THE RESURRECTION PLANT TRIPOGON SPICATUS (POACEAE) HARBORS A DIVERSITY OF PLANT GROWTH PROMOTING BACTERIA IN NORTHEASTERN BRAZILIAN CAATINGA

Plant species that naturally occur in the Brazilian Caatinga(xeric shrubland) adapt in several ways to these harsh conditions, and that can be exploited to increase crop production. Among the strategic adaptations to confront low water availability, desiccation tolerance stands out. Up to now, the association of those species with beneficial soil microorganisms is not well understood. The aim of this study was to characterize Tripogon spicatusdiazotrophic bacterial isolates from the Caatingabiome and evaluate their ability to promote plant growth in rice. Sixteen bacterial isolates were studied in regard to their taxonomic position by partial sequencing of the 16S rRNA gene, putative diazotrophic capacity, in vitro indole-acetic acid (IAA) production and calcium phosphate solubilization, metabolism of nine different C sources in semi-solid media, tolerance to different concentrations of NaCl to pHs and intrinsic resistance to nine antibiotics. Finally, the ability of the bacterial isolates to promote plant growth was evaluated using rice (Oryza sativa) as a model plant. Among the 16 isolates evaluated, eight of them were classified as Enterobacteriaceae members, related to Enterobacter andPantoeagenera. Six other bacteria were related toBacillus, and the remaining two were related toRhizobiumand Stenotrophomonas.The evaluation of total N incorporation into the semi-solid medium indicated that all the bacteria studied have putative diazotrophic capacity. Two bacteria were able to produce more IAA than that observed for the strain BR 11175Tof Herbaspirillum seropedicae.Bacterial isolates were also able to form a microaerophilic pellicle in a semi-solid medium supplemented with different NaCl concentrations up to 1.27 mol L-1. Intrinsic resistance to antibiotics and the metabolism of different C sources indicated a great variation in physiological profile. Seven isolates were able to promote rice growth, and two bacteria were more efficient than the reference strainAzospirillum brasilense, Ab-V5. The results indicate the potential of T. spicatus as native plant source of plant growth promoting bacteria.

Detection of siderophores in endophytic bacteria Methylobacterium spp. associated with Xylella fastidiosa subsp. pauca

The objective of this work was to study the production of siderophores by endophytic bacteria Methylobacterium spp., which occupy the same ecological niche as Xylella fastidiosa subsp. pauca (Xfp) in citrus plants. The siderophore production of Methylobacterium strains was tested according to chromeazurol agar assay test (CAS), Csáky test (hydroxamate-type) and Arnow test (catechol-type). In addition, the ability of Xfp to use siderophores, in vitro, produced by endophytic bacteria as source of iron, was evaluated. All 37 strains of Methylobacterium spp. tested were CAS-positive for siderophore production. Methylobacterium spp. produced hydroxamate-type, but not catechol-type siderophores. In vitro growth of Xfp was stimulated by the presence of supernatant siderophores of endophytic Methylobacterium mesophilicum.

Bacterial spot and early blight biocontrol by epiphytic bacteria in tomato plants

The objective of this work was to evaluate in vitro and in vivo biocontrol of bacterial spot (Xanthomonas vesicatoria) and early blight (Alternaria solani) by the epiphytic bacteria Paenibacillus macerans and Bacillus pumilus. Tomato plants were previously sprayed with epiphytic bacteria, benzalkonium chloride and PBS buffer and, after four days, they were inoculated with A. solani and X. vesicatoria. To determine the phytopathogenic bacteria population, leaflet samples were collected from each treatment every 24 hours, for seven days, and plated on semi-selective medium. The effect of epiphytic bacteria over phytopathogens was performed by the antibiosis test and antagonistic activity measured by inhibition zone diameter. The epiphytic and benzalkonium chloride drastically reduced the severity of early blight and bacterial spot in comparison to the control (PBS). In detached leaflets, the epiphytic bacteria reduced in 70% the number of phytopathogenic bacteria cells in the phylloplane. The antibiosis test showed that the epiphytic bacteria efficiently inhibit the phytopathogens growth. In all the bioassays, the epiphytic bacteria protect tomato plants against the phytopathogens

Tannin-tolerant bacteria from crossbred Holstein x Zebu cows

The objective of this work was to isolate and characterize tannin-tolerant ruminal bacteria from crossbred Holstein x Zebu cows fed a chopped mixture of elephant grass (Pennisetum purpureum), young stems of "angico-vermelho" (Parapiptadenia rigida), and banana tree (Musa sp.) leaves. A total of 117 bacteria strains were isolated from enrichment cultures of rumen microflora in medium containing tannin extracts. Of these, 11 isolates were able to tolerate up to 3 g L-1 of tannins. Classical characterization procedures indicated that different morphological and physiological groups were represented. Restriction fragments profiles using Alu1 and Taq1 of 1,450 bp PCR products from the 16S rRNA gene grouped the 11 isolates into types I to VI. Sequencing of 16S rRNA PCR products was used for identification. From the 11 strains studied, seven were not identifiable by the methods used in this work, two were strains of Butyrivibrio fibrisolvens, and two of Streptococcus bovis.

Cultivable mushroom growth-promoting bacteria and their impact on Agaricus blazei productivity

The objective of this work was to identify growth-promoting bacteria isolated from Agaricus blazei and to evaluate their effect on mushroom mycelial growth and productivity. A total of 56 A. blazei-associated bacterial isolates were obtained from casing soil and identified by 16S rRNA gene sequencing. Bacteria were evaluated as to phosphate-solubilization ability, nitrogen-fixation capability, and secretion of cellulase. Superior isolates were tested for their to effect on A. blazei productivity, micelial growth, and on the contents of the polysaccharide-protein complex and of N, P, K, Ca, and Mg. Bacterial isolates were identified as actinobacteria (60%), firmicutes (20%), and proteobacteria (20%). Among them, ten isolates had phosphate-solubilization ability, eight showed nitrogen-fixation capability, and 12 isolates promoted A. blazei mycelium growth. Bacterial inoculation reduces time till harvest in up to 26 days, increases fresh mushroom yield up to 215%, and increases total polysaccharide-protein complex content twofold when compared to the non-inoculated control. The actinobacteria group is the predominant A. blazei-associated phylum.

In vitro selection of bacteria with potential for use as probiotics in marine shrimp culture

The objective of this work was to isolate strains of lactic acid bacteria with probiotic potential from the digestive tract of marine shrimp (Litopenaeus vannamei), and to carry out in vitro selection based on multiple characters. The ideotype (ideal proposed strain) was defined by the highest averages for the traits maximum growth velocity, final count of viable cells, and inhibition halo against nine freshwater and marine pathogens, and by the lowest averages for the traits duplication time and resistance of strains to NaCl (1.5 and 3%), pH (6, 8, and 9), and biliary salts (5%). Mahalanobis distance (D²) was estimated among the evaluated strains, and the best ones were those with the shortest distances to the ideotype. Ten bacterial strains were isolated and biochemically identified as Lactobacillus plantarum (3), L. brevis (3), Weissella confusa (2), Lactococcus lactis (1), and L. delbrueckii (1). Lactobacillus plantarum strains showed a wide spectrum of action and the largest inhibition halos against pathogens, both Gram-positive and negative, high growth rate, and tolerance to all evaluated parameters. In relation to ideotype, L. plantarum showed the lowest Mahalanobis (D²) distance, followed by the strains of W. confusa, L. brevis, L. lactis, and L. delbrueckii. Among the analyzed bacterial strains, those of Lactobacillus plantarum have the greatest potential for use as a probiotic for marine shrimp.

Immobilization of laccase on hybrid layered double hydroxide

Crystals of Mg/Al layered double hydroxide were synthesized by alkaline precipitation and treated in an aqueous solution of glutamic acid. The glutamate ions were not intercalated into the interlayer space, but were detected in the material by Fourier transform infrared spectroscopy, suggesting that only the external surfaces of crystals were modified with glutamate ions. The resulting hybrid material was tested as a support for immobilization of the enzyme laccase (Myceliophthora thermophila). The immobilized enzyme preparation was characterized by electronic paramagnetic resonance spectroscopy and by assays of catalytic activity. The activity of the immobilized laccase was 97% of the activity in the free enzyme. Layered double hydroxide is a suitable support for use in remediation of soil studies.

Metabolic response induced by endophytic fungi and bacteria in H. marrubioides Epling in vitro microplants

Hyptis marrubioides Epling is a native plant from Brazilian Cerrado. In this paper, the response of in vitro microplants of this species to inoculation with bacterial and fungal endophytic isolates is evaluated. HPLC-DAD analysis showed the presence of 3,4-O-(Z)-dicaffeoylquinic acid and quercetin-7-O-glucoside as the main components. GC/MS analysis demonstrated that the sesquiterpenes τ-cadinol and caryophyllene oxide were only produced in microplants inoculated with endophytic bacteria, while methyl hexadecanoate, methyl heptadecanoate and methyl (Z,Z,Z) 9,12,15-octadecatrienoate and the triterpene methyl 3β-hydroxy-urs-12-en-28-oate were overexpressed only when the microplant was treated with endophytic fungi.

Preparation and immobilization of diNOsarcobalt(III) complex in zeolite y for the catalyzed production of hydrogen peroxide

A complex cation, diNOsarcobalt(III), [Co(diNOsar)]3+, (diNOsar = 1,8-dinitro-3,6,10,13,16,19-hexaazabicyclo-[6.6.6]eicosane), was synthesized and immobilized in the cavities of a Y zeolite by the reaction of precursor species in the pores of the zeolite. The encapsulated material was compared to the compound diNOsarcobalt(III) chloride, [Co(diNOsar)]Cl3. Both diNOsarcobalt(III) chloride and the zeolite-encapsulated complex, [Co(diNOsar)]3+/zeolite, were obtained in high yield and characterized by ultraviolet-visible and infrared spectroscopy. X-ray diffraction demonstrated the incorporation of the complex cation into the pores of the zeolite. The catalytic production of hydrogen peroxide from oxygenated water confirmed the successful synthesis of the complex diNOsarcobalt(III) immobilized in the zeolite.

A new approach to evaluate immobilization of β-galactosidase on Eupergit® C: structural, kinetic, and thermal characterization

This study aimed to evaluate β-galactosidase immobilization. For this purpose, the ionic strength of the buffer, reaction time, amount of the immobilization support, and pH were evaluated by a central composite design. Assay 8, which consisted of 1.5 mol L-1 phosphate buffer (pH 7.5) and a reaction time of 2 h, produced the maximum yield. Eupergit® C (400 mg) was subsequently used as an immobilization support. Immobilization kinetics wereinvestigated, and a significant increase in the yield was obtained after immobilization compared with that obtained from assay 8 (22.0 U mL-1 vs. 15.6 U mL-1). The enzyme efficiency of actuation was evaluated using o-nitrophenyl-β-D-galactopyranoside and lactose, with lactose providing better results. The reuse of β-galactosidase was evaluated, and more than 50% of the initial enzyme activity was maintained after five cycles of use. Enzyme characterization revealed that immobilization improved some aspects of the thermostability of β-galactosidase.

CARBOXYLATION OF SILVER NANOPARTICLES FOR THE IMMOBILIZATION OF β-GALACTOSIDASE AND ITS EFFICACY IN GALACTO-OLIGOSACCHARIDES PRODUCTION

The present study investigated the carboxylation of silver nanoparticles (AgNPs) by 1:3 nitric acid-sulfuric acid mixtures for immobilizing Aspergillus oryzae β-galactosidase. Carboxylated AgNPs retained 93% enzyme upon immobilization and the enzyme did not leach out appreciably from the modified nanosupport in the presence of 100 mmol L-1 NaCl. Atomic force micrograph revealed the binding of β-galactosidase on the modified AgNPs. The optimal pH for soluble and carboxylated AgNPs adsorbed β-galactosidase (IβG) was observed at pH 4.5 while the optimal operating temperature was broadened from 50 ºC to 60 ºC for IβG. Michaelis constant, Km was increased two and a half fold for IβG while Vmax decreases slightly as compared to soluble enzyme. β-galactosidase immobilized on surface functionalized AgNPs retained 70% biocatalytic activity even at 4% galactose concentration as compared to enzyme in solution. Our study showed that IβG produces greater amount of galacto-oligosaccharides at higher temperatures (50 ºC and 60 ºC) from 0.1 mol L-1 lactose solution at pH 4.5 as compared to previous reports.

Efficiency of phylloplane bacteria in controlling aerial tomato diseases under field conditions

The capacity of two bacteria isolated from the tomato phylloplane to control late blight (Phytophthora infestans) was investigated in the field, and compared against the effectiveness of spraying with the fungicide chlorothalonil (1.5 g a.i. L-1) or water (control). A 55% reduction in late blight intensity was observed in the leaves of the middle of the plant and 62% in those of the upper leaves when using the antagonist UFV-STB 6 (Novosphingobium capsulatum) as compared to the control. Isolate UFV-IEA 6 (Bacillus cereus) was able to reduce disease intensity by 55%, but only in the upper leaves of the tomato plants. Treatment with isolate UFV-STB 6 also led to a significant reduction in the percentage of fruits with late blight symptoms. The results demonstrate the potential of these two bacteria in controlling this disease.

Antimicrobial activity of extracellular metabolites from antagonistic bacteria isolated from potato (Solanum phureja) crops

Microorganisms for biological control are capable of producing active compounds that inhibit the development of phytopathogens, constituting a promising tool toob tain active principles that could replace synthetic pesticides. This study evaluatedtheability of severalpotentialbiocontrol microorganismsto produce active extracellular metabolites. In vitro antagonistic capability of 50 bacterial isolates from rhizospheric soils of "criolla" potato (Solanum phureja) was tested through dual culture in this plant with different plant pathogenic fungi and bacteria. Isolates that showed significantly higher antagonistic activity were fermented in liquid media and crude extracts from the supernatants had their biological activities assessed by optical density techniques. Inhibitory effecton tested pathogens was observed for concentrations between 0.5% and 1% of crude extracts. There was a correlation between the antimicrobial activity of extracts and the use of nutrient-rich media in bacteria fermentation. Using a bioguided method, a peptidic compound, active against Fusarium oxysporum, was obtained from the 7ANT04 strain (Pyrobaculum sp.). Analysis by nuclear magnetic resonance and liquid chromatography coupled to mass detector evidenced an 11-amino acid compound. Bioinformatic software using raw mass data confirmed the presence of a cyclic peptide conformed by 11 mostly non-standard amino acids.

Adherence and experimental infection of bacteria associated with periodontal infections of young cattle in Brazil ("Cara inchada")

In vitro- and in vivo-assays were conducted, to study the possible role of streptomycin- and actinomycin-producing soil actinomycetes for the pathogenesis of "Cara inchada" in cattle (CI). Adherence of Bacteroides spp. to epithelial cells of the bovine gingiva, known to be associated with the progressive lesions of CI, was significantly increased by the addition of streptomycin, actinomycin or antibiotic culture supernatants of the soil actinomycetes. Applications of these mixtures together with Actinomyces pyogenes to the marginal gingiva of the upper premolar teeth of about 1 month old Holstein Friesian calves did not lead to progressive lesions of CI. Only one calf exhibited a slight diarrhea and a temporary retraction of the gingiva at the site of application.

Mouse inoculation for the detection of non-cultivable gastric tightly spiralled bacteria

In the present study we compared the inoculation of swine gastric mucus into the stomach of mice, the urease test and carbolfuchsin-stained smears for the diagnosis of the infection with "Gastrospirillum suis" ("Helicobacter heilmannii" type 1), an uncultivated tightly spiralled gastric bacterium. Fragments obtained from the antral and oxyntic mucosa of the stomach of 50 slaughtered pigs were used for urease test, for carbolfuchsin-stained smears and for obtaining scrapings of mucus for mouse inoculation. The mice were killed by spinal dislocation 10 days after inoculation and fragments of the antral and oxyntic mucosa were used for spiral bacterium identification (urease test and carbolfuchsin-stained smears). Among the methods employed for the diagnosis of "H. heilmannii" infection, the inoculation of gastric mucus into the stomach of mice was the most sensitive and demonstrated bacterial positivity in 31 (62.0%) swine. Direct examination showed tightly spiralled bacteria in the gastric mucosa of only 4 (8.0%) of the 50 pigs studied. Among them, 3 (6.0%) presented a positive preformed urease test. Spiral bacteria were not seen in the gastric mucosa of any control mice. These results show that the use of the mouse inoculation method improved the detection of "H. heilmannii" in swine