The elite strain INPA03-11B approved as a cowpea inoculant in Brazil represents a new Bradyrhizobium species and it has high adaptability to stressful soil conditions.

The strain INPA03-11BT, isolated in the 1980s from nodules of Centrosema sp. collected in Manaus, Amazonas, Brazil, was approved by the Brazilian Ministry of Agriculture as a cowpea inoculant in 2004. Since then, several studies have been conducted regarding its phenotypic, genetic, and symbiotic characteristics under axenic and field conditions. Phenotypic features demonstrate its high adaptability to stressful soil conditions, such as tolerance to acidity, high temperatures, and 13 antibiotics, and, especially, its high symbiotic efficiency with cowpea and soybean, proven in the field. The nodC and nifH phylogenies placed the INPA strain in the same clade as the species B. macuxiense BR 10303T which was also isolated from the Amazon region. The sequencing of the 16S rRNA ribosomal gene and housekeeping genes, as well as BOX-PCR profiles, showed its potential as a new species, which was confirmed by a similarity percentage of 94.7% and 92.6% in Average Nucleotide Identity with the closest phylogenetically related species Bradyrhizobium tropiciagri CNPSo1112T and B. viridifuturi SEMIA690T, respectively. dDDH values between INPA03-11BT and both CNPSo 1112T and SEMIA690T were respectively 58.5% and 48.1%, which are much lower than the limit for species boundary (70%). Therefore, we propose the name Bradyrhizobium amazonense for INPA03-11BT (= BR3301 = SEMIA6463).

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.

In Vivo Modulation of Arbuscular Mycorrhizal Symbiosis and Soil Quality by Fungal P Solubilizers.

Phosphorus (P) is an essential nutrient with low bioavailability in soils for plant growth. The use of P solubilization fungi (PSF) has arisen as an eco-friendly strategy to increase this nutrient's bioavailability. The effect of PSF inoculation and its combination with P-transporting organisms (arbuscular mycorrhizal fungi, AMF) on plant growth has been previously studied. However, these studies did not evaluate the combined effect of PSF and AMF inoculation on plant growth, symbiosis, and soil quality. Therefore, the aim of this study is to assess the impact of PSF on the AMF-wheat symbiosis establishment and efficiency, considering the effect on plant growth and soil quality. We performed a greenhouse experiment with wheat under different treatments (+/-AMF: Rhizophagus irregularis; +/-PSF strains: Talaromyces flavus, T. helicus L7B, T. helicus N24, and T. diversus) and measured plant growth, AMF root colonization, symbiotic efficiency, and soil quality indicators. No interaction between PSF and R. irregularis was found in wheat growth, showcasing that their combination is not better than single inoculation. T. helicus strains did not interfere with the AMF-wheat symbiosis establishment, while T. diversus and T. flavus decreased it. The symbiotic efficiency was increased by T. flavus and T. helicus N24, and unchanged with T. helicus L7B and T. diversus inoculation. The soil quality indicators were higher with microbial co-inoculation, particularly the alkaline phosphatases parameter, showing the beneficial role of fungi in soil. This work highlights the importance of microbial interactions in the rhizosphere for crop sustainability and soil quality improvement, assessing the effects of PSF on AMF-wheat symbiosis.

Identification of soybean Bradyrhizobium strains used in commercial inoculants in Brazil by MALDI-TOF mass spectrometry.

Biological nitrogen fixation (BNF) with the soybean crop probably represents the major sustainable technology worldwide, saving billions of dollars in N fertilizers and decreasing water pollution and the emission of greenhouse gases. Accordingly, the identification of strains occupying nodules under field conditions represents a critical step in studies that are aimed at guaranteeing increased BNF contribution. Current methods of identification are mostly based on serology, or on DNA profiles. However, the production of antibodies is restricted to few laboratories, and to obtain DNA profiles of hundreds of isolates is costly and time-consuming. Conversely, the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique might represent a golden opportunity for replacing serological and DNA-based methods. However, MALDI-TOF databases of environmental microorganisms are still limited, and, most importantly, there are concerns about the discrimination of protein profiles at the strain level. In this study, we investigated four soybean rhizobial strains carried in commercial inoculants used in over 35 million hectares in Brazil and also in other countries of South America and Africa. A supplementary MALDI-TOF database with the protein profiles of these rhizobial strains was built and allowed the identification of unique profiles statistically supported by multivariate analysis and neural networks. To test this new database, the nodule occupancy by Bradyrhizobium strains in symbiosis with soybean was characterized in a field experiment and the results were compared with serotyping of bacteria by immuno-agglutination. The results obtained by both techniques were highly correlated and confirmed the viability of using the MALDI-TOF MS technique to effectively distinguish bacteria at the strain level.

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.

Microbial inoculants as plant growth stimulating and soil nutrient availability enhancing options for cucumber under protected cultivation.

Protected cultivation of vegetables is often hampered by declining nutrient availability in soil due to year-around farming, which in turn, leads to poor quality and yields, causing serious concern. Our study aimed towards evaluating the potential of novel biofilm formulations-Anabaena or Trichoderma as matrices with Azotobacter sp. as Anabaena-Azotobacter (An-Az) and Trichoderma-Azotobacter (Tr-Az) or together as Anabaena-Trichoderma (An-Tr), on the growth, physiological activities, yield, and changes in the profiles of soil microbial communities in two cultivars (cv. DAPC-6 and cv. Kian) of cucumber (Cucumis sativus). Photosynthetic pigments, evaluated as an index of growth showed two-threefold increase, while elicited activity of defense and antioxidant enzymes was stimulated; this facilitated significant improvement in the plants belonging to the inoculated treatments. Microbial biomass carbon and polysaccharides in soil enhanced by two-threefolds in treatments receiving microbial formulations. Available N in soil increased by 50-90% in An-Az and An-Tr biofilm inoculated treatments, while the availability of P and organic C content of soil improved by 40-60%, over control. PCR-DGGE profiles generated revealed signification modulation of cyanobacterial communities and cultivar-specific differences. Significant enhancement in leaf chlorophyll pigments, soil microbiological parameters and nutrient bio-availabilities along with positive correlation among the analysed parameters, and distinct profiles generated by PCR-DGGE analyses illustrated the promise of these novel inoculants for cucumber.

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.

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.

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.

Soil Inoculation with Bacillus spp. Modifies Root Endophytic Bacterial Diversity, Evenness, and Community Composition in a Context-Specific Manner.

The use of microbial inoculants containing plant growth-promoting rhizobacteria as a promoter of plant fitness and health is becoming increasingly popular in agriculture. However, whether and how these bacteria affect indigenous bacterial communities in field conditions is sparsely explored. We studied the effects of seed inoculation and field soil application of ubiquitous soil bacteria, B. cereus, B. subtilis, and B. amyloliquefaciens, on the diversity, evenness, and richness of endophytic bacterial communities in sprouting broccoli roots using high-throughput metagenome sequencing. The multiple operational taxonomic units (OTUs) assigned to different bacterial taxa clearly showed changes in ecological measures and relative abundances of certain taxa between control and treatment groups. The Bacillus inocula, themselves, failed to flourish as endophytes; however, the effects they extended on the endophytic bacterial community were both generic as well as species specific. In each case, Pseudomonadales, Rhizobiales, Xanthomonadales, and Burkholderiales were the most abundant orders in the endosphere. B. amyloliquefaciens drastically reduced the most abundant genus, Pseudomonas, while increasing the relative abundance of a range of minor taxa. The Shannon-Weiner diversity and Buzas and Gibson's evenness indices showed that the diversity and evenness were increased in both B. amyloliquefaciens and mixed treated plants. The UniFrac measurement of beta diversity showed that all treatments affected the specific composition of the endophytic bacterial community, with an apparent interspecies competition in the mixed treatment. Taken together, Bacillus species influenced the diversity, evenness, and composition of the endophytic bacterial community. However, these effects varied between different Bacillus spp. in a context-specific manner.

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.

Phytobeneficial Properties of Bacteria Isolated from the Rhizosphere of Maize in Southwestern Nigerian Soils.

Biocontrol agents isolated outside Africa have performed inconsistently under field conditions in Africa. The development of indigenous phytobeneficial microbial strains that suit local environments may help enhance competitiveness with in situ microorganisms and effectiveness at suppressing local pathogen strains. We isolated bacteria from the rhizosphere of maize growing in southwestern Nigeria and assessed them for growth-promoting characteristics. The best isolates were characterized using 16S rRNA genes and were further evaluated in the greenhouse on maize seedlings. Four isolates (EBS8, IGBR11, EPR2, and ADS14) were outstanding in in vitro assays of antagonistic activity against a local strain of Fusarium verticillioides, phosphate solubilization efficiency, chitinase enzyme activity, and indole-3-acetic acid production. Inoculation of maize seeds with these isolates resulted in ≥95% maize seed germination and significantly enhanced radicle and plumule length. In the greenhouse, maize seedling height, stem girth, number of leaves, leaf area, shoot mass (dry matter), and nutrient contents were significantly enhanced. The bioprotectant and phytobeneficial effects were strongest and most consistent for isolate EBS8, which was identified as a Bacillus strain by 16S rRNA gene analysis. As a bacterial strain that exhibits multiple growth-promoting characteristics and is adapted to local conditions, EBS8 should be considered for the development of indigenous biological fertilizer treatments.

Systematics of the Trichoderma harzianum species complex and the re-identification of commercial biocontrol strains.

Trichoderma harzianum is known as a cosmopolitan, ubiquitous species associated with a wide variety of substrates. It is possibly the most commonly used name in agricultural applications involving Trichoderma, including biological control of plant diseases. While various studies have suggested that T. harzianum is a species complex, only a few cryptic species are named. In the present study the taxonomy of the T. harzianum species complex is revised to include at least 14 species. Previously named species included in the complex are T. guizhouense, T. harzianum, and T. inhamatum. Two new combinations are proposed, T. lentiforme and T. lixii. Nine species are described as new, T. afarasin, T. afroharzianum, T. atrobrunneum, T. camerunense, T. endophyticum, T. neotropicale, T. pyramidale, T. rifaii and T. simmonsii. We isolated Trichoderma cultures from four commercial biocontrol products reported to contain T. harzianum. None of the biocontrol strains were identified as T. harzianum s. str. In addition, the widely applied culture 'T. harzianum T22' was determined to be T. afroharzianum. Some species in the T. harzianum complex appear to be exclusively endophytic, while others were only isolated from soil. Sexual states are rare. Descriptions and illustrations are provided. A secondary barcode, nuc translation elongation factor 1-α (TEF1) is needed to identify species in this complex.

Soil type-dependent effects of a potential biocontrol inoculant on indigenous bacterial communities in the rhizosphere of field-grown lettuce.

Bacterial biocontrol strains used as an alternative to chemical fungicides may influence bacterial communities in the rhizosphere and effects might differ depending on the soil type. Here we present baseline data on the effects of Pseudomonas jessenii RU47 on the bacterial community composition in the rhizosphere of lettuce grown in diluvial sand, alluvial loam and loess loam at the same field site. 16S rRNA gene fragments amplified from total community DNA were analyzed by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. DGGE fingerprints revealed that in three consecutive years (2010-2012) RU47 had a slight but statistically significant effect on the bacterial community composition in one (2010), two (2011) or all the three soils (2012). However, these effects were much less pronounced compared with the influence of soil types. Additional pyrosequence analysis of samples from 2011 showed that significant changes in bacterial community compositions in response to RU47 inoculation occurred only in alluvial loam. Different taxonomic groups responded to the RU47 application depending on the soil type. Most remarkable was the increased relative abundance of OTUs belonging to the genera Bacillus and Paenibacillus in alluvial loam. Pyrosequencing allowed side-effects of the application of bacterial inoculants into the rhizosphere to be identified.

Nodulation competitiveness of Ensifer meliloti alfalfa nodule isolates and their potential for application as inoculants.

Alfalfa (Medicago sativa) is a widely cultivated legume, which enters into nitrogen-fixing symbiosis with Ensifer (Sinorhizobium) spp. In this study, an autochthonous rhizobial population of Ensifer sp. occupying alfalfa nodules grown in arable soil was used as the basis for selection of potential inoculants. Alfalfa nodule isolates were identified as Ensifer meliloti by partial 16S rDNA, recA, atpD and nodC nucleotide sequencing. The sampled isolates displayed different symbiotic performance and diversity in the number of plasmids and molecular weight. Isolates that were the most efficient in symbiotic nitrogen fixation were tagged with a constitutively expressed gusA gene carried by a stable plasmid vector pJBA21Tc and used in competition experiments in soil under greenhouse conditions. Two E. meliloti strains LU09 and LU12, which effectively competed with indigenous soil rhizobia, were selected. The metabolic profiles of these selected strains showed differences in the use of carbon and energy sources. In addition, the LU09 strain exhibited bacteriocin production and LU12 mineral phosphate solubilization, which are valuable traits for soil survival. These strains may be considered as potential biofertilizers for alfalfa cultivation.

A new PGPR co-inoculated with Bradyrhizobium japonicum enhances soybean nodulation.

A new PGPR (plant growth promoting rhizobacteria) strain was isolated from soybean seeds and the bacterial mechanisms related to plant growth promotion were evaluated and characterized. Isolates were genotypically compared and identified by amplification of partial sequences of 16S DNAr as Bacillus amyloliquefaciens strain LL2012. Isolates were grown until exponential growth phase to evaluate the atmospheric nitrogen fixation, enzymatic activities, phosphate solubilization, siderophores and phytohormones production. LL2012 strain was able to grow and to produce high levels of auxin, gibberellins and salicylic acid in chemically defined medium. Co-inoculation of soybean plants with LL2012 strain and the natural symbiont (Bradyrhizobium japonicum) altered plant growth parameters and significantly improved nodulation. Our results show that the association of LL2012 with B. japonicum, enhanced the capacity of the latter to colonize plant roots and increase the number of nodules, which make the co-inoculation technique attractive for use in commercial inoculant formulations following proper field evaluation.

Inoculation with a mixed degrading culture improves the pesticide removal of an on-farm biopurification system.

To investigate whether the pesticide removal in on-farm biopurification systems (BPS) filled with two different types of substrata (biomix and plastic carriers) is affected by inoculation with a pesticide-degrading strain or mixed culture, lab-scale BPS used to treat chloropropham point source contaminations were bioaugmented with either a specialized chloropropham-degrading strain or a chloropropham-degrading enrichment culture. Application of both inoculum types leads to an accelerated degradation activity in the columns filled with plastic carriers. For both substratum types, inoculation with the mixed culture resulted in a lower breakthrough of the toxic intermediate 3-chloroaniline at high hydraulic loads, compared to inoculation with the pure isolate and no inoculation. This study suggests that the use of plastic carrier materials could be a proficient alternative to the use of a conventional biomix as a substratum in on-farm BPS and that inoculation with a mixed degrading culture can reduce the leaching of more mobile toxic intermediates.