Bradyrhizobium japonicum, B. elkanii and B. diazoefficiens Interact with Rice (Oryza sativa), Promote Growth and Increase Yield.

Bradyrhizobium is a genus of plant growth-promoting rhizobacteria (PGPR) that have been studied for several decades mainly for the ability to fix diazotrophic nitrogen after having been established endosymbiotically inside root nodules of the legumes of Fabaceae. The aim of this work was to evaluate the capability of Bradyrhizobium to promote the growth of crops belonging to other families, in this case, rice (Oryza sativa), both in laboratory and in field trials. For laboratory test, surface-sterilized rice seeds were soaked with cultures of each strain and planted in pots. Plant length and dry weight were measured after 35 days. For the field test, rice seeds of varieties Yeruá La Plata and Gurí INTA were inoculated with the three best strains observed in the laboratory test and planted in plots. After 60 days of growth, plant length and dry weight were measured. At harvest time, we measured the dry weight of the aerial part, yield and thousand-grain weight. Inoculation with any of the three species described provoked significant increments compared to the uninoculated control at least in one of the parameters measured, both in the laboratory and in the field tests. Bradyrhizobium japonicum E109 was the strain that promoted rice growth the most in the lab while Bradyrhizobium elkanii SEMIA 587 was the strain that promoted rice growth the most in the field, with increments in yield of approximately 1000 kg/ha. Data obtained suggest that the Bradyrhizobium species promoted all rice growth and yield.

Phenotypic and genotypic characterization of endophytic bacteria associated with transgenic and non-transgenic soybean plants.

Endophytic bacteria isolated from non-transgenic and transgenic Roundup Ready® glyphosate-resistant (GR) soybean plants were investigated to analyze the correspondence between phenotypic and genotypic characteristics and to determine whether or not the strains could be grouped based on the source of isolation in transgenic or non-transgenic plants, respectively. Most of the strains recovered from GR plants have shown the ability for plant growth promotion (PGP) by means of IAA production and inorganic phosphate solubilization, and 100% of the strains showed great motility (swarm or swim); in addition, 90% of the strains were able to metabolize the majority of carbon sources tested. GR soybean fields showed higher endophytes abundance than non-transgenic; however, analyzing the phylogenetic trees constructed using the partial 16SrRNA gene sequences, higher diversity was observed in non-transgenic soybean fields. Overall the majority of isolated endophytes could utilize multiple patterns of carbon sources and express resistance to antibiotics, while isolates varied widely in the PGP ability. The greater pattern and frequency of utilization of carbon sources and frequency and intensity of antibiotic resistance compared with PGP ability within the soybean endophytes community suggest that carbon sources metabolism and antibiotic resistance confer a greater relative fitness benefit than PGP ability. In conclusion, cluster analysis of the phenotypes and 16SrRNA gene sequences reveals lack of correspondence between the pattern of bacterial isolates and the transgenic character of plants, and the heterogeneity of clustering suggested that various adaptive processes, such as stress response, could have contributed to generate phenotypic variability to enhance endophytes overall fitness.

Screening of bacterial endophytes as potential biocontrol agents against soybean diseases.

AIMS: This research was aimed at identifying and characterizing endophytic micro-organisms associated with soybean that have antimicrobial activity towards soybean pathogens. METHODS AND RESULTS: Soybean plants were collected from field trials in four locations of southern Brazil that were cultivated with conventional (C) and transgenic glyphosate-resistant (GR) soybeans. Endophytic bacteria isolated from roots, stems and leaves of soybeans were evaluated for their capacity to inhibit fungal and bacterial plant pathogens and 13 micro-organisms were identified with antagonistic activity. Approximately 230 bacteria were isolated and identified based on the 16S rRNA and rpoN gene sequences. Bacteria isolated from conventional and transgenic soybeans were significantly different not only in population diversity but also in their antagonistic capacity. Thirteen isolates showed in vitro antagonism against Sclerotinia sclerotiorum, Phomopsis sojae and Rhizoctonia solani. Bacillus sp. and Burkholderia sp. were the most effective isolates in controlling bacterial and fungal pathogens in vitro. Extracts and precipitates from culture supernatants of isolates showed different patterns of inhibitory activity on growth of fungal and bacterial pathogens. CONCLUSIONS: Bacillus sp. and Burkholderia sp. were the most effective isolates in controlling fungal pathogens in vitro, and the activity is mainly due to peptides. However, most of the studied bacteria showed the presence of antimicrobial compounds in the culture supernatant, either peptides, bacteriocins or secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be significant to develop tools for the biological control of soybean diseases. The work brought to the identification of micro-organisms such as Bacillus sp. and Burkholderia sp. that have the potential to protect crops in order to enhance a sustainable management system of crops. Furthermore, the study provides the first evidences of the influence of management as well as the genetics of glyphosate-resistant soybean on the diversity of bacterial endophytes of soybean phytobiome.

Structural insights into a novel interkingdom signaling circuit by cartography of the ligand-binding sites of the homologous quorum sensing LuxR-family.

Recent studies have identified a novel interkingdom signaling circuit, via plant signaling molecules, and a bacterial sub-family of LuxR proteins, bridging eukaryotes and prokaryotes. Indeed pivotal plant-bacteria interactions are regulated by the so called Plant Associated Bacteria (PAB) LuxR solo regulators that, although closely related to the quorum sensing (QS) LuxR family, do not bind or respond to canonical quorum sensing N-acyl homoserine lactones (AHLs), but only to specific host plant signal molecules. The large body of structural data available for several members of the QS LuxR family complexed with different classes of ligands (AHLs and other compounds), has been exploited to dissect the cartography of their regulatory domains through structure-based multiple sequence alignments, structural superimposition and a comparative analysis of the contact residues involved in ligand binding. In the absence of experimentally determined structures of members of the PAB LuxR solos subfamily, an homology model of its prototype OryR is presented, aiming to elucidate the architecture of its ligand-binding site. The obtained model, in combination with the cartography of the regulatory domains of the homologous QS LuxRs, provides novel insights into the 3D structure of its ligand-binding site and unveils the probable molecular determinants responsible for differences in selectivity towards specific host plant signal molecules, rather than to canonical QS compounds.

In vitro antibacterial activity of sphaeropsidins and chemical derivatives toward Xanthomonas oryzae pv. oryzae, the causal agent of rice bacterial blight.

Sphaeropsidin A, the main phytotoxin produced by Diplodia cupressi, as well as the two natural analogues sphaeropsidins B and C and 14 derivatives obtained by chemical modifications were assayed for antibacterial activity against Xanthomonas oryzae pv. oryzae, Pseudomonas fuscovaginae, and Burkholderia glumae, the causal agents of severe bacterial rice diseases. The results showed a strong and specific activity of sphaeropsidin A against X. oryzae pv. oryzae, while no activity was observed against the other two pathogens. The results of structure-activity relationship studies showed that structural features important to impart this antibacterial activity are the presence of the C-7 carbonyl group and the hemiketalic lactone functionality. The C-13 vinyl group, the double bond of ring C, and/or the tertiary C-9 hydroxy group, as well as the pimarane arrangement of the tricylic carbon skeleton, were also important for the antibacterial activity. These findings may be useful in designing novel compounds for practical applications in agriculture.

The chitinolytic activity of the Curtobacterium sp. isolated from field-grown soybean and analysis of its genome sequence.

Curtobacterium sp. GD1 was isolated from leaves of conventionally grown soybean in Brazil. It was noteworthy that among all bacteria previously isolated from the same origin, only Curtobacterium sp. GD1 showed a strong chitinase activity. The enzyme was secreted and its production was induced by the presence of colloidal chitin in the medium. The chitinase was partially purified and characterized: molecular weight was approximately 37 kDa and specific activity 90.8 U/mg. Furthermore, Curtobacterium sp. GD1 genome was sequenced and analyzed. Our isolate formed a phylogenetic cluster with four other Curtobacterium spp. strains, with ANIb/ANIm ≥ 98%, representing a new, still non described Curtobacterium species. The circular genome visualization and comparison of genome sequences of strains forming new cluster indicated that most regions within their genomes were highly conserved. The gene associated with chitinase production was identified and the distribution pattern of glycosyl hydrolases genes was assessed. Also, genes associated with catabolism of structural carbohydrates such as oligosaccharides, mixed polysaccharides, plant and animal polysaccharides, as well as genes or gene clusters associated with resistance to antibiotics, toxic compounds and auxin biosynthesis subsystem products were identified. The abundance of putative glycosyl hydrolases in the genome of Curtobacterium sp. GD1 suggests that it has the tools for the hydrolysis of different polysaccharides. Therefore, Curtobacterium sp. GD1 isolated from soybean might be a bioremediator, biocontrol agent, an elicitor of the plant defense responses or simply degrader.

Xanthomonas oryzae pv. oryzae XKK.12 contains an AroQgamma chorismate mutase that is involved in rice virulence.

Chorismate mutase (CM) is a key enzyme in the shikimate pathway which is responsible for the synthesis of aromatic amino acids. There are two classes of CMs, AroQ and AroH, and several pathogenic bacteria have been reported to possess a subgroup of CMs designated AroQ(gamma). These CMs are usually exported to the periplasm or outside the cell; in a few cases, they have been reported to be involved in virulence and their precise role is currently unknown. Here, we report that the important rice pathogen Xanthomonas oryzae pv. oryzae XKK.12 produces an AroQ(gamma) CM which we have purified and characterized from spent supernatants. This enzyme is synthesized in planta and X. oryzae pv. oryzae knock-out mutants are hypervirulent to rice. The role of this enzyme in X. oryzae pv. oryzae rice virulence is discussed.

Many plant pathogenic Pseudomonas savastanoi pv glycinea isolates possess an inactive quorum sensing ahlR gene via a point mutation.

Many plant bacterial pathogens monitor their group behaviour and their population density via production of N-acyl homoserine lactone signals which regulate the expression of several genes via the LuxI/R homologs. This regulatory network, termed quorum sensing (QS), is present in the soybean bacterial pathogen Pseudomonas savastanoi pv glycinea (Psg). The sequenced genomes of two strains of Psg, race 4 and B076, contain an N-acyl homoserine lactone (AHL) based LuxI/R QS system named AhlI/R. While studying the QS system of Psg strains race 4 and B076 isolated in USA, LMG5066 in New Zealand and IBSBF355 in Brazil, we found that B076, LMG5066 and IBSBF355 possess a point mutation in the ahlR gene that causes a frameshift resulting in a truncated AhlR protein. Psg race 4 does not possess the mutation in ahlR and the QS system is functional. The same mutation in the ahlR gene was found to be also present in 9 of 19 Psg strains isolated from diseased soybean in Illinois. Phenotypic analysis of strains showed that swarming motility is repressed whereas phosphate solubilisation was activated by QS in Psg. Analysing the secretome, we also found that four proteins were under QS regulation.

Plant-Growth Promotion and Biocontrol Properties of Three Streptomyces spp. Isolates to Control Bacterial Rice Pathogens.

Bacterial Panicle Blight caused by Burkholderia glumae is a major disease of rice, which has dramatically affected rice production around the world in the last years. In this study we describe the assessment of three Streptomyces isolates as biocontrol agents for B. glumae. Additionally, the presence of other plant-growth promoting abilities and their possible beneficial effects upon their inoculation on rice plants was evaluated as an ecological analysis for their future inoculation in rice crops. Two isolates (A20 and 5.1) inhibited growth of virulent B. glumae strains, as well as a wide range of bacterial and fungal species, while a third strain (7.1) showed only antifungal activity. In vitro tests demonstrated the ability of these strains to produce siderophores, Indoleacetic acid (IAA), extracellular enzymes and solubilizing phosphate. Greenhouse experiments with two rice cultivars indicated that Streptomyces A20 is able to colonize rice plants and promote plant growth in both cultivars. Furthermore, an egfp tagged mutant was generated and colonization experiments were performed, indicating that Streptomyces A20 -GFP was strongly associated with root hairs, which may be related to the plant growth promotion observed in the gnotobiotic experiments. In order to characterize the antimicrobial compounds produced by strain A20 bacteria, mass spectrometry analyses were performed. This technique indicated that A20 produced several antimicrobial compounds with sizes below 3 kDa and three of these molecules were identified as Streptotricins D, E and F. These findings indicate the potential of Streptomyces A20 as a biocontrol inoculant to protect rice plants against bacterial diseases.

Identification, characterization and regulation of two secreted polygalacturonases of the emerging rice pathogen Burkholderia glumae.

Burkholderia glumae is an emerging seed-borne rice pathogen in many areas around the world. Previous studies have demonstrated that B. glumae produces two major virulence factors: the phytotoxin toxoflavin and a secreted lipase. This synthesis of both of these factors is regulated by an N-acyl homoserine lactone (AHL)-dependent, cell-density-dependent quorum-sensing regulation system. This study reports the production and secretion of two highly similar endo-polygalacturonases (designated PehA and PehB) by B. glumae. The two enzymes were purified to homogeneity and the corresponding genetic determinants were identified and characterized. When either polygalacturonase gene was inactivated, B. glumae retained rice virulence comparable to that of the wild-type parent strain. Furthermore, the role of AHL-dependent quorum sensing and of plant cell wall degradation compounds in their regulation was investigated.

Oryza sativa rice plants contain molecules that activate different quorum-sensing N-acyl homoserine lactone biosensors and are sensitive to the specific AiiA lactonase.

Gram-negative bacteria most often use N-acyl homoserine lactones (AHLs) as intercellular quorum-sensing signal molecules. In this study, it was demonstrated that rice plants contain AHL mimic molecules that are very sensitive to the highly specific AiiA lactonase enzyme and can activate three different AHL bacterial biosensors, indicating that the compounds have a homoserine lactone structure and could be AHLs. The possible source and biological significance of this finding are discussed.

Heterologous expression, purification, crystallization, X-ray analysis and phasing of the acetyl xylan esterase from Bacillus pumilus.

Bacillus pumilus PS213 acetyl xylan esterase (AXE) acts as an accessory enzyme in the plant cell wall hemicellulose biodegradation pathway. It belongs to the carbohydrate esterase family 7 and hydrolyses the ester linkages of the acetyl groups in position 2 and/or 3 of the xylose moieties of the acetylated xylan fragments from hardwood. The enzyme displays activity towards a broad range of acetylated compounds including the antibiotic cephalosporin-C. In this study we report the heterologous expression, purification, physicochemical characterization and crystallization of the recombinant B. pumilus AXE. Remarkable improvement of the crystal quality was achieved by setting up crystallization conditions, at first established using the hanging drop vapor diffusion method, in a micro-batch experiment. Rod-like diffraction quality crystals were obtained using 10% PEG 6000, 0.1 M MES pH 6.0 and a wide range of LiCl concentrations (0.2-1.0 M) as precipitant agent. Two different crystal forms, both belonging to space group P2(1), were characterized, diffracting X-rays to 2.5 and 1.9 angstrom resolution. Successful molecular replacement showed 12 molecules in the asymmetric unit of either crystal forms that are arranged as two doughnut-like hexamers, each one encompassing a local 32 symmetry. A catalytic inactive mutant Ser181Ala of B. pumilus AXE was also engineered, expressed, purified and crystallized for functional and structural studies.

Involvement of quorum sensing and RpoS in rice seedling blight caused by Burkholderia plantarii.

Burkholderia plantarii is a plant pathogen responsible for causing rice seedling blight. The molecular mechanisms responsible for this pathogenicity are currently unknown. In this study, we report the identification and characterization of N-acyl homoserine lactone quorum sensing and the stationary phase RpoS sigma factor of B. plantarii. Both global regulatory systems are involved in causing rice seedling blight. This is the first report of gene regulators of B. plantarii implicated in the disease.

The plant pathogen Erwinia amylovora produces acyl-homoserine lactone signal molecules in vitro and in planta.

We report for the first time the production of acyl homoserine lactones (AHLs) by Erwina amylovora, an important quarantine bacterial pathogen that causes fire blight in plants. E. amylovora produces one N-acyl homoserine lactone [a N-(3-oxo-hexanoyl)-homoserine lactone or a N-(3-hydroxy-hexanoyl)-homoserine lactone] quorum sensing signal molecule both in vitro and in planta (pear plant). Given the involvement of AHLs in plant pathogenesis, we speculate that AHL-dependent quorum sensing could play an important role in the regulation of E. amylovora virulence.

A thermostable alpha-arabinofuranosidase from xylanolytic Bacillus pumilus: purification and characterisation.

Bacillus pumilus PS213 secretes an alpha-L-arabinofuranosidase (AF) when grown in the presence of arabinogalactan or oat meal. The enzyme has been purified to homogeneity and characterised. Its molecular mass, as determined by gel filtration, is 220 kDa, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band of approximately 60 kDa. According to the result of the mass spectrometry analysis showing a molecular mass of 56 kDa, the enzyme should be a homotetramer. The isoelectric point was found to be 5.2, the enzyme activity was optimal at 55 degrees C and pH 7.0. The enzyme retained 80% of its activity after 2 h at 65 degrees C and lost 50% of activity at 75 degrees C after 135 min. The Michaelis constant K(m) and V(max) for p-nitrophenylarabinofuranoside at 37 degrees C were 1.7 mM and 52.9 U mg(-1), respectively. N-terminal sequence analysis and internal peptide fragments showed homology with glycosyl hydrolases of family 51.

High-level secretory expression of penicillin amidase from Providencia rettgeri in Saccharomyces cerevisiae: purification and characterization.

Heterologous production of the heterodimeric penicillin G amidase (PAC) from Providencia rettgeri was optimized in Saccharomyces cerevisiae. Several factors, including the effect of different growth and induction conditions, were identified to be critical for the enzyme overproduction and secretion. The PAC yield was significantly increased by more than 500-fold compared to that obtained in the native bacterium, and the recombinant enzyme was almost entirely secreted. Electrophoretic characterization of the secreted rPAC(Pr), which was purified over 20-fold by a combination of hydrophobic interaction and ion-exchange chromatography, demonstrated a microheterogeneity of the recombinant enzyme. The recombinant PAC(Pr) was further characterized in terms of specific activity, pH, and temperature profiles and kinetic parameters. The data presented here suggest that by overexpressing rPAC(Pr) in S.cerevisiae and purifying secreted enzyme from culture medium one can readily obtain a large amount of an alternative source of penicillin amidase with properties comparable to that of todays main industrial source of enzyme.

Bacterial LuxR solos have evolved to respond to different molecules including signals from plants.

A future challenge will be understanding the extensive communication that most likely takes place in bacterial interspecies and interkingdom signaling between plants and bacteria. A major bacterial inter-cellular signaling system in Gram-negative bacteria is LuxI/R quorum sensing (QS) based on the production (via the LuxI-family proteins) and detection (via the LuxR-family proteins) of N-acyl homoserine lactones (AHLs) signaling molecules. LuxR proteins which have the same modular structure of QS LuxRs but are devoid of a cognate LuxI AHL synthase are called solos. LuxR solos have been shown to be responsible to respond to exogenous AHLs produced by neighboring cells as well endogenously produced AHLs. It is now also evident that some LuxR proteins have evolved from the ability to binding AHLs and respond to other molecules/signals. For example, recent research has shown that a sub-family of LuxR solos responds to small molecules produced by plants. This indicates the presence of a uni-directional interkingdom signaling system occurring from plants to bacteria. In addition LuxR solos have now been also implicated to respond to endogenously produced signals which are not AHLs. In this Mini Review article we will discuss current trends and implications of the role of LuxR solos in bacterial responses to other signals using proteins related to AHL QS systems.

A proteomic study of Xanthomonas oryzae pv. oryzae in rice xylem sap.

Xanthomonas oryzae pv. oryzae (Xoo) is the second most important rice pathogen, causing a disease called bacterial leaf blight. Xoo colonizes and infects the vascular tissue resulting in tissue necrosis and wilting causing significant yield losses worldwide. In this study Xoo infected vascular fluid (xylem sap) was recovered and analyzed for secreted Xoo proteins. Three independent experiments resulted in the identification of 324 different proteins, 64 proteins were found in all three samples which included many of the known virulence-associated factors. In addition, 10 genes encoding for the identified proteins were inactivated and one mutant displayed statistically a significant loss in virulence when compared to the wild type Xoo, suggesting that a new virulence-associated factor has been revealed. The usefulness of this approach in understanding the lifestyle and unraveling the virulence-associated factors of phytopathogenic vascular bacteria is discussed.

Key elements in a strategic approach to capacity building in the biosafety of genetically modified organisms.

In recent times, it has become imperative for countries to define and implement policy in biosafety due to the widespread adoption of genetically modified crops. As such, countries wishing to utilise transgenic technologies in the development of advanced agricultural products must have regulations in place coupled with trained personnel in national competent authorities able to contribute effectively to the decision-making process. Capacity building initiatives play an important role in supporting such individuals, institutions and governmental authorities by providing training and/or physical structures/equipment and technical assistance. There are many types of capacity building activities; however not all have the same relevance in different regions of the world. For capacity building to be effective, a strategic approach incorporating a variety of forms and disciplines is desired. This commentary discusses the importance of factors such as: the targeting of support to relevant beneficiary(ies); the identification of specific needs and the incorporation of socio-economic conditions when elaborating effective strategies designed to help building capacity. Moreover, the importance of interaction and collaboration amongst the various capacity builders is also discussed such that unnecessary duplication of efforts and best use of available human and economic resources results.

Isolation, heterologous expression and characterization of an endo-polygalacturonase produced by the phytopathogen Burkholderia cepacia.

Endo-polygalacturonases (endoPGs) belong to the glycoside hydrolase family 28 and hydrolyze the alpha-1,4 glycosidic bond present in the smooth regions of pectins. Pectic substances are among the principal macromolecular components of the primary plant cell walls and are subjected to enzymatic degradation not only in the course of important physiological processes such as plant senescence and ripening, but also during infection events by plant pathogens. Here we report, for the first time, the isolation and the purification of an endoPG (PehA) from the supernatant of the plant pathogen Burkholderia cepacia strain ATCC 25416. In order to obtain adequate amounts of protein required for structural and functional studies, the gene coding for pehA was PCR-amplified and cloned in Escherichia coli cells. The recombinant protein was purified to homogeneity and characterized. PehA exhibited a pI value of 8.0 and an optimal activity at pH 3.5. Far-UV circular dichroism (CD) measurements show that PehA assumes a beta-helix fold super-secondary structural motif.