Search

Gymnostoma sumatranum, a member of the Casuarinaceae family, is a resilient tree species well-adapted to nutrient-deficient soils. In Sabah, it stands out among the three and widely distributed Casuarina species, holding promise for land reclamation initiatives in degraded ultramafic forests. This comprehensive study addresses three primary objectives for effective G. sumatranum propagation, 1) sprig selection for rooted cutting, 2) impact of commercial rooting hormones on growth and nodulation, and 3) identification of suitable potting media for seedling development. The results indicated that the middle-part sprig is preferable for rooted cutting, with a higher rooting percentage (75.9%) compared to the upper-part sprig (57.5%). Additionally, the use of Seradix-3 as a growth hormone treatment yields significant improvements in explant length and root production. However, variations in root nodulation, influenced by different treatments, suggested limitations in commercial rooting hormones, with only 31% of rooted explants exhibiting nodulation. This underscores the need for further research to comprehend factors influencing nodulation in this context. In terms of potting media, river sand (R-sand) emerges as the most effective for G. sumatranum seedling development in the nursery stage based on S-index (healthy seedlings = 31.5%) and growth index (Height = 51.14±14.82 mm; N-Branch = 51.14±14.82), emphasising the critical role of proper potting medium selection for optimal seedling growth and survival. The study also highlights previous restoration efforts by the Sabah Forestry Department, utilising G. sumatranum and other pioneer species to restore degraded ultramafic areas in Sabah. Overall, these results contribute to the long-term goal of restoring the functioning capacity of valuable ultramafic ecosystems and promoting sustainable reforestation practices.

Cu+ -chaperones are a diverse group of proteins that allocate Cu+ ions to specific copper proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu+ -chaperone encoded in the Medicago truncatula genome, with a N-terminus Atx1-like domain that can bind Cu+ with picomolar affinities. MtNCC1 is able to interact with nodule-specific Cu+ -importer MtCOPT1. MtNCC1 is expressed primarily from the late infection zone to the early fixation zone and is located in the cytosol, associated with plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation, ncc1 mutants have a severe reduction in nitrogenase activity and a 50% reduction in copper-dependent cytochrome c oxidase activity. A subset of the copper proteome is also affected in the ncc1 mutant nodules. Many of these proteins can be pulled down when using a Cu+ -loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.

Type IVc Pili (T4cP), also known as Tad or Flp pili, are long thin microbial filaments that are made up of small-sized pilins. These appendages serve different functions in bacteria, including attachment, biofilm formation, surface sensing, motility, and host colonization. Despite their relevant role in diverse microbial lifestyles, knowledge about T4cP in bacteria that establish symbiosis with legumes, collectively referred to as rhizobia, is still limited. Sinorhizobium meliloti contains two clusters of T4cP-related genes: flp-1 and flp-2, which are located on the chromosome and the pSymA megaplasmid, respectively. Bundle-forming pili associated with flp-1 are involved in the competitive nodulation of alfalfa plants, but the role of flp-2 remains elusive. In this work, we have performed a comprehensive bioinformatic analysis of T4cP genes in the highly competitive S. meliloti GR4 strain and investigated the role of its flp clusters in pilus biogenesis, motility, and in the interaction with alfalfa. Single and double flp-cluster mutants were constructed on the wild-type genetic background as well as in a flagellaless derivative strain. Our data demonstrate that both chromosomal and pSymA flp clusters are functional in pili biogenesis and contribute to surface translocation and nodule formation efficiency in GR4. In this strain, the presence of flp-1 in the absence of flp-2 reduces the competitiveness for nodule occupation.

Rhizobia are bacteria that form nitrogen-fixing nodules in legume plants. The sets of genes responsible for both nodulation and nitrogen fixation are carried in plasmids or genomic islands that are often mobile. Different strains within a species sometimes have different host specificities, while very similar symbiosis genes may be found in strains of different species. These specificity variants are known as symbiovars, and many of them have been given names, but there are no established guidelines for defining or naming them. Here, we discuss the requirements for guidelines to describe symbiovars, propose a set of guidelines, provide a list of all symbiovars for which descriptions have been published so far, and offer a mechanism to maintain a list in the future.

The inclusion of soybean in Swedish crop rotations would enable an increase in legume production in Sweden. Previous attempts to introduce soybean as a crop in Sweden have failed, but in recent years, new attempts have been made under experimental conditions. Soybean cultivars suitable for cultivation in Sweden belong to maturity group (MG) 000, as well as historically Swedish-bred soybean cultivars, such as Bråvalla. However, for these cultivars to fix N, compatible rhizobia such as Bradyrhizobium japonicum are needed. These are not native to Swedish soils and have to be introduced through inoculation. Inoculating seeds can have several beneficial effects on soybean traits, but this varies with the interaction between soybean cultivar, rhizobial strain- or species and environmental factors. Inoculants can have different formulations which have different properties and may affect rhizobial survival and inoculation effect. The aim of this thesis was to evaluate the effects the inoculation methods no-, liquid- or peat inoculation had on phenotypic traits and N-fixation in soybean cultivars suitable for cultivation in Swedish climate in both field- and greenhouse conditions. The peat- and liquid inoculants containing B. japonicum, as well as uninoculated control, were tested in a field trial on Gotland, Sweden, on the soybean cultivars Abaca, Gallec, Sussex, and Todeka (all MG 000), and in a greenhouse experiment in Uppsala, Sweden, on the cultivars Abaca, Gallec, Sussex and Bråvalla (historic). Inoculation of the seeds resulted in higher N content and %Ndfa in both the field trial (significant increase) and the greenhouse experiment compared to the uninoculated plants. The peat – and liquid formulations did not differ in performance in the greenhouse experiment, but the peat inoculant did overall perform better than the liquid inoculant in the field trial. This may be an effect of the protective properties of peat supporting rhizobial survival in field conditions. Ino

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.

Common bean (Phaseolus vulgaris L.) yield is limited by prolonged drought conditions and nutrients scarcity in the soil. The Colombian Agricultural Research Corporation (AGROSAVIA) selected five common bean genotypes for presenting biofortification characteristics, good grain size and water deficit tolerance, however, sustainable techniques are required that contribute to the adaptation of these genotypes, and mitigate the impact of drought and nutrient scarcity on the crop production. Plant growth promoting rhizobacteria is an alternative to improve nutrients absorption and plants adaptation to adverse conditions. In this work, the efficiency of rhizobia in biological nitrogen fixation (BNF) in selected common bean genotypes was evaluated. Soil samples were taken in the rhizosphere and roots of the five bean genotypes at the Motilonia Research Center, as well as two alternative samplings in other areas of the Cesar department. Bean seeds were used as a trap to obtain nodules in the laboratory. The nodulation and biological nitrogen fixation of 11 strains from the AGROSAVIA bank in five bean genotypes were evaluated and the symbiotic efficiency of 7 of the strains in four of the genotypes was measured. Three isolates with rhizobia characteristics, symbionts of the 77-SMG22 genotype, were obtained. The most efficient strains in BNF were P17 and P22 in genotype 45-HTA10-2, strains C229 and P37 in 48-HTA14-1, in genotype 56-DAB295 all symbiont strains were efficient (C229, P03, P22 and P37), and in the 77-SMG22 genotype the most efficient treatment was the commercial inoculant Rhizobiol®. Therefore, the strains from the AGROSAVIA bank promote the development of some common bean genotypes reflected in the growth of the plants., El rendimiento de frijol común (Phaseolus vulgaris L.) se ve limitado por condiciones de sequía prolongada y escasez de nutrientes en el suelo. La Corporación Colombiana de Investigación Agropecuaria (AGROSAVIA) seleccionó cinco genotipos de frijol común por presentar características de biofortificación, buen tamaño de grano y tolerancia al déficit hídrico, sin embargo, se requiere técnicas sostenibles que contribuyan a la adaptación de estos genotipos, y mitiguen el impacto de la sequía y escasez de nutrientes en la producción de este cultivo. Las rizobacterias promotoras de crecimiento vegetal son una alternativa para mejorar la absorción de nutrientes y la adaptación de las plantas a condiciones adversas. En este trabajo se evaluó la eficiencia de rizobios en la fijación biológica de nitrógeno (FBN) en los genotipos de frijol común seleccionados. Se tomaron muestras de suelo en la rizósfera y raíces de los cinco genotipos de frijol en el Centro de Investigación Motilonia, como también dos muestreos alternativos en otras zonas del departamento del Cesar. Se utilizaron las semillas de frijol como trampa para obtener nódulos en el laboratorio. También se evaluó la nodulación y la FBN de 11 cepas del banco de AGROSAVIA en los cinco genotipos de frijol y se midió la eficiencia simbiótica de 7 de las cepas en cuatro de los genotipos. Se obtuvieron 3 aislados con características de rizobios, simbiontes del genotipo 77-SMG22. Las cepas más eficientes en la FBN fueron la P17 y P22 en el genotipo 45-HTA10-2, las cepas C229 y P37 en el 48-HTA14-1, en el genotipo 56-DAB295 todas las cepas simbiontes fueron eficientes (C229, P03, P22 Y P37), y en el genotipo 77-SMG22 el tratamiento más eficiente fue el inoculante comercial Rhizobiol®. Por tanto, las cepas del banco de AGROSAVIA promueven el desarrollo de algunos genotipos de frijol común reflejado en el crecimiento de las plantas.

Approximately 77% of global arable land use is devoted to forage cultivation, which is the basis for animal production and global food security. Therefore a sustainable and biologically diverse forage cultivation is needed. Feed for cattle needs to contain a good balance between fibre, energy and protein. Legumes are important for forage quality and fix their own nitrogen, reducing the need for N-fertilization. Medicago sativa L. (lucerne) is an agronomically important legume species that is cultivated on over 30 million hectares worldwide. Lucerne fixates N² in symbiosis with bacterial species E.meliloti, which attaches to the root and forms nodules. E. meliloti is acid sensitive and at pH<6 nodulation is reduced. This leads to decreased biomass production and quality. The aim of this study was to evaluate the pH tolerance of two lucerne subspecies; Blue lucerne (BL) and yellow lucerne (YL). The hypotheses were 1) YL will be more tolerant than BL against low pH, but BL will produce more biomass than YL in high pH conditions; 2) A low pH will decrease the nodulation formation and quality for BL and YL; and 3) A low pH will decrease biomass quantity for BL and YL. A mineral soil with different rates of lime was used to create six treatments with pH 5.7-6.5. 60 pots containing YL and BL cultivars inoculated with Nitragin Gold, a commercial E. meliloti mix were placed in a randomized block design in a greenhouse chamber. After 75-81 days the pots were harvested. Non-destructive measurements were made during growth and destructive measurements were made at harvest. The significance of four factors; cultivar, pH, cultivar*pH and time*cultivar, were statistically tested for 11 response variables connected to biomass and nodulation. The effect of liming on biomass and nodulation was also evaluated in a regression analysis using lime rate as an explanatory variable. The soil pH kept rising after sowing, even for the non-limed soil, and ended up higher than intended at 6.5-6, Vall odlas på ungefär 77% av jordens åkermark. Vallodling är grunden till all djurproduktion och en viktig del av människans livsmedelförsörjning. Därför är det viktigt att vallodlingen är så hållbar och biologiskt mångfacetterad som möjligt. Det är viktigt att det finns en god balans mellan fiber-, energi- och proteininnehåll i djurföda. Baljväxter är viktiga för en god vallfoderkvalitet och de förser sig själva med kväve, vilket minskar behovet av att kvävegödsla. Medicago sativa L. (lusern) odlas på över 30 miljoner hektar globalt och är en viktig baljväxt för jordbruket. Lusern fixerar kväve i symbios med bakteriearten E. meliloti, som bildar rhizobiumknölar (noduler) på lusernrötter. E. meliloti är känslig för sura miljöer och vid pH<6 minskar noduleringen. Detta leder till minskad tillväxt och sämre näringskvalitet hos lusern. Målet med den här studien var att undersöka och jämföra pH-känsligheten hos två underarter av lusern, blålusern (BL) och gullusern (YL). Studien utgick från tre hypoteser: 1) YL tål lägre pH än BL men vid höga pH-värden producerar BL mer biomassa än YL; 2) Noduleringen minskar med lägre pH för både BL och YL; och 3) Lågt pH minskar biomassaproduktionen för BL och YL.En mineraljord kalkades med olika mängder kalk för att ta fram sex behandlingar med pH-värden mellan 5.7-6.5. BL- och YL-frön inockulerades med Nitragin Gold (en kommersiell blandning av olika E. meliloti-sorter) och planterades i 60 krukor. Krukorna placerades ut i randomized block design i en växthuskammare och skördades 75-81 dagar efter sådd. Icke-destruktiva mätningar gjordes under tillväxt och vid skörd utfördes destruktiva mätningar. I en statistisk analys testades signifikansen för fyra faktorer; lusernsort, pH, lusernsort*pH och tid*lusernsort, för 11 responsvariabler kopplade till biomassa och nodulering. Kalkningseffekt på biomassa och nodulering utvärderades i en regressionsanalys med kalkmängd som förklaringsvariabel. Under experimentets gång ökade pH i jordarna

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 t

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., 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

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.

[EN] Background: The introduction of species with tolerance to salinity is one of the strategies to reduce the harmful effect of salts in the eastern region of Cuba. The species Vigna unguiculata L., shows rusticity and capability of adaptation, however, it is necessary to search for cultivars with greater tolerance and symbiotic nitrogen fixation possibilities to increase yields. Objective: To determine the behavior of the bradyrizobium-cowpea bean relationship under salinity conditions, based on variables related to symbiotic nitrogen fixation, growth and development. Methodology: An experiment was carried out under controlled glass house conditions at the CSIC, Madrid, Spain. Two cultivars were used; IITA-Precoz and IT 86 D-715, which were inoculated with Strain VIBA-1 of Bradyrhizobum liaoningense. Treatments were Hoagland nutrient solution (control) and nutrient solution with 150 mM NaCl (saline). A completely randomized design with eight replications was used. At 40 days after germination, variables related to symbiotic nitrogen fixation, growth and development, protein concentration of the cytosolic soluble fraction of leaves and nodules were evaluated. Results: Although salinity negatively affected both cultivars, the damage did not manifest with the same intensity. IT 86 D-715 was less affected by showing less damage on the variables number of nodules, ARA, protein concentration in nodules and leaves, accumulation of leaf and root biomass, so its tolerance was greater. The IITA-Precoz cultivar was more susceptible, all the variables were notably affected under stress conditions, therefore, its tolerance level is lower. Implications: The cultivar IT 86 D-715 can be used in salinized soils due to its high tolerance, which is evidenced in variables of growth development and atmospheric nitrogen fixation. Conclusions: The cultivar IT 86 D-715 presents a greater capacity for symbiotic nitrogen fixation under salinity conditions and the tolerance index in all the, [ES] Antecedentes: La introducción de especies con tolerancia a la salinidad es una de las estrategias para disminuir el efecto dañino de las sales en la región oriental del Cuba. La especie Vigna unguiculata L., muestra rusticidad y poder de adaptación, sin embargo, es necesaria la búsqueda de cultivares con mayor tolerancia y fijación simbiótica de nitrógeno para incrementar los rendimientos ante estas condiciones. Objetivo: Determinar el comportamiento de la relación bradyrizobio-frijol Caupí en condiciones de salinidad, a partir de variables relacionadas con la fijación simbiótica de nitrógeno, crecimiento y desarrollo. Metodología: Se desarrolló un experimento en condiciones controladas de casa de cristal en el CSIC, Madrid, España. Se utilizaron dos cultivares; IITA-Precoz y IT 86 D-715, que fueron inoculados con la Cepa VIBA-1 de Bradyrhizobum liaoningense. Los tratamientos fueron solución nutritiva Hoagland (control) y solución nutritiva con 150 mM NaCl (salino). Se utilizó un diseño completamente aleatorizado con ocho repeticiones. A los 40 días después de la germinación, se evaluaron variables relacionadas con la fijación simbiótica de nitrógeno, crecimiento y desarrollo, concentración de proteína de fracción soluble citosólica de hojas y nódulos. Resultados: La salinidad provocó un efecto dañino sobre la relación bradyrizobio-frijol Caupí en ambos cultivares, aunque estos no se manifestaron con la misma intensidad. IT 86 D-715 fue menos afectado al evidenciarse menor perjuicio sobre las variables número de nódulos, ARA, concentración de proteínas en nódulos y hojas, acumulación de biomasa foliar y radical, por lo que su tolerancia fue mayor. El cultivar IITA-Precoz fue más susceptible, todas las variables fueron notablemente afectadas en condiciones de estrés, por tanto, su nivel de tolerancia es menor. Implicaciones: El cultivar IT 86 D-715 puede ser utilizado en suelos salinizados por su alta tolerancia, que se evidencia en variables del crecimiento, de

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., Peer Reviewed

The plant hormone gibberellin (GA) is required at different stages of legume nodule development, with its spatiotemporal distribution tightly regulated. Transcriptomic and bioinformatic analyses established that several key GA biosynthesis and catabolism enzyme encoding genes are critical to soybean (Glycine max) nodule formation. We examined the expression of several GA oxidase genes and used a Förster resonance energy transfer-based GA biosensor to determine the bioactive GA content of roots inoculated with DsRed-labelled Bradyrhizobium diazoefficiens. We manipulated the level of GA by genetically disrupting the expression of GA oxidase genes. Moreover, exogenous treatment of soybean roots with GA3 induced the expression of key nodulation genes and altered infection thread and nodule phenotypes. GmGA20ox1a, GmGA3ox1a, and GmGA2ox1a are upregulated in soybean roots inoculated with compatible B. diazoefficiens. GmGA20ox1a expression is predominately localized to the transient meristem of soybean nodules and coincides with the spatiotemporal distribution of bioactive GA occurring throughout nodule organogenesis. GmGA2ox1a exhibits a nodule vasculature-specific expression pattern, whereas GmGA3ox1a can be detected throughout the nodule and root. Disruptions in the level of GA resulted in aberrant rhizobia infection and reduced nodule numbers. Collectively, our results establish a central role for GAs in root hair infection by symbiotic rhizobia and in nodule organogenesis.

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.

The improvement of pea as a crop over many decades has been employing the use of mutants. Several hundreds of different mutations are known in pea (Pisum sativum subsp. sativum), some of which are valuable for breeding. Breeding strategies may be diverse in different countries depending on different obstacles. In Russia, numerous spontaneous and induced mutations have been implemented in breeding. To our knowledge some of these, are not used in pea breeding beyond Russia. This review describes the use of mutations in pea breeding in Russia. The paper provides examples of cultivars created on the basis of mutations affecting the development of seeds (def), inflorescence (det, deh), compound leaves (af, af unitac), and symbiotic nitrogen fixation (various alleles of Sym and Nod loci). Novel mutations which are potentially promising for breeding are currently being investigated. Together with numerous cultivars of dry and fodder pea carrying commonly known mutations, new ‘chameleon’ and ‘lupinoid’ morphotypes, both double mutants, are under study. A cultivar Triumph which increases the effectiveness of interactions with beneficial soil microbes, was bred in Russia for the first time in the history of legume breeding.

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

Background: Cowpea is a grain legume of major importance in sub-Saharan Africa where it is cultivated by smallholder farmers on poor soils and production is often constrained by the parasitic weed Striga gesnerioides. Method: Experiments were conducted to assess the potential of rhizobium inoculation to mitigate Striga infection and increase cowpea productivity. We infested soils with S. gesnerioides and assessed the impact of treatments combining cowpea genotypes and bradyrhizobium inoculation on Striga dynamics and cowpea yield. In total, 20 cowpea genotypes were included, of which nine were resistant to Striga and 11 were susceptible. In the first experiment these were factorially combined with three inoculation options (two bradyrhizobium strains USDA3384 and IRJ2180A, and uninoculated control) in a screen-house using potted sterile soils. Second, the same trial was repeated in the field with basal phosphorus applied at sowing and a fourth treatment of fertilizer-N (urea) included testing whether N was limiting cowpea growth. The field trial also included a separate treatment with no input that served as a negative check. Result: Significant genotype x treatment interactions were observed in nodule counts, Striga attachment, emergence, and cowpea shoot growth in the screen-house. There were few nodules across all cowpea lines. Striga counts were the lowest for resistant varieties with no emerged plants. Rhizobial inoculants depressed Striga counts with consistent differences across cowpea genotypes. Inoculation with IRJ2180A performed the best against Striga attachment in resistant genotypes, and against Striga emergence in susceptible genotypes. In the field trial, cowpea grown without inputs had the least number of nodules. The genotype x treatment interaction was significant: resistant cowpea genotypes were free of emerged Striga while there was much more Striga emergence without input addition with susceptible genotypes. A significant genotype x treatment in

Background: Nodule symbiosis with diazotrophic Frankia or rhizobium occurs in plant species belonging to ten taxonomic lineages within the related orders Fabales, Fagales, Cucurbitales, and Rosales. Phylogenomic studies indicate that this nitrogen-fixing nodulation trait has a single evolutionary origin. In legume model plants, the molecular interaction between plant and rhizobium microsymbiont is mapped to a significant degree. A specific LysM-type receptor kinase, LjEPR3 in Lotus japonicus and MtLYK10 in Medicago truncatula, was found to act in a secondary identity-based mechanism, controlling intracellular rhizobium infection. Furthermore, LjEPR3 showed to bind surface exopolysaccharides of Mesorhizobium loti, the diazotrophic microsymbiont of L. japonicus. EPR3 orthologous genes are not unique to legumes. Surprisingly, however, its ortholog EXOPOLYSACCHARIDE RECEPTOR (EPR) is pseudogenized in Parasponia, the only lineage of non-legume plants that nodulate also with rhizobium. Results: Analysis of genome sequences showed that EPR3 orthologous genes are highly conserved in nodulating plants. We identified a conserved retrotransposon insertion in the EPR promoter region in three Parasponia species, which associates with defected transcriptional regulation of this gene. Subsequently, we studied the EPR gene of two Trema species as they represent the sister genus of Parasponia for which it is assumed it lost the nitrogen-fixing nodulation trait. Both Trema species possess apparently functional EPR genes that have a nodulation-specific expression profile when introduced into a Parasponia background. This indicates the EPR gene functioned in nodulation in the Parasponia-Trema ancestor. Conclusion: We conclude that nodule-specific expression of EPR3 orthologous genes is shared between the legume and Parasponia-Trema lineage, suggesting an ancestral function in the nitrogen-fixing nodulation trait. Pseudogenization of EPR in Parasponia is an exceptional case in nodulatin

Soybean (Glycine max L.) is one of the valuable leguminous crops with grain used for human consumption, livestock feeding, bio-fuel (bio-diesel) production, vegetable oil and is a protein resource. The crop also fixes atmospheric nitrogen. The study aimed to evaluate the performance of two soybean varieties to a combination of inoculation and seaweed extract. The research trial was conducted at Syferkuil farm during 2017/2018 summer season in which two soybean varieties (TGx 1937-1F promiscuous) and (PAN 1583R specific-nodulating) were evaluated for their growth, nodulation and yield responses to rhizobium inoculation and seaweed extract (0 % rate (0ml/12L), 50 % rate (30ml/12L) and 100 % (60ml/12L). The research trial was laid out as a split-split plot arrangement fitted in RCBD with four replications. Inoculation significantly influenced grain yield at P≤0.05, seed nutrient content and total above-ground biomass, except for seed potassium. No inoculation significantly achieved higher primary branches per plant at P≤0.05, pod number per plant, stem diameter (P≤0.05), grain yield (P≤0.05), harvest index and total above-ground biomass at P≤0.001. The variety TGx 1937-1F had significantly (P≤0.001) higher nodule number per plant, effective nodules per plant, nodule dry weight, dried shoot biomass, leaf number per plant, pod number per plant, primary branches per plant, stem diameter, plant height, leaf chlorophyll content, total above-ground biomass, grain yield and seed iron (Fe) content. Application of full rate seaweed extract significantly (P≤0.001) increased primary branches per plant, stem diameter, leaf number per plant, plant height, shelling percentage, total above-ground biomass, grain yield, and seed content of calcium (Ca), potassium, magnesium (Mg), manganese (Mn) and sodium (Na) all at P≤0.05. Seaweed extract rate at 0 % obtained the highest harvest index (P≤0.001). Inoculation and variety TGx 1937-1F interaction exhibited a significant increase on leaf, National Research Foundation (NRF)

[ES] Las leguminosas son capaces de colonizar ambientes desfavorables para otras especies vegetales por lo que suelen utilizarse con éxito para la repoblación y restauración de ambientes áridos y degradados. El establecimiento y crecimiento de estas plantas se ve favorecido por la simbiosis efectiva con una variedad de microorganismos del suelo comúnmente conocidos como rhizobios. De este modo, juegan un rol importante en la disponibilidad de nitrógeno y en su potencial utilización como biofertilizantes para restaurar e incrementar la fertilidad de suelos degradados (Requena et al. 2001; Rodríguez-Echevarría y Pérez-Fernández 2005). Para el desarrollo de este trabajo, se cultivaron, en macetas, plantas de diferentes especies de leguminosas del ambiente mediterráneo, en dos tipos de suelos; Sierra Calderona y Cañada. Transcurrido el tiempo necesario para un adecuado crecimiento, se extrajo un determinado número de plantas y se estudió la nodulación que se produjo en las raíces, así como las características macro y microscópicas de los nódulos. A partir de dichos nódulos se realizó una caracterización genética de los microrganismos nodulantes mediante una amplificación del gen rRNA 16S y nodC. La caracterización de las bacterias implicadas facilitaría la futura selección de cepas eficientes para establecer simbiosis, aumentar la disponibilidad de nitrógeno y mejorar el desarrollo vegetal., [EN] Leguminous plants are capable of colonizing environments unfavourable to other plant species and are therefore often used successfully for the repopulation and restoration of arid and degraded environments. The establishment and growth of these plants is favoured by effective symbiosis with a variety of soil microorganisms commonly known as rhizobia. In this way, they play an important role in the availability of nitrogen and its potential use as biofertilizers to restore and increase the fertility of degraded soils (Requena et al. 2001; Rodríguez-Echevarría and Pérez-Fernández 2005). For the development of this work, plants of different leguminous species from the Mediterranean environment were cultivated in pots in two types of soils: Sierra Calderona and Cañada. Once the time required for adequate growth has elapsed, a certain number of plants was extracted and the nodulation produced in the roots was studied, as well as the macro and microscopic characteristics of the nodules. From these nodules, a genetic characterization of the nodulating microorganisms was carried out by means of an amplification of the 16S rRNA gene. The characterization of the bacteria involved would facilitate the future selection of efficient strains to establish symbiosis, increase nitrogen availability and improve plant development.

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.

To support the adaption of soybean [Glycine max (L) Merrill] cultivation across Central Europe, the availability of compatible soybean nodulating Bradyrhizobia (SNB) is essential. Little is known about the symbiotic potential of indigenous SNB in Central Europe and the interaction with an SNB inoculum from commercial products. The objective of this study was to quantify the capacity of indigenous and inoculated SNB strains on the symbiotic performance of soybean in a pot experiment, using soils with and without soybean history. Under controlled conditions in a growth chamber, the study focused on two main factors: a soybean cropping interval (time since the last soybean cultivation; SCI) and inoculation with commercial Bradyrhizobia strains. Comparing the two types of soil, without soybean history and with 1–4 years SCI, we found out that plants grown in soil with soybean history and without inoculation had significantly more root nodules and higher nitrogen content in the plant tissue. These parameters, along with the leghemoglobin content, were found to be a variable among soils with 1–4 years SCI and did not show a trend over the years. Inoculation in soil without soybean history showed a significant increase in a nodulation rate, leghemoglobin content, and soybean tissue nitrogen concentration. The study found that response to inoculation varied significantly as per locations in soil with previous soybean cultivation history. An inoculated soybean grown on loamy sandy soils from the location Müncheberg had significantly more nodules as well as higher green tissue nitrogen concentration compared with non-inoculated plants. No significant improvement in a nodulation rate and tissue nitrogen concentration was observed for an inoculated soybean grown on loamy sandy soils from the location Fehrow. These results suggest that introduced SNB strains remained viable in the soil and were still symbiotically competent for up to 4 years after soybean cultivation. However, t, Peer Reviewed

Biostimulants promote physiological and morphological changes in the plant, leading to better adaptation in adverse conditions and increases in growth and productivity. In this work, we evaluated the biostimulatory effect of Bacillus mycoides BSC25 and humic acids derived from goat manure vermicompost (HA-V) and a lignite low rank coal (HA-C), on guajiro beans (Vigna unguiculata L. Walp). The work included: 1) checking in the soil the presence of rhizobia capable of forming nodules on V. unguiculata, and molecular typing of an Rhizobium isolated strain, 2) experiments under controlled conditions in a plant growth chamber to check the promotion of bean seedlings early growth treated separately with HA-V, HA-C or B. mycoides and joint application trials HA-B. mycoides, 3) development under a field conditions bioestimulation trial with HA-V,HA-C and B. mycoides in a semi-arid soil of the mean Guajira.. It was found that a population of Rhizobium sp. strain persistent in the soil was able to nodulate guajiro bean; in addition, treatment with both biostimulants, HA or B. mycoides, promote bean growth and increases nodulation degree of the rhizobia native population, these findings suggests the convenience of promote this technology to improve the production of guajiro bean crop., La bioestimulación es una tecnología pertinente en apoyo a la intensificación de la agricultura ecológica, principalmente en suelos de zonas marginales donde los tensores ambientales mantienen a las plantas bajo condiciones de estrés permanente en detrimento de la productividad. Los bioestimulantes promueven en la planta cambios fisiológicos y morfológicos conducentes a una mejor adaptación en condiciones adversas e incrementos en el crecimiento y productividad. En este trabajo se evaluó el efecto bioestimulante de Bacillus mycoides BSC25 y ácidos húmicos derivados de lombricompost de estiércol de caprino (AH-L) y de un carbón pobre tipo lignito (AH-C), sobre el frijol guajiro (Vigna unguiculata L. Walp). El trabajo comprendió: 1) la comprobación de la presencia de rizóbios en el suelo, con capacidad de nodular V. unguiculata, y caracterización molecular de una cepa aislada, 2) experimentos bajo condiciones controladas en cámara de crecimiento vegetal, para comprobar la estimulación del crecimiento temprano del frijol tratado con AH-L, AH-C o B. mycoides y la aplicación conjunta AH-B. mycoides, 3) un experimento de bioestimulacion con AH-L, AH-C y B. mycoides bajo condiciones de campo, en un suelo semiárido de la media Guajira. Adicionalmente se determinó la identidad química de los AH, relacionada con su bioactividad. Se encontró que en el suelo persistía una población de Rhizobium sp capaz de nodular el frijol guajiro, el tratamiento con los agentes bioestimulantes favorece el crecimiento de la planta e incrementa el grado de nodulación por la población nativa de rizóbios, lo cual sugiere la conveniencia de promover esta tecnología para mejorar la producción del cultivo de frijol guajiro.

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 LMG28514 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.

Bacteria belonging to the genus Aminobacter are metabolically versatile organisms thriving in both natural and anthropized terrestrial environments. To date, the taxonomy of this genus is poorly defined due to the unavailability of the genomic sequence of A. anthyllidis LMG 26462T and the presence of unclassified Aminobacter strains. Here, we determined the genome sequence of A. anthyllidis LMG 26462T and performed phylogenomic, average nucleotide identity and digital DNA-DNA hybridization analyses of 17 members of genus Aminobacter. Our results indicate that 16S rRNA-based phylogeny does not provide sufficient species-level discrimination, since most of the unclassified Aminobacter strains belong to valid Aminobacter species or are putative new species. Since some members of the genus Aminobacter can utilize certain C1 compounds, such as methylamines and methyl halides, a comparative genomic analysis was performed to characterize the genetic basis of some degradative/assimilative pathways in the whole genus. Our findings suggest that all Aminobacter species are heterotrophic methylotrophs able to generate the methylene tetrahydrofolate intermediate through multiple oxidative pathways of C1 compounds and convey it in the serine cycle. Moreover, all Aminobacter species carry genes implicated in the degradation of phosphonates via the C-P lyase pathway, whereas only A. anthyllidis LMG 26462T contains a symbiosis island implicated in nodulation and nitrogen fixation.

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-chitooligosaccharideinduced 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.

Quorum sensing (QS) is a bacterial cell-to-cell signaling mechanism that collectively regulates and synchronizes behaviors by means of small diffusible chemical molecules. In rhizobia, QS systems usually relies on the synthesis and detection of N-acyl-homoserine lactones (AHLs). In the model bacterium Sinorhizobium meliloti functions regulated by the QS systems TraI-TraR and SinI-SinR(-ExpR) include plasmid transfer, production of surface polysaccharides, motility, growth rate and nodulation. These systems are also present in other bacteria of the Sinorhizobium genus, with variations at the species and strain level. In Sinorhizobium fredii NGR234 phenotypes regulated by QS are plasmid transfer, growth rate, sedimentation, motility, biofilm formation, EPS production and copy number of the symbiotic plasmid (pSym). The analysis of the S. fredii HH103 genomes reveal also the presence of both QS systems. In this manuscript we characterized the QS systems of S. fredii HH103, determining that both TraI and SinI AHL-synthases proteins are responsible of the production of short- and long-chain AHLs, respectively, at very low and not physiological concentrations. Interestingly, the main HH103 luxR-type genes, expR and traR, are split into two ORFs, suggesting that in S. fredii HH103 the corresponding carboxy-terminal proteins, which contain the DNA-binding motives, may control target genes in an AHL-independent manner. The presence of a split traR gene is common in other S. fredii strains.

The symbiotic interaction between leguminous plants and their cognate rhizobia allows for the fixation of gaseous dinitrogen into bioavailable ammonia. The perception of host-derived flavonoids is a key initial step for the signaling events that must occur preceding the formation of the nitrogen-fixing organ. Past work investigating chemotaxis – the directed movement of bacteria through chemical gradients – of Bradyrhizobium japonicum, Rhizobium leguminosarum, and Rhizobium meliloti discovered chemotaxis to various organic compounds, but focused on chemotaxis to flavonoids because of their relevance to the symbiosis biochemistry. The current work sought to replicate and further examine Sinorhizobium (Ensifer) meliloti chemotaxis to the flavonoids previously thought to act as the principal attractant molecules prior to the initial signaling stage. Exudate from germinating alfalfa seedlings was analyzed for composition and quantities of different flavonoid compounds using mass spectrometry. The abundance of four prevalent flavonoids in germinating alfalfa seed exudates (SEs) was at a ratio of 200:5:5:1 for hyperoside, luteolin, luteolin-7-glucoside, and chrysoeriol. Using quantitative chemotaxis capillary assays, we did not detect chemotaxis of motile S. meliloti cells to these, and two other flavonoids identified in seed exudates. In support of these findings, the flavonoid fraction of seed exudates was found to be an insignificant attractant relative to the more hydrophilic fraction. Additionally, we observed that cosolvents commonly used to dissolve flavonoids confound the results. We propose that the role flavonoids play in S. meliloti chemotaxis is insignificant relative to other components released by alfalfa seeds.

Results GunA 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, GunAwas also detected in soybean and cowpea developed nodules. Finally, nodulation assays indicate that GunA is beneficial for symbiosis with soybean but detrimental with cowpea. Conclusion Secretion 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

The broad bean (Vicia faba L.) originated in the Near East, and is cultivated around the world, however, its cultivation is affected by drought stress in several central growing regions of the globe. The present study was designed to determine the effect of biochar on bean plant growth, acquisition of nitrogen (N), phosphorus (P), and potassium (K) and on soil nutrient contents under drought and irrigated conditions. Pyrolysis char from maize (MBC) at 2 and 4% concentrations was used for pot experiments. The shoot and/or root biomass of bean grown in soil amended with 2 and 4% MBC under irrigated condition was increased. Furthermore, increased nodule numbers of bean grown at 4% MBC amendment was observed under both irrigated and drought conditions. P and K uptake of plants under drought conditions increased by 14% and 23% under 2% MBC amendment, and by 23% and 34% under 4% MBC amendment as compared to plants grown without biochar application, respectively. This study demonstrated beneficial effects of biochar produced from maize on growth and nutrient uptake of broad bean, by improving the nodule formation and soil nutritional contents in a sandy loam soil., Peer Reviewed

Cowpea (Vigna unguiculata) is the most important leguminous crop raised as pulse as well as a vegetable crop in India. The slow initial growth and wider spacing necessitate weed control in the early stages of crop growth. The critical period of crop weed competition in cowpea was 20 to 30 DAS. Season-long crop weed competition resulted in a yield reduction of 40 to 90 per cent in cowpea depending on the intensity of weed infestation, weed flora, season, location, and the climatic condition prevailed. Mulching, manual weeding and stale seedbed are the different cultural methods which can be adopted for weed control in cowpea. Chemical method of weed control is the cheapest and economically viable option for weed control in cowpea. Both Pre-emergence herbicides viz., pendimethalin, diclosulam and imazethapyr and post-emergence herbicides viz., imazethapyr, quizalofop-p-ethyl can be used for weed control in cowpea. Studies revealed that pre-emergence application of herbicide (imazethapyr/pendimethalin or diclosulam) followed by manual weeding or post-emergence application of herbicides (quizalofop-p-ethyl or imazethapyr) or mulching followed by post-emergence application of herbicides (quizalofop-p-ethyl or imazethapyr) were found to be more effective for the broad-spectrum weed control in cowpea. The integrated approach of weed control has resulted in a higher yield, net returns and B: C ratio in cowpea.

Plants and arbuscular mycorrhizal fungi (AMF) engage in mutually beneficial symbioses based on a reciprocal exchange of nutrients. The beneficial character of the symbiosis is maintained through a mechanism called autoregulation of mycorrhization (AOM). AOM includes root-to-shoot-to-root signaling; however, the molecular details of AOM are poorly understood. AOM shares many features of autoregulation of nodulation (AON) where several genes are known, including the receptor-like kinase SUPER NUMERIC NODULES (SUNN), root-to-shoot mobile CLAVATA3/ENDOSPERM SURROUNDING REGION (ESR)-RELATED (CLE) peptides, and the hydroxyproline O-arabinosyltransferase ROOT DETERMINED NODULATION1 (RDN1) required for post-translational peptide modification. In this work, CLE53 was identified to negatively regulate AMF symbiosis in a SUNN- and RDN1-dependent manner. CLE53 expression was repressed at low phosphorus, while it was induced by AMF colonization and high phosphorus. CLE53 overexpression reduced AMF colonization in a SUNN- and RDN1 dependent manner, while cle53, rdn1, and sunn mutants were more colonized than the wild type. RNA-sequencing identified 700 genes with SUNN-dependent regulation in AMFcolonized plants, providing a resource for future identification of additional AOM genes. Disruption of AOM genes in crops potentially constitutes a novel route for improving AMF-derived phosphorus uptake in agricultural systems with high phosphorus levels.

Leucaena leucocephala is a Mimosoid legume tree indigenous to America that has spread to other continents, although it is not still present in some European countries such as Portugal. Nevertheless, we found that this legume can be nodulated in this country by slow-growing rhizobial strains which were identified as Bradyrhizobium canariense trough the analysis of the core genes recA and glnII. The analysis of the symbiotic gene nodC showed that these strains belong to the symbiovar genistearum, which commonly nodulates Genistoid legumes. Although two strains nodulating L. leucocephala in China and Brazil were classified within the genus Bradyrhizobium, they belong to undescribed species and to the symbiovars glycinearum and tropici, respectively. Therefore, we report here for the first time the ability of L. leucocephala to establish symbiosis with strains of B. canariense sv genistearum confirming the high promiscuity of L. leucocephala, that allows it to establish symbiosis with rhizobia native to different continents increasing its invasiveness potential.

© 2020 The Authors Mesorhizobium is a genus of soil bacteria, some isolates of which form an endosymbiotic relationship with diverse legumes of the Loteae tribe. The symbiotic genes of these mesorhizobia are generally carried on integrative and conjugative elements termed symbiosis islands (ICESyms). Mesorhizobium strains that nodulate Lotus spp. have been divided into host-range groupings. Group I (GI) strains nodulate L. corniculatus and L. japonicus ecotype Gifu, while group II (GII) strains have a broader host range, which includes L. pedunculatus. To identify the basis of this extended host range, and better understand Mesorhizobium and ICESym genomics, the genomes of eight Mesorhizobium strains were completed using hybrid long- and short-read assembly. Bioinformatic comparison with previously sequenced mesorhizobia genomes indicated host range was not predicted by Mesorhizobium genospecies but rather by the evolutionary relationship between ICESym symbiotic regions. Three radiating lineages of Loteae ICESyms were identified on this basis, which correlate with Lotus spp. host-range grouping and have lineage-specific nod gene complements. Pangenomic analysis of the completed GI and GII ICESyms identified 155 core genes (on average 30.1 % of a given ICESym). Individual GI or GII ICESyms carried diverse accessory genes with an average of 34.6 % of genes unique to a given ICESym. Identification and comparative analysis of NodD symbiotic regulatory motifs - nod boxes - identified 21 branches across the NodD regulons. Four of these branches were associated with seven genes unique to the five GII ICESyms. The nod boxes preceding the host-range gene nodZ in GI and GII ICESyms were disparate, suggesting regulation of nodZ may differ between GI and GII ICESyms. The broad host-range determinant(s) of GII ICESyms that confer nodulation of L. pedunculatus are likely present amongst the 53 GII-unique genes identified.

Organisms rely on symbiotic associations for metabolism, protection, and energy. However, these intimate partnerships can be vulnerable to exploitation. What prevents microbial mutualists from parasitizing their hosts? In legumes, there is evidence that hosts have evolved sophisticated mechanisms to manage their symbiotic rhizobia, but the generality and evolutionary origins of these control mechanisms are under debate. Here, we focused on the symbiosis between Parasponia hosts and N2-fixing rhizobium bacteria. Parasponia is the only non-legume lineage to have evolved a rhizobial symbiosis and thus provides an evolutionary replicate to test how rhizobial exploitation is controlled. A key question is whether Parasponia hosts can prevent colonization of rhizobia under high nitrogen conditions, when the contribution of the symbiont becomes nonessential. We grew Parasponia andersonii inoculated with Bradyrhizobium elkanii under four ammonium nitrate concentrations in a controlled growth chamber. We measured shoot and root dry weight, nodule number, nodule fresh weight, nodule volume. To quantify viable rhizobial populations in planta, we crushed nodules and determined colony forming units (CFU), as a rhizobia fitness proxy. We show that, like legumes and actinorhizal plants, P. andersonii is able to control nodule symbiosis in response to exogenous nitrogen. While the relative host growth benefits of inoculation decreased with nitrogen fertilization, our highest ammonium nitrate concentration (3.75 mM) was sufficient to prevent nodule formation on inoculated roots. Rhizobial populations were highest in nitrogen free medium. While we do not yet know the mechanism, our results suggest that control mechanisms over rhizobia are not exclusive to the legume clade.

Nitrogen‐fixing nodulation occurs in ten taxonomic lineages, either with rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume‐rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF‐Y) complex.It is hypothesized that nodulation has a single evolutionary origin ~ 110 million years ago, followed by many independent losses. Despite a significant body of knowledge of the legume‐rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. We used Parasponia andersonii to investigate the role of NIN and NF‐YA genes in rhizobium nodulation in a non‐legume system.Consistent with legumes, P. andersonii PanNIN and PanNF‐YA1 are co‐expressed in nodules. By analyzing single, double and higher‐order CRISPR‐Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF‐YA1 are PanNIN‐dependent and that PanNF‐YA1 is specifically required for intracellular rhizobium infection.This demonstrates that NIN and NF‐YA1 commit conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF‐YA1 represent core transcriptional regulators in this symbiosis.

In the late 19th century, it was discovered that legumes can establish a root nodule endosymbiosis with nitrogen-fixing rhizobia. Soon after, the question was raised whether it is possible to transfer this trait to non-leguminous crops. In the past century, an ever-increasing amount of knowledge provided unique insights into the cellular, molecular, and genetic processes controlling this endosymbiosis. In addition, recent phylogenomic studies uncovered several genes that evolved to function specifically to control nodule formation and bacterial infection. However, despite this massive body of knowledge, the long-standing objective to engineer the nitrogen-fixing nodulation trait on non-leguminous crop plants has not been achieved yet. In this review, the unsolved questions and engineering strategies toward nitrogen-fixing nodulation in non-legume plants are discussed and highlighted.

The Vigna unguiculata (L.) Walp crop is one of the most important legumes due to its potential as food and fodder in agroecosystems, in addition to being considered a drought tolerant crop and with the ability to grow in poor soils. In the current context of Cuban agriculture, the search for fertilization alternatives with chemical reduction is a new and necessary issue for the sustainability of agroecosystems. In the investigation, the effect of the doses and timing of application of the FerKiASerT biopreparation, an agroecological alternative for fertilizing the cowpea bean crop cv. INIFAT 93. A design with factorial structure was used, considering as factors: dose and time of application. The results to highlight were obtained with doses of 50 and 75 kg / ha of the biopreparation, in addition, with the application of 75 kg / ha of FerKiASerT, a significant increase in nodulation due to rhizobacteria was achieved., El cultivo Vigna unguiculata (L.) Walp es una de las leguminosas más importantes debido a sus potencialidades como alimento y forraje en los agroecosistemas, además de considerarse un cultivo tolerante a las sequías y con capacidad para crecer en suelos pobres. En el contexto actual de la agricultura cubana, la búsqueda de alternativas de fertilización con reducción de productos químicos constituye un tema novedoso y necesario para la sostenibilidad de los agroecosistemas. En la investigación, se determinó el efecto de las dosis y momento de aplicación del biopreparado FerKiASerT, una alternativa agroecológica para la fertilización del cultivo de frijol caupí cv. INIFAT 93. Se empleó un diseño con estructura factorial, considerando como factores: dosis y momento de aplicación. Los resultados ha destacar se obtuvieron con dosis de 50 y 75 kg/ha del biopreparado, además, con la aplicación de 75 kg/ha de FerKiASerT, se logró un incremento significativo en la nodulación por rizobacterias.

В обзоре приведены данные о наиболее распространенных видах тиреоидной патологии — субклиническом, манифестном гипотиреозе (особенно у лиц пожилого возраста) и гипотиреозе как осложнении после проведения гемитиреоидэктомии. Приведены данные по послеоперационному гипотиреозу (субклиническому и манифестному): частота возникновения, диагностирование после операции, факторы, влияющие на развитие после­операционного гипотиреоза, среди основных — предоперационный уровень тиреотропного гормона, возраст и наличие антитиреоидных антител. Также проанализированы риски узлообразования в викарной доле щитовидной железы после гемитиреоидэктомии. В целом в краткосрочной перспективе развитие узлов в остаточной доле не отмечалось., В огляді наведені дані про найбільш поширені види тиреоїдної патології — субклінічний, маніфестний гіпотиреоз (особливо в осіб похилого віку) і гіпотиреоз як ускладнення після проведення гемітиреоїдектомії. Наведено дані щодо післяопераційного гіпотиреозу (субклінічного й маніфестного): частота виникнення, час діагностування після операції, фактори, що впливають на розвиток післяопераційного гіпотиреозу, серед основних — передопераційний рівень тиреотропного гормону, вік і наявність антитирео­їдних антитіл. Також були проаналізовані ризики вузлоутворення у вікарній частці щитоподібної залози після гемітиреоїдектомії. У цілому в короткостроковій перспективі розвиток вузлів у залишковій частці не відзначався., The review presents data of the most common complication after hemithyroidectomy — hypothyroidism, as well as postoperative hypothyroidism: its subclinical and manifest forms, incidence, the time of occurrence after the surgery, factors affecting the development of postoperative hypothyroidism, among the main ones — the preoperative level of thyroid-stimulating hormone, the age and presence of antithyroid antibodies. The risks of nodulation in the vicarious part of the thyroid gland after hemithyroidectomy were also analyzed. In general, there was no nodule development in the remaining lobe in the short-term period.

Nitrogen (N) in soybean (Glycine maxL.) plants derived from biological nitrogen fixationwas shown to be a sustainable N resource to substitute for N fertilizer. However, the limitedwater supply in sandy soil is a critical factor for soybean nodulation and crop growth. This studyinvestigated the potential mechanism of the effect of biochar and irrigation on the soybean-Rhizobiumsymbiotic performance and soil biological activity in a field trial. In the absence of N fertilizer,10 t ha−1of black cherry wood-derived biochar were applied under irrigated and rainfed conditionson an experimental, sandy field site. The plant biomass, plant nutrient concentrations, nodulenumber, nodule leghemoglobin content, soil enzyme activities, and soil-available nutrients wereexamined. Our results show that biochar application caused a significant increase in the nodulenumber by 35% in the irrigated condition. Shoot biomass and soil fluorescein diacetate hydrolyticactivity were significantly increased by irrigation in comparison to the rainfed condition. The activityof soil protease reduced significantly, by 8%, with the biochar application in the irrigated condition.Further, a linear correlation analysis and redundancy analysis performed on the plant, nodule, andsoil variables suggested that the biochar application may affect soybean N uptake in the sandy field.Nodulation was enhanced with biochar addition, however, the plant N concentration and nodule Lbcontent remained unaffected., Peer Reviewed

Increasing awareness to global climate change has drastically focused attention on finding solutions to reduce environmental impacts while still providing sufficient food for the increasing world population. Beneficial Nitrogen Fixing (BNF) microbes provide a possible solution by delivering biological nitrogen to plants resulting in reduced environmental impacts due to fertilizer runoff and eutrophication. One well studied model is that of Sinorhizobium meliloti and its legume host Medicago sativa (alfalfa), the fourth largest USA crop used for animal feed. Advancing research for this symbiosis model can provide solutions to enhance yield while minimizing environmental impacts. Chapter 2 focuses on the deviation of the S. meliloti chemotaxis system from the enteric paradigm. Quantitative immunoblots determined the cellular amounts of chemotaxis proteins. Overall, chemotaxis protein levels were approximately 10-fold lower in S. meliloti compared to B. subtilis and E. coli. Focusing on cellular stoichiometric ratios, S. meliloti generally exhibits drastically higher values for CheB, CheR, and CheY to the histidine kinase CheA monomer compared to E. coli and B. subtilis. Chapter 3 characterizes the role of McpX to quaternary ammonium compound (QAC) sensing. QACs are exuded by germinating alfalfa seeds. In vitro binding assays were performed to determine ligand binding characteristics. S. meliloti chemotaxis behavior to QACs was analyzed in in vivo capillary assays under real-time imaging. These studies strengthen our knowledge of the chemotaxis system in the symbiosis model of S. meliloti and alfalfa. The data can further be used to create a mathematical model of the dynamics of bacteria-host interaction. The results can be used to optimize chemotaxis to host plants to improve crop yield and protect watersheds.

Aims The 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. Methods A 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.

Sinorhizobium fredii HH103 RifR, a broad-host-range rhizobial strain, forms ineffective nodules with Lotus japonicus but induces nitrogen-fixing nodules in Lotus burttii roots that are infected by intercellular entry. Here we show that HH103 RifR nolR or nodD2 mutants gain the ability to induce infection thread formation and to form nitrogen-fixing nodules in L. japonicus Gifu. Microscopy studies showed that the mode of infection of L. burttii roots by the nodD2 and nolR mutants switched from intercellular entry to infection threads (ITs). In the presence of the isoflavone genistein, both mutants overproduced Nod-factors. Transcriptomic analyses showed that, in the presence of Lotus japonicus Gifu root exudates, genes related to Nod factors production were overexpressed in both mutants in comparison to HH103 RifR. Complementation of the nodD2 and nolR mutants provoked a decrease in Nod-factor production, the incapacity to form nitrogen-fixing nodules with L. japonicus Gifu and restored the intercellular way of infection in L. burttii. Thus, the capacity of S. fredii HH103 RifR nodD2 and nolR mutants to infect L. burttii and L. japonicus Gifu by ITs and fix nitrogen L. japonicus Gifu might be correlated with Nod-factor overproduction, although other bacterial symbiotic signals could also be involved

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

We studied symbiotic performance of factorial combinations of diverse rhizobial genotypes (GR) and East African common bean varieties (GL) that comprise Andean and Mesoamerican genetic groups. An initial wide screening in modified Leonard jars (LJ) was followed by evaluation of a subset of strains and genotypes in pots (contained the same, sterile medium) in which fixed nitrogen was also quantified. An additive main effect and multiplicative interaction (AMMI) model was used to identify the contribution of individual strains and plant genotypes to the GL × GR interaction. Strong and highly significant GL × GR interaction was found in the LJ experiment but with little evidence of a relation to genetic background or growth habits. The interaction was much weaker in the pot experiment, with all bean genotypes and Rhizobium strains having relatively stable performance. We found that R. etli strain CFN42 and R. tropici strains CIAT899 and NAK91 were effective across bean genotypes but with the latter showing evidence of positive interaction with two specific bean genotypes. This suggests that selection of bean varieties based on their response to inoculation is possible. On the other hand, we show that symbiotic performance is not predicted by any a priori grouping, limiting the scope for more general recommendations. The fact that the strength and pattern of GL × GR depended on growing conditions provides an important cautionary message for future studies.IMPORTANCE The existence of genotype-by-strain (GL × GR) interaction has implications for the expected stability of performance of legume inoculants and could represent both challenges and opportunities for improvement of nitrogen fixation. We find that significant genotype-by-strain interaction exists in common bean (Phaseolus vulgaris L.) but that the strength and direction of this interaction depends on the growing environment used to evaluate biomass. Strong genotype and strain main effects, combined with a lack of

Simultaneous quantification of transcripts of the whole bacterial genome allows the analysis of the global transcriptional response under changing conditions. RNA-seq and microarrays are the most used techniques to measure these transcriptomic changes, and both complement each other in transcriptome profiling. In this review, we exhaustively compiled the symbiosis-related transcriptomic reports (microarrays and RNA sequencing) carried out hitherto in rhizobia. This review is specially focused on transcriptomic changes that takes place when five rhizobial species, Bradyrhizobium japonicum (=diazoefficiens) USDA 110, Rhizobium leguminosarum biovar viciae 3841, Rhizobium tropici CIAT 899, Sinorhizobium (=Ensifer) meliloti 1021 and S. fredii HH103, recognize inducing flavonoids, plant-exuded phenolic compounds that activate the biosynthesis and export of Nod factors (NF) in all analysed rhizobia. Interestingly, our global transcriptomic comparison also indicates that each rhizobial species possesses its own arsenal of molecular weapons accompanying the set of NF in order to establish a successful interaction with host legumes.