and adaptation to different environments. Diversity in root morphology and architecture enables plants to acquire water and nutrients in contrasting substrate conditions, resist biotic and abiotic stress, and develop symbiotic associations. At its most fundamental level, morphology is determined by discrete changes in tissue patterning. Differences in the number and arrangement of the cell layers in the root can change tissue structure, as well as root length and girth, affecting important productivity traits. Therefore, understanding the molecular mechanisms controlling variation in developmental patterning is an important goal in biology. The ground tissue (GT) system is an ideal model to study the genetic basis of morphological diversity because it displays great interspecific variability in cell layer number. In addition, the genetic circuit controlling GT patterning in Arabidopsis thaliana has been well described, although little is known about species with more complex root anatomies. In this review, we will describe the Arabidopsis model for root radial patterning and present recent progress in elucidating the genetic circuitry controlling GT patterning in monocots and the legume Medicago truncatula (Mt), species that develop roots with more complex anatomies and multilayered cortex.

Many Bradyrhizobium strains are able to establish a Nod factor-independent symbiosis with the leguminous plant Aeschynomene indica by the use of a type III secretion system (T3SS). Recently, an important advance in the understanding of the molecular factors supporting this symbiosis has been achieved by the in silico identification and functional characterization of 27 putative T3SS effectors (T3Es) of Bradyrhizobium vignae ORS3257. In the present study, we experimentally extend this catalog of T3Es by using a multi-omics approach. Transcriptome analysis under non-inducing and inducing conditions in the ORS3257 wild-type strain and the ttsI mutant revealed that the expression of 18 out of the 27 putative effectors previously identified, is under the control of TtsI, the global transcriptional regulator of T3SS and T3Es. Quantitative shotgun proteome analysis of culture supernatant in the wild type and T3SS mutant strains confirmed that 15 of the previously determined candidate T3Es are secreted by the T3SS. Moreover, the combined approaches identified nine additional putative T3Es and one of them was experimentally validated as a novel effector. Our study underscores the power of combined proteome and transcriptome analyses to complement in silico predictions and produce nearly complete effector catalogs. The establishment of the ORS3257 effectome will form the basis for a full appraisal of the symbiotic properties of this strain during its interaction with various host legumes via different processes.

Here, we report an improved and complete genome sequence of Sinorhizobium (Ensifer) meliloti strain WSM1022, a microsymbiont of Medicago species, revealing its tripartite structure. This improved genome sequence was generated combining Illumina and Oxford nanopore sequencing technologies to better understand the symbiotic properties of the bacterium. The 6.75 Mb WSM1022 genome consists of three scaffolds, corresponding to a chromosome (3.70 Mb) and the pSymA (1.38 Mb) and pSymB (1.66 Mb) megaplasmids. The assembly has an average GC content of 62.2% and a mean coverage of 77X. Genome annotation of WSM1022 predicted 6058 protein coding sequences (CDSs), 202 pseudogenes, 9 rRNAs (3 each of 5S, 16S, and 23S), 55 tRNAs, and 4 ncRNAs. We compared the genome of WSM1022 to two other rhizobial strains, closely related Sinorhizobium (Ensifer) meliloti Sm1021 and Sinorhizobium (Ensifer) medicae WSM419. Both WSM1022 and WSM419 species are high-efficiency rhizobial strains when in symbiosis with Medicago truncatula, whereas Sm1021 is ineffective. Our findings report significant genomic differences across the three strains with some similarities between the meliloti strains and some others between the high efficiency strains WSM1022 and WSM419. The addition of this high-quality rhizobial genome sequence in conjunction with comparative analyses will help to unravel the features that make a rhizobial symbiont highly efficient for nitrogen fixation.

The highly conserved plant microRNA, miR156, affects root architecture, nodulation, symbiotic nitrogen fixation, and stress response. In Medicago sativa, transcripts of eleven SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE, SPLs, including SPL12, are targeted for cleavage by miR156. Our previous research revealed the role of SPL12 and its target gene, AGL6, in nodulation in alfalfa. Here, we investigated the involvement of SPL12, AGL6 and AGL21 in nodulation under osmotic stress and different nitrate availability conditions. Characterization of phenotypic and molecular parameters revealed that the SPL12/AGL6 module plays a negative role in maintaining nodulation under osmotic stress. While there was a decrease in the nodule numbers in WT plants under osmotic stress, the SPL12-RNAi and AGL6-RNAi genotypes maintained nodulation under osmotic stress. Moreover, the results showed that SPL12 regulates nodulation under a high concentration of nitrate by silencing AGL21. AGL21 transcript levels were increased under nitrate treatment in WT plants, but SPL12 was not affected throughout the treatment period. Given that AGL21 was significantly upregulated in SPL12-RNAi plants, we conclude that SPL12 may be involved in regulating nitrate inhibition of nodulation in alfalfa by targeting AGL21. Taken together, our results suggest that SPL12, AGL6, and AGL21 form a genetic module that regulates nodulation in alfalfa under osmotic stress and in response to nitrate.

Sunnhemp (Crotalaria juncea L.) is an important legume cover crop used in tree cropping systems, where there is increased interest by growers to identify rhizobia to maximize soil nitrogen (N) inputs. We aimed to isolate and identify native rhizobia and compare their capabilities with non-native rhizobia from commercial inoculants to fix atmospheric dinitrogen (N2), produce and reduce nitrous oxide (N2O), and improve plant growth. Phylogenetic analyses of sequences of the 16S rRNA and recA, atpD, and glnII genes showed native rhizobial strains belonged to Rhizobium tropici and the non-native strain to Bradyrhizobium japonicum. Plant nodulation tests, sequencing of nodC and nifH genes, and the acetylene-dependent ethylene production assay confirmed the capacity of all strains to nodulate sunnhemp and fix N2. Inoculation with native rhizobial strains resulted in significant increases in root and shoot weight and total C and N contents in the shoots, and showed greater N2-fixation rates and lower emissions of N2O compared to the non-native rhizobium. Our results suggest that native rhizobia improve plant growth, fix N2, and reduce greenhouse emissions of sunnhemp more than commercial rhizobia inoculants in Florida citrus orchards.

Red clover (Trifolium pratense L.) is an important forage crop and serves as a major contributor of nitrogen input in pasture settings because of its ability to fix atmospheric nitrogen. During the legume-rhizobial symbiosis, the host plant undergoes a large number of gene expression changes, leading to development of root nodules that house the rhizobium bacteria as they are converted into nitrogen-fixing bacteroids. Many of the genes involved in symbiosis are conserved across legume species, while others are species-specific with little or no homology across species and likely regulate the specific plant genotype/symbiont strain interactions. Red clover has not been widely used for studying symbiotic nitrogen fixation, primarily due to its outcrossing nature, making genetic analysis rather complicated. With the addition of recent annotated genomic resources and use of RNA-seq tools, we annotated and characterized a number of genes that are expressed only in nodule forming roots. These genes include those encoding nodule-specific cysteine rich peptides (NCRs) and nodule-specific Polycystin-1, Lipoxygenase, Alpha toxic (PLAT) domain proteins (NPDs). Our results show that red clover encodes one of the highest number of NCRs and ATS3-like/NPDs, which are postulated to increase nitrogen fixation efficiency, in the Inverted-Repeat Lacking Clade (IRLC) of legumes. Knowledge of the variation and expression of these genes in red clover will provide more insights into the function of these genes in regulating legume-rhizobial symbiosis and aid in breeding of red clover genotypes with increased nitrogen fixation efficiency.

Winter cover crops are used in organic olive production to increase N-supply and yields, and to reduce weed competition. However, there is limited information on the effect of different cover crops on weed suppression, soil fertility and productivity of organic olive orchards. Here, we compared the relative effect of four contrasting cover crops established from (i) untreated vetch seed, (ii) vetch seed inoculated with a commercial Rhizobium seed inoculum, (iii) an untreated vetch/barley/pea seed mixture and (iv) untreated seed of Medicago polymorpha L. (a native legume species which establishes naturally in olive orchards in Crete) in a 35-year-old experimental table olive orchard. The use of a vetch/barley/pea mixture resulted in the greatest suppression of the dominant weed species Oxalis pes-caprae. Rhizobium inoculation of vetch seed resulted in significantly lower vetch establishment and significantly higher Oxalis suppression but had no significant effect on the root nodulation of vetch plants. There was no significant difference in fruit yield between cover crop treatments, but the fruit weight was significantly higher when cover crops were established from un-treated vetch seeds and the vetch/barley/pea seed mixture compared with the cover crops based on inoculated vetch or Medicago seed. However, although Medicago establishment was very low (

The stable genetic transformation of soybean is time-consuming and inefficient. As a simple and practical alternative method, hairy root transformation mediated by Agrobacterium rhizogenes is widely applied in studying root-specific processes, nodulation, biochemical and molecular functions of genes of interest, gene editing efficiency of CRISPR/Cas9, and biological reactors and producers. Therefore, many laboratories have developed unique protocols to obtain hairy roots in composite plants composed of transgenic roots and wild-type shoots. However, these protocols still suffer from the shortcomings of low efficiency and time, space, and cost consumption. To address this issue, we developed a new protocol efficient regeneration and transformation of hairy roots (eR&T) in soybean, by integrating and optimizing the main current methods to achieve high efficiency in both hairy root regeneration and transformation within a shorter period and using less space. By this eR&T method, we obtained 100% regeneration of hairy roots for all explants, with an average 63.7% of transformation frequency, which promoted the simultaneous and comparative analysis of the function of several genes. The eR&T was experimentally verified Promoter:GUS reporters, protein subcellular localization, and CRISPR/Cas9 gene editing experiments. Employing this approach, we identified several novel potential regulators of nodulation, and nucleoporins of the Nup107-160 sub-complex, which showed development-dependent and tissue-dependent expression patterns, indicating their important roles in nodulation in soybean. Thus, the new eR&T method is an efficient and economical approach for investigating not only root and nodule biology, but also gene function.

Improving legumes crops’ performance under dense stands shade environment (e.g., intercropped oats–clover) is needed to promote agroecological practices. Previous studies have revealed that ethylene produced by plants under dense standing conditions is among other factors that affect crops’ growth performance and reduce legumes’ ability to fix nitrogen (N). Here, we identified a Pseudomonas thivervalensis strain T124 as a high ACC deaminase-producing bacterium and evaluated its potential ability to alleviate the effects of reduced light (RL) and exogenous ethylene applied as ACC (ethylene precursor) on clover growth and development under controlled conditions and field conditions at dense stands of clover and oats intercrops. RL decreases clover root and shoots biomass, whereas the T124 strain counteracted RL effects, enhancing clover tolerance to shade. Exogenous ACC reduced clover growth and chlorophyll content while inducing overaccumulation of reactive oxygen species (H2O2 and O2•−). ACC-elicited cellular stress was suppressed by strain T124, suggesting the role of bacterial ACC deaminase activity. Combined with Rhizobium leguminosarum strain T618 (the strain identified as being able to fix N in symbiosis with clover), T124 prevents early nodule senescence by improving nodule leghemoglobin and reducing nodule nitric oxide levels. Co-inoculation with T124 + T618 increased shoot N content (+24%) more than T618 alone. Field experiments revealed that intercropping decreases Photosynthetic Active Radiation (PAR) at the top of clover due to oats, affecting clover photosynthesis assimilation. Interestingly, under T124 inoculation treatments, clover net photosynthetic rate (Anet) and stomatal conductance (Gs) were found to improve relative to the control and T618 inoculation treatments. Clover exhibits improved growth performance in terms of branching and nodulation after T124 inoculation. Most significant improvements occurred with the mixing of the two strains. Data suggest that co-inoculation with R. leguminosarum T618 and P. thivervalensis T124 potentially decreases the interspecific competition between clover and oats intercrops by reducing ACC (ethylene precursor) levels. Our study revealed that co-inoculation of legumes with competitive rhizobia and ACC deaminase-producing PGPRs is an eco-friendly approach to improving intercropping systems’ performance.

Commercial legume varieties vary in terms of their drought tolerance when grown as sole crops, though relatively little is known about how legume variety selection affects cereal–legume intercrop performance under drought conditions. This study aims to test the hypothesis that positive rhizosphere interactions in faba bean–wheat intercrops will confer a “buffering capacity” on faba bean and wheat performance under water stress and that this effect will (i) depend on faba bean varietal selection and (ii) be enhanced with increasing faba bean varietal diversity. In a greenhouse experiment, three commercial faba bean (Vicia faba L.) varieties [Gloria (G), Alexia (A), Julia (J)] were grown in sole crop or intercropped with spring wheat (Triticum aestivum L.) under well-watered or water-stress conditions. Under intercropping, either one, two, or all three faba bean varieties were grown together with wheat to test the effect of intraspecific diversity on a cereal–legume intercrop performance. Consistent with the proposed hypothesis, we found that, under well-watered and water-stress conditions, wheat and faba bean shoot biomass production and nitrogen (N) acquisition improved with intercropping and that faba bean variety and variety mixture strongly modulated the intercropping effect. Interestingly, in both well-watered and water-stress conditions, wheat dry biomass and N accumulation were greatest in intercrops containing Gloria, while nodule number, nodule weight, and N accumulation in faba bean were greatest for intercrops containing Alexia and Julia (AJ). The effect of varietal diversity was inconsistent. Intercrops with two faba bean varieties tended to have positive or neutral effects on measured wheat and faba bean variables. However, overall performance under intercropping was generally reduced when all three faba bean varieties were planted with wheat. The effect of faba bean species diversity can buffer faba bean–wheat intercrop performance against water stress, and intercropping tended to have positive or neutral effects on the measured wheat and faba bean variables, notably with two-varietal faba bean mixtures.

Bradyrhizobium elkanii utilizes the type III effector Bel2-5 for nodulation in host plants in the absence of Nod factors (NFs). In soybean plants carrying the Rj4 allele, however, Bel2-5 causes restriction of nodulation by triggering immune responses. Bel2-5 shows similarity with XopD of the phytopathogen Xanthomonas campestris pv. vesicatoria and possesses two internal repeat sequences, two ethylene (ET)-responsive element-binding factor-associated amphiphilic repression (EAR) motifs, a nuclear localization signal (NLS), and a ubiquitin-like protease (ULP) domain, which are all conserved in XopD except for the repeat domains. By mutational analysis, we revealed that most of the putative domains/motifs in Bel2-5 were essential for both NF-independent nodulation and nodulation restriction in Rj4 soybean. The expression of soybean symbiosis- and defense-related genes was also significantly altered by inoculation with the bel2-5 domain/motif mutants compared with the expression upon inoculation with wild-type B. elkanii, which was mostly consistent with the phenotypic changes of nodulation in host plants. Notably, the functionality of Bel2-5 was mostly correlated with the growth inhibition effect of Bel2-5 expressed in yeast cells. The nodulation phenotypes of the domain-swapped mutants of Bel2-5 and XopD indicated that both the C-terminal ULP domain and upstream region are required for the Bel2-5-dependent nodulation phenotypes. These results suggest that Bel2-5 interacts with and modifies host targets via these multiple domains to execute both NF-independent symbiosis and nodulation restriction in Rj4 soybean.

Summary The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR‐Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR‐Cas9 as a mutant screening tool. Here, we report a pooled CRISPR‐Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR‐Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1‐1/1‐2/1‐3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops.

During the legume-rhizobium symbiotic interaction, rhizobial invasion of legumes is primarily mediated by a plant-made tubular invagination called an infection thread (IT). Here, we identify a gene in Lotus japonicus encoding a leucine-rich repeat receptor-like kinase (LRR-RLK), RINRK1 (Rhizobial Infection Receptor-like Kinase1), that is induced by Nod factors (NFs) and is involved in IT formation but not nodule organogenesis. A paralog, RINRK2, plays a relatively minor role in infection. RINRK1 is required for full induction of early infection genes, including Nodule Inception (NIN), encoding an essential nodulation transcription factor. RINRK1 displayed an infection-specific expression pattern, and NIN bound to the RINRK1 promoter, inducing its expression. RINRK1 was found to be an atypical kinase localized to the plasma membrane and did not require kinase activity for rhizobial infection. We propose RINRK1 is an infection-specific RLK, which may specifically coordinate output from NF signaling or perceive an unknown signal required for rhizobial infection.

Groundnut (Arachis hypogaea L.) is an important food legume in tropical and subtropical areas because of its ability to adapt to a wide range of agro-climatic regions. Groundnut is usually cultivated in nutrient-poor soil and rain-fed conditions, so average yield tends to be very low relative to potential yield. Even though the nitrogen (N) requirement of groundnut is much higher than cereals due to its high protein content, it has the capacity to meet 60-80% of N-based requirements through symbiotic N fixation via its root nodules. In its symbiotic relationship with legumes, Rhizobium fixes N, thereby positively impacting the content of this nutrient. This study aimed to isolate, characterize and purify microbial strains of Rhizobium specific to groundnut in a bid to increase this legume’s productivity. The research was conducted in the AICRP-Groundnut laboratory and greenhouse of the Directorate of Research, BCKV, in Kalyani, India during October 2016 to March 2017. Two Rhizobium isolates (RhBC and NRA1) were isolated and selected from groundnut pot cultures. After 45 days, NRA1 produced higher plant biomass, longer roots and shoots, more nodules and higher nodule dry weight than RhBC. NRA1 was selected for a future field trial. The two isolated microbial strains will aid in the screening of additional local isolates to test their effectiveness when co-cultured with local groundnut cultivars to increase yield in soil with low fertility.

In the current study, the impact of the preceding crops on growth, fresh pod yield, nitrogen fixation efficiency, and nitrogen nutrition of faba bean (Vicia faba L.) was investigated for two years in both organic and conventional crops. As preceding crops served cabbage, pea, and faba bean. The pod number per plant (PN) and the total fresh pod yield (TFPY) were significantly lower with cabbage compared to pea and faba bean as preceding crops in both cropping systems and both experimental years. However, in the organic farming system, pea increased significantly in PN and TFPY compared to faba bean as a preceding crop, while in the conventional system, there was no significant difference between the two legumes. The greater yield performance with the two legumes as preceding crops was associated with higher soil NO3-N and total-N concentrations at the beginning of the subsequent faba bean crop. The higher soil N availability when the preceding crop was a legume resulted partly from the higher biomass of crop residues left by these crops on the field after harvest, compared to cabbage. However, it was also associated with a more extensive nodulation of the faba bean roots by rhizobia and a higher percentage of N derived from atmosphere (%Ndfa) in their plant tissues, as determined through the natural abundance of the 15N isotope, when the preceding crop was a legume. The cropping system had no impact on pod yield, but organic farming increased the %Ndfa in both years.

International audience; Low phosphorus (P) availability is a primary factor that limits the production of common bean (Phaseolus vulgaris L.) in many parts of the world, especially when plant growth depends on N2 fixation. In order to understand how common bean copes with this nutritional constraint, two recombinant inbred lines (RILs) of common bean namely RILs 115 and 147 were studied in the field conditions during three growing seasons from 2012 to 2014. At flowering stage, plants were harvested and analyzed for their nodulation, growth, P content and yield. Results showed that for RIL115, the nodulation (43%), shoot (28%) and root growth (32%) was higher than for RIL147 in a low-P availability soil whatever the growing season. In addition, RIL115 had better growth and efficiency in use of P for the rhizobial symbiosis (7.29 mg nodule dry weight mg−1 nodule P) about twice higher may be involved in tolerance to low-P, and to be a useful marker for field data. At harvest, the RIL115 showed higher grain yield (1146 kg ha−1) than the RIL147 (1045 kg ha−1). It is concluded that the genotype of RIL 115 had a higher yield and could best adapt to low-P availability under field condition.

Nitrogen fixation by rhizobia is a highly energy-demanding process. Therefore, nodule initiation in legumes is tightly regulated. Environmental nitrate is a potent inhibitor of nodulation. However, the precise mechanism by which this agent (co)regulates the inhibition of nodulation is not fully understood. Here, we demonstrate that in Medicago truncatula the lipo-chitooligosaccharide-induced accumulation of cytokinins is reduced in response to the application of exogenous nitrate. Under permissive nitrate conditions, perception of rhizobia-secreted signalling molecules leads to an increase in the level of four cytokinins (i.e., iP, iPR, tZ, and tZR). However, under high-nitrate conditions, this increase in cytokinins is reduced. The ethylene-insensitive mutant Mtein2/sickle, as well as wild-type plants grown in the presence of the ethylene biosynthesis inhibitor 2-aminoethoxyvinyl glycine (AVG), is resistant to the inhibition of nodulation by nitrate. This demonstrates that ethylene biosynthesis and perception are required to inhibit nodule organogenesis under high-nitrate conditions.

In this work, we analyzed the diversity of the nodule-forming bacteria associated with Lupinus luteus and Lupinus cosentinii grown in the Maamora Cork oak forest acidic soils in Morocco. The phenotypic analysis showed the high diversity of the strains nodulating the two lupine's species. The strains were not tolerant to acidity or high alkalinity. They do not tolerate salinity or high temperatures either. The strains isolated from L. luteus were more tolerant to antibiotics and salinity than those isolated from L. cosentinii. The plant growth promoting (PGP) activities of our strains are modest, as among the 28 tested isolates, only six produced auxins, six produced siderophores, whereas three solubilized phosphates. Only two strains possess the three activities. The rrs gene sequences from eight representative strains selected following ARDRA and REP-PCR results revealed that they were members of the genus Bradyrhizobium. Six strains were then retained for further molecular analysis. The glnII, recA, gyrB, dnaK, and rpoB housekeeping gene sequence phylogeny showed that some strains were close to B. lupini LMG28514T whereas others may constitute new genospecies in the genus Bradyrhizobium. The strains were unable to nodulate Glycine max and Phaseolus vulgaris and effectively nodulated L. luteus, L. cosentinii, L. angustifolius, Chamaecytisus albidus, and Retama monosperma. The nodC and nodA symbiotic gene phylogenies showed that the strains are members of the genistearum symbiovar.

Commercial inoculants are often used to inoculate field-grown soybean in Europe. However, nodulation efficiencies in these areas are often low. To enhance biological nitrogen (N) fixation and increase domestic legume production, indigenous strains that are adapted to local conditions could be used to develop more effective inoculants. The objective of this study was to assess the ability of locally isolated Bradyrhizobium strains to enhance soybean productivity in different growing conditions of Northeast Germany. Three indigenous Bradyrhizobium isolates (GMF14, GMM36, and GEM96) were tested in combination with different soybean cultivars of different maturity groups and quality characteristics in one field trial and two greenhouse studies. The results showed a highly significant strain × cultivar interactions on nodulation response. Independent of the Bradyrhizobium strain, inoculated plants in the greenhouse showed higher nodulation, which corresponded with an increased N uptake than that in field conditions. There were significantly higher nodule numbers and nodule dry weights following GMF14 and GMM36 inoculation in well-watered soil, but only minor differences under drought conditions. Inoculation of the soybean cultivar Merlin with the strain GEM96 enhanced nodulation but did not correspond to an increased grain yield under field conditions. USDA110 was consistent in improving the grain yield of soybean cultivars Sultana and Siroca. On the other hand, GMM36 inoculation to Sultana and GEM96 inoculation to Siroca resulted in similar yields. Our results demonstrate that inoculation of locally adapted soybean cultivars with the indigenous isolates improves nodulation and yield attributes. Thus, to attain optimal symbiotic performance, the strains need to be matched with specific cultivars.

Rhizobia are soil proteobacteria able to engage in a nitrogen‐fixing symbiotic interaction with legumes that involves the rhizobial infection of roots and the bacterial invasion of new organs formed by the plant in response to the presence of appropriate bacterial partners. This interaction relies on a complex molecular dialogue between both symbionts. Bacterial N‐acetyl‐glucosamine oligomers called Nod factors are indispensable in most cases for early steps of the symbiotic inter-action. In addition, different rhizobial surface polysaccharides, such as exopolysaccharides (EPS), may also be symbiotically relevant. EPS are acidic polysaccharides located out of the cell with little or no cell association that carry out important roles both in free‐life and in symbiosis. EPS production is very complexly modulated and, frequently, co‐regulated with Nod factors, but the type of co‐regulation varies depending on the rhizobial strain. Many studies point out a signalling role for EPS‐derived oligosaccharides in root infection and nodule invasion but, in certain symbiotic cou-ples, EPS can be dispensable for a successful interaction. In summary, the complex regulation of the production of rhizobial EPS varies in different rhizobia, and the relevance of this polysaccharide in symbiosis with legumes depends on the specific interacting couple. Polish National Science Centre 2018/31/B/NZ9/00663 Ministerio de Ciencia, Innovación y Universidades PID2019-107634RB-I00

Background Like most legumes, chickpeas form specialized organs called root nodules. These nodules allow for a symbiotic relationship with rhizobium bacteria. The rhizobia provide fixed atmospheric nitrogen to the plant in a usable form. It is of both basic and practical interest to understand the host plant genetics of legume root nodulation. Chickpea lines PM233 and PM405, which harbor the mutationally identified nodulation genes rn1 and rn4, respectively, both display nodulation-deficient phenotypes. Previous investigators identified the rn1 mutation with the chickpea homolog of Medicago truncatula nodulation gene NSP2, but were unable to define the mutant rn1 allele. We used Illumina and Nanopore sequencing reads to attempt to identify and characterize candidate mutation sites responsible for the PM233 and PM405 phenotypes. Results We aligned Illumina reads to the available desi chickpea reference genome, and did a de novo contig assembly of Nanopore reads. In mutant PM233, the Nanopore contigs allowed us to identify the breakpoints of a ~ 35 kb deleted region containing the CaNSP2 gene, the Medicago truncatula homolog of which is involved in nodulation. In mutant PM405, we performed variant calling in read alignments and identified 10 candidate mutations. Genotyping of a segregating progeny population narrowed that pool down to a single candidate gene which displayed homology to M. truncatula nodulation gene NIN. Conclusions We have characterized the nodulation mutation sites in chickpea mutants PM233 and PM405. In mutant PM233, the rn1 mutation was shown to be due to deletion of the entire CaNSP2 nodulation gene, while in mutant PM405 the rn4 mutation was due to a single base deletion resulting in a frameshift mutation between the predicted RWP-RK and PB1 domains of the NIN nodulation gene. Critical to characterization of the rn1 allele was the generation of Nanopore contigs for mutant PM233 and its wild type parent ICC 640, without which the deletional boundaries could not be defined. Our results suggest that efforts of prior investigators were hampered by genomic misassemblies in the CaNSP2 region of both the desi and kabuli reference genomes.

Simple Summary Eicosanoids are oxygenated polyunsaturated fatty acids containing 20 carbons and subdivided into prostaglandin, leukotriene, and epoxyeicosatrienoic acid. They mediate immune responses in insects as well as mammals. Excessive eicosanoids cause severe inflammatory diseases in humans. However, a lack of eicosanoids induces immunosuppressive conditions in insects, which are highly susceptible to various entomopathogens. Despite the physiological significance of eicosanoids, their molecular action was not clearly understood in insects. This study was focused on Repat (Response to Pathogen) gene family, which might be regulated by eicosanoids. Among 44 members of Repat family genes, Repat33 was highly inducible to Gram-negative bacteria. Its expression was dependent on eicosanoids. Loss of function of Repat33 by RNA interference (RNAi) caused significant immunosuppression of a lepidopteran insect, Spodoptera exigua. Larvae of S. exigua treated by RNAi did not exhibit efficient cellular immune responses. They also failed to express antimicrobial peptide genes at high inducible levels in response to bacterial challenges. These results suggest that Repat33 is a downstream component of eicosanoid immune mediation. Abstract Repat (=response to pathogen) is proposed for an immune-associated gene family from Spodoptera exigua, a lepidopteran insect. In this gene family, 46 members (Repat1–Repat46) have been identified. They show marked variations in their inducible expression patterns in response to infections by different microbial pathogens. However, their physiological functions in specific immune responses and their interactions with other immune signaling pathways remain unclear. Repat33 is a gene highly inducible by bacterial infections. The objective of this study was to analyze the physiological functions of Repat33 in mediating cellular and humoral immune responses. Results showed that Repat33 was expressed in all developmental stages and induced in immune-associated tissues such as hemocytes and the fat body. RNA interference (RNAi) of Repat33 expression inhibited the hemocyte-spreading behavior which impaired nodule formation of hemocytes against bacterial infections. Such RNAi treatment also down-regulated expression levels of some antimicrobial genes. Interestingly, Repat33 expression was controlled by eicosanoids. Inhibition of eicosanoid biosynthesis by RNAi against a phospholipase A2 (PLA2) gene suppressed Repat33 expression while an addition of arachidonic acid (a catalytic product of PLA2) to RNAi treatment recovered such suppression of Repat33 expression. These results suggest that Repat33 is a downstream component of eicosanoids in mediating immune responses of S. exigua.

Selection of a suitable carrier material for rhizobial inoculants is essential for biofertilizers production. Locally available wastes or by-products as carrier material will increase the cost effectiveness of the inoculants preparation. Here, were evaluated four such waste materials from local ground viz. charcoal, saw dust, garden soil and sugarcane bagasse with carrier based inoculums (108 viable cells/ml) and kept at room temperature (30 ± 20C). The colony forming unit (CFU) count of each strain in different carriers was monitored every month. The charcoal, garden soil and saw dust resulted to allow a better survival of the inoculums. The viable counts in charcoal, soil, saw dust and sugarcane bagasse after 240 days of storage was recorded as 107, 106, 105 and 103 for MPR8 and 107, 105, 105 and 103 for TFR3 strains respectively. The effects of storage of carrier on plant productivity showed better plant biomass accumulation and nodulation in cases of charcoal, sawdust and garden soil. However it was insignificant with the sugarcane bagasse based inoculants.

Nitrogen fixing symbiosis between rhizobia and legumes contributes significant amounts of N to agricultural and natural environments. In natural soils, rhizobia compete with indigenous bacterial communities to colonize legume roots, which leads to symbiotic interactions. However, limited information is currently available on the effects of the rhizobial symbiont on the resident microbial community in the legume rhizosphere, rhizoplane, and endosphere, which is partly due to the presence of native nodulating rhizobial strains. In the present study, we used a symbiotic system comprised of Paraburkholderia phymatum and Mimosa pudica to examine the interaction of an inoculant strain with indigenous soil bacteria. The effects of a symbiont inoculation on the native bacterial community was investigated using high throughput sequencing and an analysis of 16S rRNA gene amplicons. The results obtained revealed that the inoculation induced significant alterations in the microbial community present in the rhizoplane+endosphere of the roots, with 13 different taxa showing significant changes in abundance. No significant changes were observed in the rhizospheric soil. The relative abundance of P. phymatum significantly increased in the rhizoplane+endosphere of the root, but significant decreased in the rhizospheric soil. While the rhizosphere, rhizoplane, and root endosphere contained a wide diversity of bacteria, the nodules were predominantly colonized by P. phymatum. A network analysis revealed that the operational taxonomic units of Streptomyces and Phycicoccus were positively associated with P. phymatum as potential keystone taxa. Collectively, these results suggest that the success of an inoculated symbiont depends on its ability to colonize the roots in the face of competition by other soil bacteria. A more detailed understanding of the mechanisms by which an inoculated strain colonizes its plant host is crucial for realizing the full potential of microbial inoculants in sustainable agriculture.

Background The formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process. Infection threads are tubes of cell wall material that transport rhizobacteria from root hair cells to developing nodules in host roots. They form in a type of reverse tip-growth from an inversion of the root hair cell wall, but the mechanism driving this growth is unknown, and the composition of the thread wall remains unclear. High resolution, 3-dimensional imaging of infection threads, and cell wall component specific labelling, would greatly aid in our understanding of the nature and development of these structures. To date, such imaging has not been done, with infection threads typically imaged by GFP-tagged rhizobia within them, or histochemically in thin sections. Results We have developed new methods of imaging infection threads using novel and traditional cell wall fluorescent labels, and laser confocal scanning microscopy. We applied a new Periodic Acid Schiff (PAS) stain using rhodamine-123 to the labelling of whole cleared infected roots of Medicago truncatula; which allowed for imaging of infection threads in greater 3D detail than had previously been achieved. By the combination of the above method and a calcofluor-white counter-stain, we also succeeded in labelling infection threads and plant cell walls separately, and have potentially discovered a way in which the infection thread matrix can be visualized. Conclusions Our methods have made the imaging and study of infection threads more effective and informative, and present exciting new opportunities for future research in the area.