Inoculation of Native Arbuscular Mycorrhizae and Bacillus subtilis Can Improve Growth in Vegetable Crops.

Arbuscular mycorrhizal fungi (AMF) and some rhizobacteria are known as plant growth-promoting microorganism (PGPM) as they play significant roles in improving soil fertility structure, plant nutrition, growth, and health. However, little is known about the PGPM potential of AMF and rhizobacteria native to the Rift Valley and highland regions of Ethiopia. Hence, this study aimed to investigate the PGPM effect of single and co-inoculation of AMF and the Bacillus subtilis ALCR46 strain, on tomato (Lycopersicum esculentum L.), onion (Allium cepa L.), and squash (Cucurbita pepo L.) plants. The experimental setup was a randomized complete block design with three replications of the following treatments: (i) inoculation with a consortium of AMF, (ii) co-inoculation with a consortium of AMF and the Bacillus subtilis, (iii) inoculation with Rhizophagus clarus, (iv) co-inoculation with R. clarus and B. subtilis, (v) inoculation with B. subtilis, (vi) plants without inoculation (negative control), and (vii) plants treated with chemical fertilizer (positive control). Plants were maintained in a greenhouse for 60 days, and after harvest, plant growth parameters, percentage of AMF root colonization, and spore number were analyzed. The result shows that the growth of crops significantly increased by co-inoculation with the consortium of AMF and B. subtilis. AMF spore density and root colonization rate were also increased in co-inoculated plants. Highest root colonization, spore number, and mycorrhizal dependency were observed in A. cepa. Our results suggest that there is a synergistic effect between the AMF and B. subtilis ALCR46, and between AMF inoculants. However, the application of present findings under field conditions is required to be confirmed by further studies.

Inoculation with Azospirillum argentinense Az19 improves the yield of maize subjected to water déficit at key stages of plant development / La inoculación con Azospirillum argentinense Az19 mejora el rendimiento del maíz expuesto a déficit hídrico durante estadios clave del desarrollo vegetal

Resumen El déficit hídrico constituye una severa limitación a la productividad agrícola. En el marco de la producción sostenible de cultivos, la biotecnología microbiana está cobrando relevancia para aumentar la tolerancia a la sequía y mejorar el rendimiento de los cultivos en condiciones adversas. El propósito de este trabajo fue comparar la acción de la cepa de Azospirillum argentinense Az19, con tolerancia in vitro a estresores abióticos, con la cepa Az39, utilizada ampliamente para la formulación de inoculantes comerciales, al inocularlas en plantas sometidas a déficit hídrico. Se realizaron ensayos de invernadero y de campo. En invernadero, la cepa Az19 evitó el impacto adverso del déficit hídrico en el estadio V2 sobre el crecimiento del maíz. Además, el porcentaje de plantas con espigas y el peso de la espiga disminuyó significativamente con la restricción hídrica aplicada en V2 y en floración en plantas inoculadas con la cepa Az39, pero no en las inoculadas con Az19. En el primer ensayo de campo con el maíz híbrido comercial DOW DS 515 PW las plantas inoculadas con Az19 fueron las que mejor toleraron la deficiencia hídrica. En el segundo ensayo de campo se utilizaron dos genotipos de maíz con sensibilidad diferencial a la sequía. La inoculación con Az19 condujo a una mayor tolerancia al déficit hídrico, con un efecto detectable en algunos componentes del rendimiento en el genotipo sensible. Sobre la base de estos resultados, proponemos el empleo de A. argentinense Az19 para la formulación de inoculantes basados en Azospirillum especialmente indicados para áreas agroecológicas que experimenten períodos de déficit hídrico.

Genome sequence of Mesorhizobium mediterraneum strain R31, a nitrogen-fixing rhizobium used as an inoculant for chickpea in Argentina.

Here, we report the complete genome sequence of Mesorhizobium mediterraneum R31, a rhizobial strain recommended and used as a commercial inoculant for chickpea in Argentina. The genome consists of 7.25 Mb, distributed into four circular replicons: a chromosome of 6.72 Mbp and three plasmids of 0.29, 0.17, and 0.07 Mbp.

Inoculation with Azospirillum argentinense Az19 improves the yield of maize subjected to water deficit at key stages of plant development.

Water deficit constitutes a severe limitation to agricultural productivity. In the context of sustainable crop production, the potential of microbial biotechnology to increase plant drought tolerance and improve crop yields under adverse conditions is gaining relevance. This work aimed to compare the performance of Azospirillumargentinense strain Az19 to that of strain Az39, the most widely used for commercial inoculants, when inoculated in maize plants exposed to water deficit. For this purpose, greenhouse and field assays were conducted. In the greenhouse experiment, strain Az19 prevented the adverse effect of water deficit at V2 stage on maize growth. Moreover, the percentage of fertile plants and the ear weight decreased significantly under water deficits imposed at V2 and flowering in Az39-inoculated plants but not in Az19-inoculated plants. In the first field trial with the commercial maize hybrid DOW DS 515 PW, Az19-inoculated plants were those which better tolerated the water deficit imposed. In the second field trial, two maize genotypes with differential drought sensitivity (LP 29×LP 2542, sensitive; LP 882 (923)×LP 4703, tolerant) were tested. Higher tolerance to water deficit was detected in plants inoculated with A. argentinense Az19, with a noticeable effect on grain yield components in the sensitive genotype. Based on these results, we propose the use of A. argentinense Az19 for the formulation of more targeted Azospirillum-based inoculants, suitable for agroecological areas subjected to seasonal water deficits.

Complete Genome Sequence of Mesorhizobium ciceri Strain R30, a Rhizobium Used as a Commercial Inoculant for Chickpea in Argentina.

We report the complete genome sequence of Mesorhizobium ciceri strain R30, a rhizobium strain recommended and used as a commercial inoculant for chickpea in Argentina. The genome consists of almost 7 Mb, distributed into two circular replicons: a chromosome of 6.49 Mb and a plasmid of 0.46 Mb.

Genome-based reclassification of Azospirillum brasilense Az39 as the type strain of Azospirillum argentinense sp. nov.

Strain Az39T of Azospirillum is a diazotrophic plant growth-promoting bacterium isolated in 1982 from the roots of wheat plants growing in Marcos Juárez, Córdoba, Argentina. It produces indole-3-acetic acid in the presence of l-tryptophan as a precursor, grows at 20-38 °C (optimal 38 °C), and the cells are curved or spiral-shaped, with diameters ranging from 0.5-0.9 to 1.8-2.2 µm. They contain C16 : 0, C18 : 0 and C18 : 1 ω7c/ω6c as the main fatty acids. Phylogenetic analysis of its 16S rRNA gene sequence confirmed that this strain belongs to the genus Azospirillum, showing a close relationship with Azospirillum baldaniorum Sp245T, Azospirillum brasilense Sp7T and Azospirillum formosense CC-Nfb-7T. Housekeeping gene analysis revealed that Az39T, together with five strains of the genus (Az19, REC3, BR 11975, MTCC4035 and MTCC4036), form a cluster apart from A. baldaniorum Sp245T, A. brasilense Sp7T and A. formosense CC-Nfb-7T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between Az39T and the aforementioned type strains revealed values below 96 %, the circumscription limit for the species delineation (ANI: 95.3, 94.1 and 94.0 %; dDDH: 62.9, 56.3 and 55.6 %). Furthermore, a phylogeny evaluation of the core proteome, including 809 common shared proteins, showed an independent grouping of Az39T, Az19, REC3, BR 11975, MTCC4035 and MTCC4036. The G+C content in the genomic DNA of these six strains varied from 68.3 to 68.5 %. Based on the combined phylogenetic, genomic and phenotypic characterization presented here, we consider that strain Az39T, along with strains Az19, REC3, BR 11975, MTCC4035 and MTCC4036, are members of a new Azospirillum species, for which the name Azospirillum argentinense sp. nov. is proposed. The type strain is Az39T (=LBPCV39T=BR 148428T=CCCT 22.01T).

Complete Genome Sequence of Bradyrhizobium sp. Strain C-145, a Nitrogen-Fixing Rhizobacterium Used as a Peanut Inoculant in Argentina.

We present the complete genome sequence of Bradyrhizobium sp. strain C-145, one of the most widely used nitrogen-fixing rhizobacteria for inoculating peanut crops in Argentina. The genome consists of 9.53 Mbp in a single circular chromosome and was determined using a hybrid long- and short-read assembly approach.

The drop plate method as an alternative for Azospirillum spp viable cell enumeration within the consensus protocol of the REDCAI network

Abstract Quality evaluation of commercial inoculants is essential to warrant an adequate cropresponse to inoculation within a biosecurity framework. In this sense, this work is aimed at standardizing and validating the drop plate method for the enumeration of Azospirillum viable cellsas an alternative to the spread plate technique, which is currently proposed in the consensusprotocol of the REDCAI network. Between 14 and 25 private and public laboratories partici-pated in three independent trials. We obtained consistent and robust results that allowed toconfirm that both techniques are equivalent, concluding that the drop plate method is an alternative enumeration technique that is adequate to be included in the abovementioned consensusprotocol.

Resumen La evaluación de la calidad de los inoculantes comerciales es fundamental para garantizar una adecuada respuesta de los cultivos a la inoculación dentro de un marco de bioseguridad. En este sentido, el objetivo de este trabajo fue la estandarización y validación de la técnica de la microgota para la cuantificación de Azospirillum como metodología alternativa a la técnica de siembra en superficie, propuesta actualmente en el protocolo consenso de la Red de Calidad de Inoculantes, REDCAI. Entre 14 y 25 laboratorios, tanto privados como públicos, participaron de tres ensayos independientes. A partir de ellos se obtuvieron resultados reproducibles y robustos que permiten confirmar que ambas técnicas son equivalentes y concluir que la técnica de recuento por la microgota es una alternativa adecuada para ser incluida dentro del mencionado protocolo consenso.

The drop plate method as an alternative for Azospirillum spp. viable cell enumeration within the consensus protocol of the REDCAI network.

Quality evaluation of commercial inoculants is essential to warrant an adequate crop response to inoculation within a biosecurity framework. In this sense, this work is aimed at standardizing and validating the drop plate method for the enumeration of Azospirillum viable cells as an alternative to the spread plate technique, which is currently proposed in the consensus protocol of the REDCAI network. Between 14 and 25 private and public laboratories participated in three independent trials. We obtained consistent and robust results that allowed to confirm that both techniques are equivalent, concluding that the drop plate method is an alternative enumeration technique that is adequate to be included in the abovementioned consensus protocol.

Desarrollo de marcadores moleculares del tipo SCAR para la identificación de Azospirillum brasilense Az39 / Development of sequence characterized amplified región markers for identification of Azospirillum brasilense Az39

Resumen Azospirillum brasilense Az39 es utilizada por empresas productoras de inoculantespara la formulación de bioinsumos en América del Sur desde hace más de 30 a˜nos. Esta cepapuede promover el crecimiento, desarrollo, así como la capacidad de tolerar diferentes tiposde estrés en las plantas inoculadas, lo que determina un aumento de la productividad de culti-vos de interés agronómico. En la actualidad, no existen protocolos en Argentina que permitanconfirmar la identidad de Az39 en productos comerciales a nivel de laboratorios de control decalidad de inoculantes. Por ello, el objetivo de este trabajo fue desarrollar una metodología enbase molecular que permita la identificación certera de A. brasilense Az39. Con la secuenciacompleta del genoma y mediante herramientas bioinformáticas, se pudieron reconocer frag-mentos de ADN presentes únicamente en el genoma de Az39. Se dise˜naron cebadores dirigidosa amplificar por PCR dichas secuencias. Como resultado se observaron los productos específicosúnicamente en la presencia de la cepa de interés. La reacción pudo detectar un título mínimode 105UFC/ml (4,5 ng/l ADN) o de 102UFC/ml (0,88 ng/l ADN) o una concentración mínimade 0,098 ng/l ADN, dependiendo del método de extracción utilizado. Los cebadores fueronevaluados en el análisis de productos comerciales obtenidos del mercado nacional, arrojandoresultados positivos, tanto en muestras directas como así también en pruebas confirmatoriasa partir de colonias aisladas de tales productos. La metodología desarrollada en este trabajo,permite la detección certera de A. brasilense Az39 en cultivos puros o mezclas complejas demicroorganismos.

Abstract Azospirillum brasilense Az39 has been used since more than 30 years by several companies in South America for biofertilizers production. This strain may promote plants growth and development, as well as the ability of inoculated plants to tolerate environmental stresses, which determines an increase in the productivity under field conditions. At present, there are no protocols in Argentina to confirm the identity of Az39 in commercial products; however, such biofertilizers are formulated almost exclusively with this strain. Therefore, the objective of this paper was to develop a molecular methodology that allows the accurate identification of A. brasilense Az39. Using the complete genome sequence and several bioinformatics tools, fragments of DNA present only in the Az39 genome were recognized. A set of PCR primers to amplify these sequences were designed, and the specific products were observed only in the strain of our interest. The sensitivity of the methodology was evaluated, where the strain could be detected up to a titer of 105 CFU/ml (4.5 ng/pl ADN) or 102 CFU/ml (0.88 ng/pl DNA) or in a minimal concentration of 0.098 ng/pl DNA, depending on the DNA extraction methodology used. Primers were tested against direct samples of commercial inoculants and cultures, in both cases there were specifics products, both in direct samples and in confirmatory tests from isolated colonies from those products. The procedure presented in this paper allows the accurate identification of A. brasilense Az39 in pure cultures, mixtures of microorganisms, and commercial biofertilizers.

[Development of sequence characterized amplified region markers for identification of Azospirillum brasilense Az39]. / Desarrollo de marcadores moleculares del tipo SCAR para la identificación de Azospirillum brasilense Az39.

Azospirillum brasilense Az39 has been used since more than 30 years by several companies in South America for biofertilizers production. This strain may promote plants growth and development, as well as the ability of inoculated plants to tolerate environmental stresses, which determines an increase in the productivity under field conditions. At present, there are no protocols in Argentina to confirm the identity of Az39 in commercial products; however, such biofertilizers are formulated almost exclusively with this strain. Therefore, the objective of this paper was to develop a molecular methodology that allows the accurate identification of A. brasilense Az39. Using the complete genome sequence and several bioinformatics tools, fragments of DNA present only in the Az39 genome were recognized. A set of PCR primers to amplify these sequences were designed, and the specific products were observed only in the strain of our interest. The sensitivity of the methodology was evaluated, where the strain could be detected up to a titer of 105 CFU/ml (4.5 ng/µl ADN) or 102 CFU/ml (0.88 ng/µl DNA) or in a minimal concentration of 0.098 ng/µl DNA, depending on the DNA extraction methodology used. Primers were tested against direct samples of commercial inoculants and cultures, in both cases there were specifics products, both in direct samples and in confirmatory tests from isolated colonies from those products. The procedure presented in this paper allows the accurate identification of A. brasilense Az39 in pure cultures, mixtures of microorganisms, and commercial biofertilizers.

Stable symbiotic nitrogen fixation under water-deficit field conditions by a stress-tolerant alfalfa microsymbiont and its complete genome sequence.

We here characterized the stress-tolerant alfalfa microsymbiont Sinorhizobium meliloti B401. B401-treated plants showed high nitrogen fixation rates under humid and semiarid environments. The production of glycine betaine in isolated bacteroids positively correlated with low precipitation levels, suggesting that this compound acts as a critical osmoprotectant under field conditions. Genome analysis revealed that strain B401 contains alternative pathways for the biosynthesis and uptake of glycine betaine and its precursors. Such genomic information will offer substantial insight into the environmental physiology of this biotechnologically valuable nitrogen-fixing bacterium.

Microevolution Rather than Large Genome Divergence Determines the Effectiveness of Legume-Rhizobia Symbiotic Interaction Under Field Conditions.

Despite the vast screening for natural nitrogen-fixing isolates by public and private consortia, no significant progresses in the production of improved nitrogen-fixing inoculants for alfalfa production have been made in the last years. Here, we present a comprehensive characterization of the nitrogen-fixing strain Ensifer meliloti B399 (originally named Rhizobium meliloti 102F34), probably the inoculant most widely used in alfalfa production since the 1960s. Complete nucleotide sequence and genome analysis of strain B399 showed that the three replicons present in this commercial strain and the model bacterium Ensifer meliloti 1021 are extremely similar to each other in terms of nucleotide identity and synteny conservation. In contrast to that observed in B399-treated plants, inoculation of plants with strain 1021 did not improve nitrogen content in different alfalfa cultivars under field conditions, suggesting that a small genomic divergence can drastically impact on the symbiotic phenotype. Therefore, in addition to the traditional screening of natural nitrogen-fixing isolates, the genome engineering of model strains could be an attractive strategy to improve nitrogen fixation in legume crops.

Complete Genome Sequence of the Model Rhizosphere Strain Azospirillum brasilense Az39, Successfully Applied in Agriculture.

We present the complete genome sequence of Azospirillum brasilense Az39, isolated from wheat roots in the central region of Argentina and used as inoculant in extensive and intensive agriculture during the last four decades. The genome consists of 7.39 Mb, distributed in six replicons: one chromosome, three chromids, and two plasmids.