Microbial inoculants enhance the persistence of antibiotic resistance genes in aerobic compost of food waste mainly by altering interspecific relationships.

The effects of microbial inoculants on ARG removal in composting are poorly understood. Here, a co-composting method for food waste and sawdust amended with different microbial agents (MAs) was designed. The results show that the compost without MA unexpectedly achieved the best ARG removal. The addition of MAs markedly increased the abundance of tet, sul and multidrug resistance genes (p < 0.05). Structural equation modeling demonstrated that MAs can enhance the contribution of the microbial community to ARG changes by reshaping community structure and altering the ecological niche, causing the proliferation of individual ARGs, an effect related to the MA component. Network analysis revealed that inoculants weakened the correlation between ARGs and community but increased the linkage between ARGs and core species, suggesting that inoculant-induced ARG proliferation may correspond with gene exchange occurring mainly between core species. The outcome provides new insights into MA application for ARG removal in waste treatment.

Isolation and Characterization of Phosphate Solubilizing Bacteria from Paddy Field Soils in Japan.

Phosphorus (P) is abundant in soil and is essential for plant growth and development; however, it is easily rendered insoluble in complexes of different types of phosphates, which may lead to P deficiency. Therefore, increases in the amount of P released from phosphate minerals using microbial inoculants is an important aspect of agriculture. The present study used inorganic phosphate solubilizing bacteria (iPSB) in paddy field soils to develop microbial inoculants. Soils planted with rice were collected from different regions of Japan. Soil P was sequentially fractionated using the Hedley method. iPSB were isolated using selective media supplemented with tricalcium phosphate (Ca-P), aluminum phosphate (Al-P), or iron phosphate (Fe-P). Representative isolates were selected based on the P solubilization index and soil sampling site. Identification was performed using 16S rRNA and rpoB gene sequencing. Effectiveness was screened based on rice cultivar Koshihikari growth supplemented with Ca-P, Al-P, or Fe-P as the sole P source. Despite the relatively homogenous soil pH of paddy field sources, three sets of iPSB were isolated, suggesting the influence of fertilizer management and soil types. Most isolates were categorized as ß-Proteobacteria (43%). To the best of our knowledge, this is the first study to describe the genera Pleomorphomonas, Rhodanobacter, and Trinickia as iPSB. Acidovorax sp. JC5, Pseudomonas sp. JC11, Burkholderia sp. JA6 and JA10, Sphingomonas sp. JA11, Mycolicibacterium sp. JF5, and Variovorax sp. JF6 promoted plant growth in rice supplemented with an insoluble P source. The iPSBs obtained may be developed as microbial inoculants for various soil types with different P fixation capacities.

Integrating 16S rRNA amplicon metagenomics and selective culture for developing thermophilic bacterial inoculants to enhance manure composting.

Composting is an important method for treating and recycling organic waste, and the use of microbial inoculants can increase the efficiency of composting. Herein, we illustrate an approach that integrate 16S rRNA amplicon metagenomics and selective culture of thermophilic bacteria for the development of inoculants to improve manure composting. The 16S rRNA amplicon sequencing analysis revealed that Firmicutes and Actinobacteria were dominant in the composting mixture, and that different microbial hubs succeeded during the thermophilic stage. All isolated thermophilic bacteria were affiliated with the order Bacillales, such as Geobacillus, Bacillus, and Aeribacillus. These isolated thermophilic bacteria were grouped into 11 phylotypes, which shared ＞99% sequence identity to 0.15% to 5.32% of 16S rRNA reads by the amplicon sequencing. Three of these phylotypes transiently enriched during the thermophilic stage. Six thermophilic bacteria were selected from the three phylotypes to obtain seven microbial inoculants. Five out of seven of the microbial inoculants enhanced the thermophilic stage of composting by 16.9% to 52.2%. Three-dimensional excitation emission matrix analysis further revealed that two inoculants (Thermoactinomyces intermedius and Ureibacillus thermophilus) stimulated humification. Additionally, the 16S rRNA amplicon sequencing analysis revealed that inoculation with thermophilic bacteria enhanced the succession of the microbial community during composting. In conclusion, 16S rRNA amplicon metagenomics is a useful tool for the development of microbial inoculants to enhance manure composting.

Efficacy of bioinoculants to control of bacterial and fungal diseases of rice (Oryza sativa L.) in northwestern Himalaya.

INTRODUCTION: Biological control holds great promise for environmentally friendly and sustainable management of the phytopathogens. The multi-function features of plant growth-promoting rhizobacteria (PGPR) enable to protect the plants from disease infections by replacing the chemical inputs. The interaction between the plant root exudates and the microbes stimulates the production of secondary metabolism and enzymes and induces systemic resistance in the plants. AIM: The aim was to identify the potential PGPR which would show an antagonistic effect against basmati rice fungal and bacterial diseases. METHODS: In the study, native originating microbes have been isolated, characterized using 16S rRNA sequencing, and used as potential antagonistic microbial isolates against diseases of rice plants. RESULTS: Rhizobacteria isolated from rhizosphere, endo-rhizosphere, and bulk soil samples of Basmati 370 exhibited promising inhibitory activity against rice pathogens. Molecular characterization of bacterial isolates based on 16S rRNA sequencing classified the bacterial isolates into different genera such as Bacillus, Pseudomonas, Streptomyces, Exiguobacterium, Aeromonas, Chryseobacterium, Enterobacter, and Stenotrophomonas. PGPRs exhibited biocontrol activities against various rice diseases like bacterial leaf blight, leaf blast, brown spot, and sheath blight and boost the plant growth traits. CONCLUSION: In the study, the potentially identified PGPRs isolates could be used as efficient bioinoculants as bio-fertilizers and biocontrol agents for sustainable rice crop production.

Inoculation of Mimosa Pudica with Paraburkholderia phymatum Results in Changes to the Rhizoplane Microbial Community Structure.

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

Integrated use of phosphate-solubilizing Bacillus subtilis strain IA6 and zinc-solubilizing Bacillus sp. strain IA16: a promising approach for improving cotton growth.

Mineral nutrition of crop plants is one of the major challenges faced by modern agriculture, particularly in arid and semi-arid regions. In alkaline calcareous soils, the availability of phosphorus and zinc is critically less due to their fixation and precipitation as complexes. Farmers use fertilizers to fulfill crop requirements, but their efficacy is less, which increases production costs. Plant growth-promoting rhizobacteria (PGPR) can improve the availability of crop nutrients through solubilizing the insoluble compounds of phosphorus and zinc in soil. In the present study, a total of 40 rhizobacterial isolates were isolated from cotton rhizosphere and screened for improving cotton growth through the solubilization of phosphorus and zinc. Out of these 40 isolates, seven isolates (IA2, IA3, IA6, IA7, IA8, IA13, and IA14) efficiently solubilized insoluble rock phosphate while seven isolates (IA10, IA16, IA20, IA23, IA24, IA28, and IA30) were more efficient in solubilizing insoluble zinc oxide. In liquid media, strain IA7 (2.75 µg/mL) solubilized the highest amount of phosphate while the highest concentration of soluble zinc was observed in the broth inoculated with strain IA20 (3.94 µg/mL). Seven phosphate-solubilizing and seven zinc-solubilizing strains were evaluated using jar trial to improve the growth of cotton seedlings, and the results were quite promising. All the inoculated treatments showed improvement in growth parameters in comparison with control. Best results were shown by the combined application of IA6 and IA16, followed by the combination of strains IA7 and IA20. Based on the jar trial, the selected isolates were further characterized by plant growth-promoting characters such as siderophores production, HCN production, ammonia production, and exopolysaccharides production. These strains were identified through 16S rRNA sequencing as Bacillus subtilis IA6 (accession # MN005922), Paenibacillus polymyxa IA7 (accession # MN005923), Bacillus sp. IA16 (accession # MN005924), and Bacillus aryabhattai IA20 (accession # MN005925). It is hence concluded that the integrated use of phosphate-solubilizing and zinc-solubilizing strains as potential inoculants can be a promising approach for improving cotton growth under semi-arid conditions.

Stage-dependent concomitant microbial fortification improves soil nutrient status, plant growth, antioxidative defense system and gene expression in rice.

Stage-dependent concomitant fortification of rice (Oryza sativa L.) varieties PB1612 and CO51 with microbial inoculants Trichoderma asperellum and Pseudomonas fluorescens as seed coating, seedling root inoculation and soil application enhanced growth, activated antioxidant enzymes and modulated defence-related genes in plants. Microbial inoculants improved shoot height, tiller numbers, fresh weight and dry biomass. Co-inoculation was more impactful in enhancing plant growth and development as compared to single inoculation. Single and co-inoculation improved organic carbon (OC) and N, P and K content in the soil substantially. Mean values between control and co-inoculation varied significantly for OC in PB1612 (p0.001) and CO51 (p0.019) and phosphorus content in PB1612 (p0.044) and CO51 (p0.021). Microbial inoculation enhanced soil nutrients and increased their bioavailability for the plants. Total polyphenolics, flavonoids and protein content increased in the leaves following microbial inoculation. Enhanced non-enzymatic antioxidant parameters (ABTS, DPPH, Fe-ion reducing power and Fe-ion chelation) was found in microbe inoculated rice reflecting high free radical scavenging activity in polyphenolics-rich leaf extracts. Increased enzyme activity of superoxide dismutase (SOD), glutathione reductase (GR), phenylalanine ammonia-lyase (PAL), peroxidase (PO), glutathione peroxidase (GPX), ascorbate peroxidase (APX) and catalase (CAT) showed improved ROS scavenging in rice plants having co-inoculation. Over-expression of PAL, cCuZn-SOD and CAT genes in microbial inoculated rice plants was recorded. The study concludes that plant stage-wise concomitant fortification by microbial inoculants could play multi-pronged manifestations at physiological, biochemical and molecular level in rice to positively influence growth, development and defense attributes in plants.

Effective microorganisms inoculant: Diversity and effect on the germination of palisade grass seeds.

Effective microorganisms (EM) are inoculants formed by fungi and bacteria isolated from soil. EM are commonly used by farmers on agronomic crops to stimulate plant growth, but their composition and their benefits has been controverted. This study aimed to analyze the diversity of microorganisms growing in three EM inoculants, as well as to evaluate their efficiency in the germination of palisade grass seeds. The total DNA of the three EM inoculants was extracted, the 16S rRNA and ITS genes were amplified by PCR and sequenced on the Illumina MiSeq platform. Germination tests were conducted with three type of the EM, in three concentration and two times of the immersion. The bacterial group was the most abundant in EM, followed by fungi. Bacterial operational taxonomic units OTUs were shared by all EMs. Pre-treatments of palisade grass seeds with EMs resulted in a higher germination percentage (% G) and germination speed index (IVG) when EM was used at concentration of 1 or 2% in water. Seed immersion for 5 min was more efficient than immersion for 24 h. We can conclude that EM of different origin can share microbial groups and diversity of microorganisms, besides being an alternative to increase palisade grass seeds germination.

Virus-Induced Flowering by Apple Latent Spherical Virus Vector: Effective Use to Accelerate Breeding of Grapevine.

Apple latent spherical virus (ALSV) was successfully used in promoting flowering (virus-induced flowering, VIF) in apple and pear seedlings. In this paper, we report the use of ALSV vectors for VIF in seedlings and in vitro cultures of grapevine. After adjusting experimental conditions for biolistic inoculation of virus RNA, ALSV efficiently infected not only progeny seedlings of Vitis spp. 'Koshu,' but also in vitro cultures of V. vinifera 'Neo Muscat' without inducing viral symptoms. The grapevine seedlings and in vitro cultures inoculated with an ALSV vector expressing the 'florigen' gene (Arabidopsis Flowering locus T, AtFT) started to set floral buds 20-30 days after inoculation. This VIF technology was successfully used to promote flowering and produce grapes with viable seeds in in vitro cultures of F1 hybrids from crosses between V. ficifolia and V. vinifera and made it possible to analyze the quality of fruits within a year after germination. High-temperature (37 °C) treatment of ALSV-infected grapevine disabled virus movement to newly growing tissue to obtain ALSV-free shoots. Thus, the VIF using ALSV vectors can be used to shorten the generation time of grapevine seedlings and accelerate breeding of grapevines with desired traits.

Components of rhizospheric bacterial communities of barley and their potential for plant growth promotion and biocontrol of Fusarium wilt of watermelon.

This work aimed to characterize antagonistic bacteria from the field-grown barley rhizosphere, and evaluate their potential for growth promotion and biocontrol of Fusarium wilt on watermelon caused by Fusarium oxysporum f. sp. Niveum (FON). Seven bacteria were isolated and screened for plant growth promoting and antagonistic traits. Based on the results of phenotypic characterization and 16S rRNA gene sequencing, the isolates were identified to be related to Bacillus methylotrophicus (DMK-1), Bacillus amyloliquefaciens subsp. plantarum (DMK-7-2), Bacillus cereus (DMK-12), Pseudomonas brassicacearum subsp. brassicacearum (DMK-2), Pseudomonas veronii (DMK-3), Paenibacillus polymyxa (DMK-8), and Ensifer adhaerens (DMK-17). All the isolates were positive for the production of indole-3-acetic acid (IAA) and ammonia (NH3), while negative for the production of hydrogen cyanide (HCN). Six bacteria strains (except DMK-17) were able to phosphate solubilization. All the bacteria strains, except DMK-8, were able to produce iron siderophore complexes, and possessed the proteolytic activity. Greenhouse experiment indicated six strains can decrease diseased percentage caused by FON. All the isolates enhanced plant biomass, six strains increased root volume, six strains increased root system activity in greenhouse test. Inoculation of mixtures of seven plant growth promoting rhizobacteria could be more effective in plant growth promotion and biocontrol of Fusarium wilt in watermelon.

Role of novel bacterial Raoultella sp. strain X13 in plant growth promotion and cadmium bioremediation in soil.

Heavy metal pollution in agricultural soils has become a widespread serious problem with the rapid industrialization and urbanization in the past two decades. Cadmium (Cd2+) is of the most concern in soils due to its high toxicity. It is necessary to develop remediation strategies to remove or neutralize its toxic effects in Cd-contaminated soil. Microbial bioremediation is a promising technology to treat heavy metal-contaminated soils. In this study, Cd-resistant bacterium, isolated from heavy metal-polluted soil in Southern China, was characterized as Raoultella sp. strain X13 on the basis of its biochemical profile and 16S rRNA. We investigated the characterization of Cd2+ distribution in different cellular compartments after Cd2+ uptake. Cd2+ uptake by strain X13 was mainly by ion exchange and chelation binding tightly to the cell wall. In addition, X13 plant growth-promoting characteristics suggested that X13 could solubilize phosphate and produce indole acetic acid. Pot experiments for the remediation of Cd-contaminated soil in situ by X13 inoculation demonstrated that X13 application to Cd-contaminated soils significantly promoted pak choi growth and improved production. We also found that X13 substantially reduced the Cd2+ bioavailability for pak choi. Therefore, strain X13 is an effective treatment for potential application in Cd2+ remediation as well as for sustainable agronomic production programs in Cd-contaminated soils.

Soybean inoculants in Brazil: an overview of quality control.

The bacterial strains SEMIA 587 and 5019 (Bradyrhizobium elkanii), 5079 (Bradyrhizobium japonicum), and 5080 (Bradyrhizobium diazoefficiens) are recommended for soybean inoculants in Brazil. In several countries, the current regulations are insufficient to induce companies for improving the quality of their products, leading to low performance and subsequent abandonment of inoculant use. From 2010 to 2014, 1086 samples coming mainly from Argentina and the southern region of Brazil were analyzed for viable cells counting, strains identification, and purity analysis according to the SDA/MAPA no. 30/2010 Normative Instruction. Most products were imported and formulated in liquid carriers with 5.0 × 109 colony-forming units (CFU)/mL. The strains most frequently used were SEMIA 5079/5080. Only 2.21% of samples had contaminants. The guaranteed concentration of viable cells in inoculants mostly ranged from 4.1 × 109 to 5.0 × 109 CFU/mL or CFU/g. The most frequently found concentration was above 1.1 × 1010 CFU/mL or CFU/g, which was higher than the product guarantee. The inoculants used for soybean crop in Brazil have excellent quality, leading the country to the leadership in taking advantage of the biological nitrogen fixation benefits for a productive and sustainable agriculture.

Colonization and Competition Dynamics of Plant Growth-Promoting/Inhibiting Bacteria in the Phytosphere of the Duckweed Lemna minor.

Despite the considerable role of aquatic plant-associated bacteria in host plant growth and nutrient cycling in aquatic environments, the mode of their plant colonization has hardly been understood. This study examined the colonization and competition dynamics of a plant growth-promoting bacterium (PGPB) and two plant growth-inhibiting bacteria (PGIB) in the aquatic plant Lemna minor (common duckweed). When inoculated separately to L. minor, each bacterial strain quickly colonized at approximately 106 cells per milligram (plant fresh weight) and kept similar populations throughout the 7-day cultivation time. The results of two-membered co-inoculation assays revealed that the PGPB strain Aquitalea magnusonii H3 consistently competitively excluded the PGIB strain Acinetobacter ursingii M3, and strain H3 co-existed at almost 1:1 proportion with another PGIB strain, Asticcacaulis excentricus M6, regardless of the inoculation ratios (99:1-1:99) and inoculation order. We also found that A. magnusonii H3 exerted its growth-promoting effect over the negative effects of the two PGIB strains even when only a small amount was inoculated, probably due to its excellent competitive colonization ability. These experimental results demonstrate that there is a constant ecological equilibrium state involved in the bacterial colonization of aquatic plants.

In vitro assessment of the factors that determine the activity of the rumen microbiota for further applications as inoculum.

BACKGROUND: The rumen microbiota has been used as inoculum for in vitro studies and as a probiotic to improve productivity in young animals. However, great variability across studies has been noted depending on the inoculum considered. The present study aims to assess the relevance of different factors (microbial fraction, collection time, donor animal diet, fermentation substrate and inoculum preservation method) to maximize the rumen inoculum activity and set the standards for further in vitro and in vivo applications. RESULTS: Rumen inoculum sampled at 3 h after feeding led to greater microbial growth and activity [+12% volatile fatty acid (VFA), +17% ammonia] compared to before feeding. Similar results were noted when rumen liquid or rumen content were used as inocula. Rumen inoculum adapted to concentrate diets increased microbial activity (+19% VFA) independently of the substrate used in vitro. Freezing-thawing the inoculum, in comparison to fresh inoculum, decreased microbial activity (-14% VFA, -96% ammonia), anaerobic fungi and protozoa, with holotrichs protozoa being particularly vulnerable. Inoculum lyophilization had a stronger negative effect on microbial activity (-51% VFA) and delayed re-activation of the microbes, leading to lower levels of methanogens and anaerobic fungi, as well as almost complete wipe out of rumen protozoa. CONCLUSIONS: Fresh rumen fluid sampled at 3 h after feeding from donor animals that were fed concentrate diets should be chosen when the aim is to provide the most diverse and active rumen microbial inoculum. © 2018 Society of Chemical Industry.

The role of Frankia inoculation in casuarina plantations in China.

Casuarina trees are planted along the coast from Hainan province in South China to the Zhoushan Islands of Zhejiang province in Southeastern China. Three key species, Casuarina equisetifolia, Casuarina cunninghamiana and Casuarina glauca, are used as windbreaks, in agroforestry systems, and for the production of timber and fuel wood. Frankia have been studied in China since 1984. Today, Frankia research fields are very wide, and cover morphology, physiology and genetic diversity, and the application of inocula for specific purposes on poor quality sites. In this paper, we review the role of Frankia inoculations in nurseries and casuarina plantations in China and discuss the benefits of inoculation.

Genomic identification and characterization of the elite strains Bradyrhizobium yuanmingense BR 3267 and Bradyrhizobium pachyrhizi BR 3262 recommended for cowpea inoculation in Brazil

ABSTRACT The leguminous inoculation with nodule-inducing bacteria that perform biological nitrogen fixation is a good example of an "eco-friendly agricultural practice". Bradyrhizobium strains BR 3267 and BR 3262 are recommended for cowpea (Vigna unguiculata) inoculation in Brazil and showed remarkable responses; nevertheless neither strain was characterized at species level, which is our goal in the present work using a polyphasic approach. The strains presented the typical phenotype of Bradyrhizobium with a slow growth and a white colony on yeast extract-mannitol medium. Strain BR 3267 was more versatile in its use of carbon sources compared to BR 3262. The fatty acid composition of BR 3267 was similar to the type strain of Bradyrhizobium yuanmingense; while BR 3262 was similar to Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi. Phylogenetic analyses based on 16S rRNA and three housekeeping genes placed both strains within the genus Bradyrhizobium: strain BR 3267 was closest to B. yuanmingense and BR 3262 to B. pachyrhizi. Genome average nucleotide identity and DNA-DNA reassociation confirmed the genomic identification of B. yuanmingense BR 3267 and B. pachyrhizi BR 3262. The nodC and nifH gene analyses showed that strains BR 3267 and BR 3262 hold divergent symbiotic genes. In summary, the results indicate that cowpea can establish effective symbiosis with divergent bradyrhizobia isolated from Brazilian soils.

Aqueous peat extract exposes rhizobia to sub-lethal stress which may prime cells for improved desiccation tolerance.

Inoculation of legume seed with rhizobia is an efficient and cost-effective means of distributing elite rhizobial strains to broad-acre crops and pastures. However, necessary drying steps after coating seed expose rhizobia to desiccation stress reducing survival and limiting potential nitrogen fixation by legumes. Rhizobial tolerance to desiccation varies with strain and with growth conditions prior to drying. Cells grown in peat generally survive desiccation better than cells grown in liquid broth. We aimed to identify peat-induced proteomic changes in rhizobia that may be linked to desiccation tolerance. Proteins expressed differentially after growth in peat extract when compared with a minimal defined medium were measured in four rhizobial strains. Proteins showing the greatest increase in abundance were those involved in amino acid and carbohydrate transport and metabolism. Proteins involved in posttranslational modification and cell defence mechanisms were also upregulated. Many of the proteins identified in this study have been previously linked to stress responses. In addition, analysis using nucleic acid stains SYTO9 and propidium iodide indicated that membranes had been compromised after growth in peat extract. We targeted the membrane repair protein PspA (ΔRL3579) which was upregulated in Rhizobium leguminosarum bv. viceae 3841 after growth in peat extract to validate whether the inability to repair membrane damage after growth in peat extract reduced desiccation tolerance. The ΔRL3579 mutant grown in peat extract had significantly lower survival under desiccation stress, whereas no difference in survival between wild-type and mutant strains was observed after growth in tryptone yeast (TY) or minimal medium (JMM) media. Staining mutant and wild-type strains with SYTO9 and propidium iodide indicated that membranes of the mutant were compromised after growth in peat extract and to a lesser extent in TY. This study shows that growth in peat extract causes damage to cell membranes and exposes rhizobia to sub-lethal stress resulting in differential expression of several stress-induced proteins. The induction of these proteins may prime and protect the cells when subjected to subsequent stress such as desiccation. Identifying the key proteins involved in desiccation tolerance and properties of peat that stimulate this response will be important to inform development of new inoculant technology that maximises survival of rhizobia during delivery to legume crops and pastures.

Genomic identification and characterization of the elite strains Bradyrhizobium yuanmingense BR 3267 and Bradyrhizobium pachyrhizi BR 3262 recommended for cowpea inoculation in Brazil.

The leguminous inoculation with nodule-inducing bacteria that perform biological nitrogen fixation is a good example of an "eco-friendly agricultural practice". Bradyrhizobium strains BR 3267 and BR 3262 are recommended for cowpea (Vigna unguiculata) inoculation in Brazil and showed remarkable responses; nevertheless neither strain was characterized at species level, which is our goal in the present work using a polyphasic approach. The strains presented the typical phenotype of Bradyrhizobium with a slow growth and a white colony on yeast extract-mannitol medium. Strain BR 3267 was more versatile in its use of carbon sources compared to BR 3262. The fatty acid composition of BR 3267 was similar to the type strain of Bradyrhizobium yuanmingense; while BR 3262 was similar to Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi. Phylogenetic analyses based on 16S rRNA and three housekeeping genes placed both strains within the genus Bradyrhizobium: strain BR 3267 was closest to B. yuanmingense and BR 3262 to B. pachyrhizi. Genome average nucleotide identity and DNA-DNA reassociation confirmed the genomic identification of B. yuanmingense BR 3267 and B. pachyrhizi BR 3262. The nodC and nifH gene analyses showed that strains BR 3267 and BR 3262 hold divergent symbiotic genes. In summary, the results indicate that cowpea can establish effective symbiosis with divergent bradyrhizobia isolated from Brazilian soils.

Potential of Rice Stubble as a Reservoir of Bradyrhizobial Inoculum in Rice-Legume Crop Rotation.

Bradyrhizobium encompasses a variety of bacteria that can live in symbiotic and endophytic associations with leguminous and nonleguminous plants, such as rice. Therefore, it can be expected that rice endophytic bradyrhizobia can be applied in the rice-legume crop rotation system. Some endophytic bradyrhizobial strains were isolated from rice (Oryza sativa L.) tissues. The rice biomass could be enhanced when supplementing bradyrhizobial strain inoculation with KNO3, NH4NO3, or urea, especially in Bradyrhizobium sp. strain SUTN9-2. In contrast, the strains which suppressed rice growth were photosynthetic bradyrhizobia and were found to produce nitric oxide (NO) in the rice root. The expression of genes involved in NO production was conducted using a quantitative reverse transcription-PCR (qRT-PCR) technique. The nirK gene expression level in Bradyrhizobium sp. strain SUT-PR48 with nitrate was higher than that of the norB gene. In contrast, the inoculation of SUTN9-2 resulted in a lower expression of the nirK gene than that of the norB gene. These results suggest that SUT-PR48 may accumulate NO more than SUTN9-2 does. Furthermore, the nifH expression of SUTN9-2 was induced in treatment without nitrogen supplementation in an endophytic association with rice. The indole-3-acetic acid (IAA) and 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase produced in planta by SUTN9-2 were also detected. Enumeration of rice endophytic bradyrhizobia from rice tissues revealed that SUTN9-2 persisted in rice tissues until rice-harvesting season. The mung bean (Vigna radiata) can be nodulated after rice stubbles were decomposed. Therefore, it is possible that rice stubbles can be used as an inoculum in the rice-legume crop rotation system under both low- and high-organic-matter soil conditions.IMPORTANCE This study shows that some rice endophytic bradyrhizobia could produce IAA and ACC deaminase and have a nitrogen fixation ability during symbiosis inside rice tissues. These characteristics may play an important role in rice growth promotion by endophytic bradyrhizobia. However, the NO-producing strains should be of concern due to a possible deleterious effect of NO on rice growth. In addition, this study reports the application of endophytic bradyrhizobia in rice stubbles, and the rice stubbles were used directly as an inoculum for a leguminous plant (mung bean). The degradation of rice stubbles leads to an increased number of SUTN9-2 in the soil and may result in increased mung bean nodulation. Therefore, the persistence of endophytic bradyrhizobia in rice tissues can be developed to use rice stubbles as an inoculum for mung bean in a rice-legume crop rotation system.

Increased nutritional value in food crops.

Modern agriculture and horticulture must combine two objectives that seem to be almost mutually exclusive: to satisfy the nutritional needs of an increasing human population and to minimize the negative impact on the environment. These two objectives are included in the Goal 2 of the 2030 Agenda for Sustainable Development of the United Nations: 'End hunger, achieve food security and improved nutrition and promote sustainable agriculture'. Enhancing the nutritional levels of vegetables would improve nutrient intake without requiring an increase in consumption. In this context, the use of beneficial rhizospheric microorganisms for improving, not only growth and yield, but also the nutrient quality of crops represents a promising tool that may respond to the challenges for modern agriculture and horticulture and represents an alternative to the genetic engineering of crops. This paper summarizes the state of the art, the current difficulties associated to the use of rhizospheric microorganisms as enhancers of the nutritional quality of food crops as well as the future prospects.