Complete genome sequence of Bradyrhizobium sp. 62B, a native nitrogen-fixing rhizobium isolated from peanut nodules.

We present the complete genome sequence of Bradyrhizobium sp. 62B, a strain isolated from the root nodules of peanut plants that grow in central Argentina. The genome consists of 8.15 Mbp, distributed into a chromosome of 7.29 Mbp and a plasmid of 0.86 Mbp.

Exploring the Differential Impact of Salt Stress on Root Colonization Adaptation Mechanisms in Plant Growth-Promoting Rhizobacteria.

Salinity inhibits plant growth by affecting physiological processes, but soil microorganisms like plant growth-promoting rhizobacteria (PGPR) can alleviate abiotic stress and enhance crop productivity. However, it should be noted that rhizobacteria employ different approaches to deal with salt stress conditions and successfully colonize roots. The objective of this study was to investigate the effect of salt stress on bacterial survival mechanisms such as mobility, biofilm formation, and the autoaggregation capacity of three plant growth-promoting strains: Pseudomonas putida SJ04, Pseudomonas simiae WCS417r, and Bacillus amyloliquefaciens GB03. These strains were grown in diluted LB medium supplemented with 0, 100, 200, or 300 mM NaCl. Swimming and swarming mobility were evaluated in media supplemented with 0.3 and 0.5% agar, respectively. Biofilm formation capacity was quantified using the crystal violet method, and the autoaggregation capacity was measured spectrophotometrically. In addition, we evaluated in vitro the capacity of the strains to ameliorate the effects of saline stress in Mentha piperita. The study found that the GB03 strain exhibited enhanced swarming mobility when the salt concentration in the medium increased, resulting in a two-fold increase in the halo diameter at 300 mM. However, high concentrations of NaCl did not affect the swimming mobility. In contrast, swimming motility was reduced in WCS417r and SJ04 under salt stress. On the other hand, exposure to 300 mM NaCl resulted in a 180% increase in biofilm formation and a 30% rise in the percentage of autoaggregation in WCS417r. Conversely, the autoaggregation percentage of the strains SJ04 and GB03 remained unaffected by saline stress. However, for GB03, biofilm formation decreased by 80% at 300 mM. Simultaneously, inoculation with the three evaluated strains alleviated the detrimental effects of salinity on plant growth. Under 150 mM salt stress, all strains showed increased fresh weight, with GB03 and WCS417r improving by 40% and SJ04 exhibiting the most remarkable effect with a 70% rise compared to non-inoculated plants. Despite their different strategies for mitigating salt stress, the application of these strains presents a promising strategy for effectively mitigating the negative consequences of salt stress on plant cultivation.

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.

Biofilm-Forming Ability of Phytopathogenic Bacteria: A Review of its Involvement in Plant Stress.

Phytopathogenic bacteria not only affect crop yield and quality but also the environment. Understanding the mechanisms involved in their survival is essential to develop new strategies to control plant disease. One such mechanism is the formation of biofilms; i.e., microbial communities within a three-dimensional structure that offers adaptive advantages, such as protection against unfavorable environmental conditions. Biofilm-producing phytopathogenic bacteria are difficult to manage. They colonize the intercellular spaces and the vascular system of the host plants and cause a wide range of symptoms such as necrosis, wilting, leaf spots, blight, soft rot, and hyperplasia. This review summarizes up-to-date information about saline and drought stress in plants (abiotic stress) and then goes on to focus on the biotic stress produced by biofilm-forming phytopathogenic bacteria, which are responsible for serious disease in many crops. Their characteristics, pathogenesis, virulence factors, systems of cellular communication, and the molecules implicated in the regulation of these processes are all covered.

Whole-Genome Sequence of Burkholderia ambifaria Strain Q53, a Potential Plant Growth Promoter Isolated from the Rhizosphere of Peanut.

We report the complete genome sequence of Burkholderia ambifaria strain Q53, an environmental rhizobacterium isolated from the rhizosphere of peanut plants. The genome consists of 7.4 Mbp distributed into three circular chromosomes and was determined using a hybrid long- and short-read assembly approach.

Coaggregative interactions between rhizobacteria are promoted by exopolysaccharides from Sinorhizobium meliloti.

Bacterial surface components and extracellular compounds such as exopolysaccharides (EPSs) are crucial for interactions between cells, tolerance to different types of stress, and host colonization. Sinorhizobium meliloti produces two EPSs: Succinoglycan (EPS I), which is involved in the establishment of symbiosis with Medicago sativa, and galactoglucan (EPS II), associated with biofilm formation and the promotion of aggregation. Here, we aimed to assess their role in aggregative interactions between cells of the same strain of a given species (auto-aggregation), and between genetically different strains of the same or different species (intra- or intergeneric coaggregation). To do this, we used S. meliloti mutants which are defective in the production of EPS I, EPS II, or both. Macroscopic and microscopic coaggregation tests were performed with combinations or pairs of different bacterial strains. The EPS II-producing strains were more capable of coaggregation than those that cannot produce EPS II. This was true both for coaggregations between different S. meliloti strains, and between S. meliloti and other common rhizobacteria of agricultural relevance, such as Pseudomonas fluorescens and Azospirillum brasilense. The exogenous addition of EPS II strongly promoted coaggregation, thus confirming the polymer's importance for this phenotype. EPS II may therefore be a key factor in events of physiological significance for environmental survival, such as aggregative interactions and biofilm development. Furthermore, it might be a connecting molecule with relevant properties at an ecological, biotechnological, and agricultural level.

Ascochyta Blight in Chickpea: An Update.

Chickpea (Cicer arietinum L.), one of the most cultivated legumes worldwide, is crucial for the economy of several countries and a valuable source of nutrients. Yields may be severely affected by Ascochyta blight, a disease caused by the fungus Ascochyta rabiei. Molecular and pathological studies have not yet managed to establish its pathogenesis, since it is highly variable. Similarly, much remains to be elucidated about plant defense mechanisms against the pathogen. Further knowledge of these two aspects is fundamental for the development of tools and strategies to protect the crop. This review summarizes up-to-date information on the disease's pathogenesis, symptomatology, and geographical distribution, as well as on the environmental factors that favor infection, host defense mechanisms, and resistant chickpea genotypes. It also outlines existing practices for integrated blight management.

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.

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.

Exopolysaccharide II Is Relevant for the Survival of Sinorhizobium meliloti under Water Deficiency and Salinity Stress.

Sinorhizobium meliloti is a soil bacterium of great agricultural importance because of its ability to fix atmospheric nitrogen in symbiotic association with alfalfa (Medicago sativa) roots. We looked into the involvement of exopolysaccharides (EPS) in its survival when exposed to different environmental stressors, as well as in bacteria-bacteria and bacteria-substrate interactions. The strains used were wild-type Rm8530 and two strains that are defective in the biosynthesis of EPS II: wild-type Rm1021, which has a non-functional expR locus, and mutant Rm8530 expA. Under stress by water deficiency, Rm8530 remained viable and increased in number, whereas Rm1021 and Rm8530 expA did not. These differences could be due to Rm8530's ability to produce EPS II. Survival experiments under saline stress showed that viability was reduced for Rm1021 but not for Rm8530 or Rm8530 expA, which suggests the existence of some regulating mechanism dependent on a functional expR that is absent in Rm1021. The results of salinity-induced stress assays regarding biofilm-forming capacity (BFC) and autoaggregation indicated the protective role of EPS II. As a whole, our observations demonstrate that EPS play major roles in rhizobacterial survival.

Exopolysaccharide production in Ensifer meliloti laboratory and native strains and their effects on alfalfa inoculation.

Bacterial surface molecules have an important role in the rhizobia-legume symbiosis. Ensifer meliloti (previously, Sinorhizobium meliloti), a symbiotic Gram-negative rhizobacterium, produces two different exopolysaccharides (EPSs), termed EPS I (succinoglycan) and EPS II (galactoglucan), with different functions in the symbiotic process. Accordingly, we undertook a study comparing the potential differences in alfalfa nodulation by E. meliloti strains with differences in their EPSs production. Strains recommended for inoculation as well as laboratory strains and native strains isolated from alfalfa fields were investigated. This study concentrated on EPS-II production, which results in mucoid colonies that are dependent on the presence of an intact expR gene. The results revealed that although the studied strains exhibited different phenotypes, the differences did not affect alfalfa nodulation itself. However, subtle changes in timing and efficacy to the effects of inoculation with the different strains may result because of other as-yet unknown factors. Thus, additional research is needed to determine the most effective inoculant strains and the best conditions for improving alfalfa production under agricultural conditions.

Cell Autoaggregation, Biofilm Formation, and Plant Attachment in a Sinorhizobium meliloti lpsB Mutant.

Bacterial surface molecules are crucial for the establishment of a successful rhizobia-legume symbiosis, and, in most bacteria, are also critical for adherence properties, surface colonization, and as a barrier for defense. Rhizobial mutants defective in the production of exopolysaccharides (EPSs), lipopolysaccharides (LPSs), or capsular polysaccharides are usually affected in symbiosis with their plant hosts. In the present study, we evaluated the role of the combined effects of LPS and EPS II in cell-to-cell and cell-to-surface interactions in Sinorhizobium meliloti by studying planktonic cell autoaggregation, biofilm formation, and symbiosis with the host plant Medicago sativa. The lpsB mutant, which has a defective core portion of LPS, exhibited a reduction in biofilm formation on abiotic surfaces as well as altered biofilm architecture compared with the wild-type Rm8530 strain. Atomic force microscopy and confocal laser microscopy revealed an increase in polar cell-to-cell interactions in the lpsB mutant, which might account for the biofilm deficiency. However, a certain level of biofilm development was observed in the lpsB strain compared with the EPS II-defective mutant strains. Autoaggregation experiments carried out with LPS and EPS mutant strains showed that both polysaccharides have an impact on the cell-to-cell adhesive interactions of planktonic bacteria. Although the lpsB mutation and the loss of EPS II production strongly stimulated early attachment to alfalfa roots, the number of nodules induced in M. sativa was not increased. Taken together, this work demonstrates that S. meliloti interactions with biotic and abiotic surfaces depend on the interplay between LPS and EPS II.

Analysis of Plant Growth-Promoting Effects of Fluorescent Pseudomonas Strains Isolated from Mentha piperita Rhizosphere and Effects of Their Volatile Organic Compounds on Essential Oil Composition.

Many species or strains of the genus Pseudomonas have been characterized as plant growth promoting rhizobacteria (PGPR). We used a combination of phenotypic and genotypic techniques to analyze the community of fluorescent Pseudomonas strains in the rhizosphere of commercially grown Mentha piperita (peppermint). Biochemical techniques, Amplified rDNA Restriction Analysis (ARDRA), and 16S rRNA gene sequence analysis revealed that the majority of the isolated native fluorescent strains were P. putida. Use of two Repetitive Sequence-based PCR (rep-PCR) techniques, BOX-PCR and ERIC-PCR, allowed us to evaluate diversity among the native strains and to more effectively distinguish among them. PGPR activity was tested for the native strains and reference strain P. fluorescens WCS417r. Micropropagated M. piperita plantlets were exposed to microbial volatile organic compounds (mVOCs) emitted by the bacterial strains, and plant biomass parameters and production of essential oils (EOs) were measured. mVOCs from 11 of the native strains caused an increase in shoot fresh weight. mVOCs from three native strains (SJ04, SJ25, SJ48) induced changes in M. pierita EO composition. The mVOCs caused a reduction of metabolites in the monoterpene pathway, for example menthofuran, and an increase in menthol production. Menthol production is the primary indicator of EO quality. The mVOCs produced by native strains SJ04, SJ25, SJ48, and strain WCS417r were analyzed. The obtained mVOC chromatographic profiles were unique for each of the three native strains analyzed, containing varying hydrocarbon, aromatic, and alogenic compounds. The differential effects of the strains were most likely due to the specific mixtures of mVOCs emitted by each strain, suggesting a synergistic effect occurs among the compounds present.

Programmed Lab Experiments for Biochemical Investigation of Quorum-Sensing Signal Molecules in Rhizospheric Soil Bacteria.

Biochemistry courses in the Department of Molecular Biology at the National University of Río Cuarto, Argentina, are designed for undergraduate students in biology, microbiology, chemistry, agronomy, and veterinary medicine. Microbiology students typically have previous coursework in general, analytical, and organic chemistry. Programmed sequences of lab experiments allow these students to investigate biochemical problems whose solution is feasible within the context of their knowledge and experience. We previously designed and reported a programmed lab experiment that familiarizes microbiology students with techniques for detection and characterization of quorum-sensing (QS) and quorum-quenching (QQ) signal molecules. Here, we describe a sequence of experiments designed to expand the understanding and capabilities of biochemistry students using techniques for extraction and identification of QS and QQ signal molecules from peanut rhizospheric soil bacteria, including culturing and manipulation of bacteria under sterile conditions. The program provides students with an opportunity to perform useful assays, draw conclusions from their results, and discuss possible extensions of the study. © 2016 by The International Union of Biochemistry and Molecular Biology, 44:256-262, 2016.

Roles of Extracellular Polysaccharides and Biofilm Formation in Heavy Metal Resistance of Rhizobia.

Bacterial surface components and extracellular compounds, particularly flagella, lipopolysaccharides (LPSs), and exopolysaccharides (EPSs), in combination with environmental signals and quorum-sensing signals, play crucial roles in bacterial autoaggregation, biofilm development, survival, and host colonization. The nitrogen-fixing species Sinorhizobium meliloti (S. meliloti) produces two symbiosis-promoting EPSs: succinoglycan (or EPS I) and galactoglucan (or EPS II). Studies of the S.meliloti/alfalfa symbiosis model system have revealed numerous biological functions of EPSs, including host specificity, participation in early stages of host plant infection, signaling molecule during plant development, and (most importantly) protection from environmental stresses. We evaluated functions of EPSs in bacterial resistance to heavy metals and metalloids, which are known to affect various biological processes. Heavy metal resistance, biofilm production, and co-culture were tested in the context of previous studies by our group. A range of mercury (Hg II) and arsenic (As III) concentrations were applied to S. meliloti wild type strain and to mutant strains defective in EPS I and EPS II. The EPS production mutants were generally most sensitive to the metals. Our findings suggest that EPSs are necessary for the protection of bacteria from either Hg (II) or As (III) stress. Previous studies have described a pump in S. meliloti that causes efflux of arsenic from cells to surrounding culture medium, thereby protecting them from this type of chemical stress. The presence of heavy metals or metalloids in culture medium had no apparent effect on formation of biofilm, in contrast to previous reports that biofilm formation helps protect various microorganism species from adverse environmental conditions. In co-culture experiments, EPS-producing heavy metal resistant strains exerted a protective effect on AEPS-non-producing, heavy metal-sensitive strains; a phenomenon termed "rescuing" of the non-resistant strain.

Water-limiting conditions alter the structure and biofilm-forming ability of bacterial multispecies communities in the alfalfa rhizosphere.

Biofilms are microbial communities that adhere to biotic or abiotic surfaces and are enclosed in a protective matrix of extracellular compounds. An important advantage of the biofilm lifestyle for soil bacteria (rhizobacteria) is protection against water deprivation (desiccation or osmotic effect). The rhizosphere is a crucial microhabitat for ecological, interactive, and agricultural production processes. The composition and functions of bacterial biofilms in soil microniches are poorly understood. We studied multibacterial communities established as biofilm-like structures in the rhizosphere of Medicago sativa (alfalfa) exposed to 3 experimental conditions of water limitation. The whole biofilm-forming ability (WBFA) for rhizospheric communities exposed to desiccation was higher than that of communities exposed to saline or nonstressful conditions. A culture-dependent ribotyping analysis indicated that communities exposed to desiccation or saline conditions were more diverse than those under the nonstressful condition. 16S rRNA gene sequencing of selected strains showed that the rhizospheric communities consisted primarily of members of the Actinobacteria and α- and γ-Proteobacteria, regardless of the water-limiting condition. Our findings contribute to improved understanding of the effects of environmental stress factors on plant-bacteria interaction processes and have potential application to agricultural management practices.

The role of bacterial biofilms and surface components in plant-bacterial associations.

The role of bacterial surface components in combination with bacterial functional signals in the process of biofilm formation has been increasingly studied in recent years. Plants support a diverse array of bacteria on or in their roots, transport vessels, stems, and leaves. These plant-associated bacteria have important effects on plant health and productivity. Biofilm formation on plants is associated with symbiotic and pathogenic responses, but how plants regulate such associations is unclear. Certain bacteria in biofilm matrices have been found to induce plant growth and to protect plants from phytopathogens (a process termed biocontrol), whereas others are involved in pathogenesis. In this review, we systematically describe the various components and mechanisms involved in bacterial biofilm formation and attachment to plant surfaces and the relationships of these mechanisms to bacterial activity and survival.

Detection, characterization, and biological effect of quorum-sensing signaling molecules in peanut-nodulating bradyrhizobia.

Bacteria of the genus Bradyrhizobium are able to establish a symbiotic relationship with peanut (Arachis hypogaea) root cells and to fix atmospheric nitrogen by converting it to nitrogenous compounds. Quorum sensing (QS) is a cell-cell communication mechanism employed by a variety of bacterial species to coordinate behavior at a community level through regulation of gene expression. The QS process depends on bacterial production of various signaling molecules, among which the N-acylhomoserine lactones (AHLs) are most commonly used by Gram-negative bacteria. Some previous reports have shown the production of QS signaling molecules by various rhizobia, but little is known regarding mechanisms of communication among peanut-nodulating strains. The aims of this study were to identify and characterize QS signals produced by peanut-nodulating bradyrhizobial strains and to evaluate their effects on processes related to cell interaction. Detection of AHLs in 53 rhizobial strains was performed using the biosensor strains Agrobacterium tumefaciens NTL4 (pZLR4) and Chromobacterium violaceum CV026 for AHLs with long and short acyl chains, respectively. None of the strains screened were found to produce AHLs with short acyl chains, but 14 strains produced AHLs with long acyl chains. These 14 AHL-producing strains were further studied by quantification of ß-galactosidase activity levels (AHL-like inducer activity) in NTL4 (pZLR4). Strains displaying moderate to high levels of AHL-like inducer activity were subjected to chemical identification of signaling molecules by high-performance liquid chromatography coupled to mass spectrometry (LC-MS/MS). For each AHL-producing strain, we found at least four different AHLs, corresponding to N-hexanoyl-DL-homoserine lactone (C(6)), N-(3-oxodecanoyl)-L-homoserine lactone (3OC(10)), N-(3-oxododecanoyl)-L-homoserine lactone (3OC(12)), and N-(3-oxotetradecanoyl)-L-homoserine lactone (3OC(14)). Biological roles of 3OC10, 3OC12, and 3OC14 AHLs were evaluated in both AHL-producing and -non-producing peanut-nodulating strains. Bacterial processes related to survival and nodulation, including motility, biofilm formation, and cell aggregation, were affected or modified by the exogenous addition of increasing concentrations of synthetic AHLs. Our results clearly demonstrate the existence of cell communication mechanisms among bradyrhizobial strains symbiotic of peanut. AHLs with long acyl chains appear to be signaling molecules regulating important QS physiological processes in these bacteria.

Molecular diversity of peanut-nodulating rhizobia in soils of Argentina.

RSalpha sequencing is a valuable tool for identification of bacterial strains, and for evaluating the genetic structure of indigenous rhizobial populations. The purpose of this study was to evaluate, qualitatively, the presence or absence of RSalpha fragment in peanut-nodulating strains isolated from plants grown at four sites in central Argentina. RSalpha fragment was found in only three of 26 indigenous strains, and in one of three inoculant strains analyzed. In contrast to results from studies of other symbiotic nitrogen-fixing bacteria, such as soybean-nodulating strains, no correlation was found between generation time and presence of RSalpha sequence. Phylogenetic analysis of the 16S rRNA gene sequence grouped peanut-nodulating strains into two clusters, Bradyrhizobium japonicum vs. B. elkanii, and showed divergence among strains positive for RSalpha sequence. Our results confirm the genetic diversity previously reported for various peanut-nodulating rhizobial strains, and indicate that the RSalpha fragment is not applicable as a marker or tool for competition assays at the field or ecological level.

Systemic induction of monoterpene biosynthesis in Origanumxmajoricum by soil bacteria.

Italian oregano (Origanumxmajoricum) was subjected to root system inoculation with three species of plant growth-promoting rhizobacteria (PGPRs) (Pseudomonas fluorescens, Bacillus subtilis, Azospirillum brasilense), and essential oil (EO) content and plant growth were measured. Composition of monoterpenes, a major EO component, was analyzed qualitative and quantitatively by gas chromatography. Total EO yield for plants inoculated with P. fluorescens or A. brasilense was 3.57 and 3.41 microg/mg fresh weight, respectively, approximately 2.5-fold higher than controls, without change of quantitative oil composition. The major EO compounds, cis- and trans-sabinene hydrate, gamma-terpinene, carvacrol, and thymol, showed increased biosynthesis. Carvacrol was the only terpene showing significant increase of R% in plants inoculated with A. brasilense. Plant growth parameters (shoot and root fresh and dry weights, numbers of leaves and nodes) were evaluated. Shoot fresh weight was significantly increased by all three PGPR species, but only P. fluorescens and A. brasilense increased root dry weight. These two species have clear commercial potential for economic cultivation of O.xmajoricum. Knowledge of the factors affecting yield and accumulation of monoterpenes is essential for improving production of these economically important plant compounds.