Characterization of plant growth-promoting alkalotolerant Alcaligenes and Bacillus strains for mitigating the alkaline stress in Zea mays.

Intensification of sodic soil due to increasing pH is an emerging environmental issue. The present study aimed to isolate and characterise alkaline stress-tolerant and plant growth-promoting bacterial strains from moderately alkaline soil (pH 8-9), strongly alkaline soil (pH 9-10), and very strongly alkaline soil (> 10). Total 68 bacteria were isolated, and screened for multiple plant growth promoting (PGP) attributes. Out of total, 42 isolates demonstrating at least three plant growth promoting PGP traits selected for further assays. Then out of 42, 15 bacterial isolates were selected based on enhanced maize plant growth under greenhouse experiment, and 16S rRNA gene sequencing revealed Bacillus spp. as a dominant genus. Furthermore, based on improved seed germination percentage and biomass of maize (Zea mays L.) under alkaline stress conditions Alcaligenes sp. NBRI NB2.5, Bacillus sp. NBRI YE1.3, and Bacillus sp. NBRI YN4.4 bacterial strains were selected, and evaluated for growth-promotion and alkaline stress amelioration under greenhouse condition. Amongst the selected 3 plant growth promoting rhizobacterial (PGPR) strains, Bacillus sp. NBRI YN4.4 significantly improved the photosynthetic pigments and soluble sugar content, and decreased proline level in inoculated maize plants as compared to uninoculated control under stress conditions. Moreover, significantly enhanced soil enzymes such as dehydrogenase, alkaline phosphatase and betaglucosidase due to inoculation of Bacillus sp. NBRI YN4.4 in maize plants grown in alkaline soil attributes to its role in improving the soil health. Therefore, alkaline stress-tolerant PGPR NBRI YN4.4 can be useful for developing strategies for the reclamation of saline/sodic soils and improving the plant growth and soil health in sustainable manner.

Rhizosphere-associated Alcaligenes and Bacillus strains that induce resistance against blast and sheath blight diseases, enhance plant growth and improve mineral content in rice.

AIMS: To examine the biocontrol activities of five rhizobacterial strains (i.e. Alcaligenes faecalis strains Bk1 and P1, Bacillus amyloliquefaciens strain Bk7 and Brevibacillus laterosporus stains B4 and S5), to control the rice blast and sheath blight diseases in greenhouse and to study their possible modes of action. METHODS AND RESULTS: Five potential plant growth-promoting rhizobacterial (PGPR) strains isolated from rice rhizospheres were tested for in vitro antifungal activities against Magnaporthe oryzae, Rhizoctonia solani, Botrytis cinerea and Fusarium graminearum. In vitro trials showed that three strains, Bk1, P1 and Bk7, were able to unanimously suppress the mycelial growth of the target pathogens. In greenhouse, the application of these three PGPR strains significantly suppressed the incidences of rice blast and sheath blight diseases. At 2 weeks after pathogen inoculation, the highest percentages of disease suppression were noted for Alc. faecalis strain Bk1 (72%) for rice blast, Alc. faecalis strain P1 (71%) for sheath blight, followed by B. amyloliquefaciens strain Bk7. Moreover, these strains significantly improved the plant growth, enriched the content of mineral nutrients in seedlings and increased the expression of major defence-related rice genes. All three strains were marked positive for phosphate solubilization, the production of indoleacetic acid, ammonia and siderophores and catalase activity. In addition, these strains were able to form biofilms and carried multiple lipopeptide biosynthetic genes as revealed by multiplex PCR. CONCLUSION: This study reports new potential biocontrol agents for blast and sheath blight diseases of rice. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to better understanding of the mechanisms involved in interaction between beneficial rhizobacteria, fungal pathogens and host plants.

Interactions between Pteris vittata L. genotypes and a polycyclic aromatic hydrocarbon (PAH)-degrading bacterium (Alcaligenes sp.) in arsenic uptake and PAH-dissipation.

The effects of two Pteris vittata L. accessions and a polycyclic aromatic hydrocarbon (PAH)-degrading bacterium (Alcaligenes sp.) on arsenic (As) uptake and phenanthrene dissipation were studied. The Alcaligenes sp. survived in the rhizosphere and improved soil As bioavailability with co-exposure. However, bacterial inoculation altered Pteris vittata L. stress tolerance, and substantially affected the As distribution in the rhizosphere of the two P. vittata accessions. Bacterial inoculation was beneficial to protect the Guangxi accession against the toxic effects, and significantly increased plant As and phenanthrene removal ratios by 27.8% and 2.89%, respectively. In contrast, As removal was reduced by 29.8% in the Hunan accession, when compared with corresponding non-inoculated treatments. We conclude that plant genotype selection is critically important for successful microorganism-assisted phytoremediation of soil co-contaminated with As and PAHs, and appropriate genotype selection may enhance remediation efficiency.

Trafficking of antigens from gut to sacroiliac joints and spine in reactive arthritis and spondyloarthropathies: Mainly through lymphatics?

Bacterial trafficking from gut to mesenteric lymph nodes is physiologic only for a few commensal species, like Alcaligenes which produces antimicrobial-substances inhibiting growth of pathogenic bacteria. In reactive arthritis, some living bacteria transiently manage to travel from gut to joints/enthesis within dendritic cells and/or macrophages. Migration of dead or dormant bacteria outside the gut in spondyloarthropathies, including those associated with Crohn's disease, can occur either through blood or lymphatics. Migration through lymphatics instead of blood depends on the host, but also on the subset of pathogen, as shown for Salmonella. Retrograde trafficking within lymphatics of immune cells infected by dormant or dead bacteria, from mesenteric lymph nodes or thoracic duct to sacroiliac joint and spine, might contribute to axial involvement in some spondyloarthropathies and related disorders, since: 1- large influxes of pathogens can overwhelm lymph nodes, and Yersinia can even replicate within lymph nodes; 2- Whipple cells have been shown to circulate in thoracic duct lymph; 3- expansion of lymphatics is a prominent feature of gastro-intestinal inflammation, and obstruction of gut lymphatics a hallmark of Crohn's disease; 4- lymph reflux has been demonstrated in models of mesenteric lymph vessel obstruction; 5- reflux to sacroiliac has been observed in patients with chyluria undergoing lymphography; 6- lymphatics are present in the outer periosteum and paraspinal ligaments surrounding intervertebral discs and connected to thoracic duct. Accordingly, further studies on the trafficking of dendritic cells, macrophages and lymphocytes from gut to joints and spine in animal models of reactive arthritis or spondyloarthropathies should also focus on lymphatic routes.

Effect of bacterial inoculation of strains of Pseudomonas aeruginosa, Alcaligenes feacalis and Bacillus subtilis on germination, growth and heavy metal (Cd, Cr, and Ni) uptake of Brassica juncea.

Bacterial inoculation may influence Brassica juncea growth and heavy metal (Ni, Cr, and Cd) accumulation. Three metal tolerant bacterial isolates (BCr3, BCd33, and BNi11) recovered from mine tailings, identified as Pseudomonas aeruginosa KP717554, Alcaligenes feacalis KP717561, and Bacillus subtilis KP717559 were used. The isolates exhibited multiple plant growth beneficial characteristics including the production of indole-3-acetic acid, hydrogen cyanide, ammonia, insoluble phosphate solubilization together with the potential to protect plants against fungal pathogens. Bacterial inoculation improved seeds germination of B. juncea plant in the presence of 0.1 mM Cr, Cd, and Ni, as compared to the control treatment. Compared with control treatment, soil inoculation with bacterial isolates significantly increased the amount of soluble heavy metals in soil by 51% (Cr), 50% (Cd), and 44% (Ni) respectively. Pot experiment of B. juncea grown in soil spiked with 100 mg kg(-1) of NiCl2, 100 mg kg(-1) of CdCl2, and 150 mg kg(-1) of K2Cr2O7, revealed that inoculation with metal tolerant bacteria not only protected plants against the toxic effects of heavy metals, but also increased growth and metal accumulation of plants significantly. These findings suggest that such metal tolerant, plant growth promoting bacteria are valuable tools which could be used to develop bio-inoculants for enhancing the efficiency of phytoextraction.

A heavy-metal tolerant novel bacterium, Alcaligenes pakistanensis sp. nov., isolated from industrial effluent in Pakistan.

Two strains, NCCP-650(T) and NCCP-667, were isolated from industrial effluent and their taxonomic positions were investigated using a polyphasic taxonomic approach. The strains were found to be Gram-stain negative, strictly aerobic, motile short rods, which are tolerant to heavy-metals (Cr(+2), As(+2), Pb(+2) and Cu(+2)). Cells were observed to grow at a temperature range of 10-37 °C (optimal 25-33 °C), pH range of 5.5-10.0 (optimal 6.5-7.5) and can tolerate 0-7 % NaCl (w/v) (optimum 0-1 %) in tryptic soya agar medium. Sequencing of the 16S rRNA gene and two housekeeping genes, gyrB and nirK, of the isolated strains revealed that both strains belong to the Betaproteobacteria showing highest sequence similarities with members of the genus Alcaligenes. The chemotaxonomic data [major quinones as Q-8; predominant cellular fatty acids as summed features 3 (C16 :1 ω7c/iso-C15 :0 2OH) and C16:0 followed by Summed features 2 (iso-C16 :1 I/C14 :0 3OH), C17:0 Cyclo and C18:1 ω7c; major polar lipids as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminolipid] also supported the affiliation of the isolated strains with the genus Alcaligenes. DNA-DNA hybridizations between the two strains and with closely related type strains of species of the genus Alcaligenes confirmed that both isolates belong to a single novel species within the genus Alcaligenes. On the basis of phylogenetic analyses, physiological, biochemical characteristics and DNA-DNA hybridization, the isolated strains can be differentiated from established Alcaligenes species and thus represent a novel species, for which the name Alcaligenes pakistanensis sp. nov. is proposed with the type strain NCCP-650(T) (=LMG 28368(T) = KCTC42083(T) = JCM 30216(T)).

Wheat Bran Enhances the Cytotoxicity of Immobilized Alcaligenes aquatilis F8 against Microcystis aeruginosa.

Algicidal bacteria offer a promising option for killing cyanobacteria. Therefore, a new Alcaligenes aquatilis strain F8 was isolated to control Microcystis aeruginosa in this study. The algicidal activity of strain F8 was dependent on the cell density of M. aeruginosa, and the maximal algicidal rate of the free bacterium reached 88.45% within 72 h. With a view to its application to the control of M. aeruginosa in the natural environment, strain F8 was immobilized in sodium alginate beads, but immobilization of the strain decreased its algicidal rate compared to that of the free bacterium. However, addition of wheat bran to the sodium alginate matrix used to immobilize strain F8 not only eliminated the adverse effects of immobilization on the bacteria but also resulted in an 8.83% higher algicidal rate of the immobilized than free bacteria. Exclusion and recovery methods were used to identify key ingredients of wheat bran and gain insight into the mechanism underlying the observed enhancement of algicidal activity. This analysis indicated that certain factors in wheat bran, including vitamins B1, B2, B9, and E were responsible for promoting bacterial growth and thereby improving the algicidal rate of immobilized strain F8. Our findings indicate that wheat bran is able to improve the algicidal efficiency of A. aquatilis strain F8 for killing M. aeruginosa and is a good source of not only carbon and nitrogen but also vitamins for bacteria.

Tyrosinase inhibitory effect and antioxidative activities of fermented and ethanol extracts of Rhodiola rosea and Lonicera japonica.

This is the first study to investigate the biological activities of fermented extracts of Rhodiola rosea L. (Crassulaceae) and Lonicera japonica Thunb. (Caprifoliaceae). Alcaligenes piechaudii CC-ESB2 fermented and ethanol extracts of Rhodiola rosea and Lonicera japonica were prepared and the antioxidative activities of different concentrations of samples were evaluated using in vitro antioxidative assays. Tyrosinase inhibition was determined by using the dopachrome method with L-DOPA as substrate. The results demonstrated that inhibitory effects (ED50 values) on mushroom tyrosinase of fermented Rhodiola rosea, fermented Lonicera japonica, ethanol extract of Lonicera japonica, and ethanol extract of Rhodiola rosea were 0.78, 4.07, 6.93, and >10 mg/ml, respectively. The DPPH scavenging effects of fermented Rhodiola rosea (ED50 = 0.073 mg/ml) and fermented Lonicera japonica (ED50 = 0.207 mg/ml) were stronger than effects of their respective ethanol extracts. Furthermore, the scavenging effect increases with the presence of high content of total phenol. However, the superoxide scavenging effects of fermented Rhodiola rosea was less than effects of fermented Lonicera japonica. The results indicated that fermentation of Rhodiola rosea and Lonicera japonica can be considered as an effective biochemical process for application in food, drug, and cosmetics.

Isolation and characterization of a phenol-degrading strain of Alcaligenes sp. AM4.

Six isolates with phenol degrading ability were obtained from marine sediments by enrichment procedures and an isolate, AM4, was identified as Alcaligenes sp. by 16S rDNA sequencing. The Plackett-Burman design was applied to estimate the significance of culture medium components and conditions for phenol degradation by Alcaligenes sp. AM4. The resulting medium formula which was predicted to be near optimal was: phenol conc. (240 µg/ml), culture volume (37.5 ml), inoculum's size (0.15 ml), NH4SO4 (0.5 g/l), K2HPO4 (0.75 g/l), KH2PO4 (0.75 g/l), MgSO4 (0.3 g/l) and NaCl (0.25 g/l). Scanning electron microscopy was applied to cells exposed to phenol, and a larger cell size was detected, resulting in a reduced cell surface. This relative reduction of the cell surface represents a cellular mechanism to reduce the toxic effect of this environmental stress factor.

Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria.

The mammalian intestinal tract is colonized by trillions of beneficial commensal bacteria that are anatomically restricted to specific niches. However, the mechanisms that regulate anatomical containment remain unclear. Here, we show that interleukin-22 (IL-22)-producing innate lymphoid cells (ILCs) are present in intestinal tissues of healthy mammals. Depletion of ILCs resulted in peripheral dissemination of commensal bacteria and systemic inflammation, which was prevented by administration of IL-22. Disseminating bacteria were identified as Alcaligenes species originating from host lymphoid tissues. Alcaligenes was sufficient to promote systemic inflammation after ILC depletion in mice, and Alcaligenes-specific systemic immune responses were associated with Crohn's disease and progressive hepatitis C virus infection in patients. Collectively, these data indicate that ILCs regulate selective containment of lymphoid-resident bacteria to prevent systemic inflammation associated with chronic diseases.

Predacious bacteria, Bdellovibrio with potential for biocontrol.

Bacteria of the genus of Bdellovibrio are highly motile Gram-negative predators of other Gram-negative bacteria causing lysis of their prey. Here we report results of studies on the interactions of Bdellovibrio with species of Alcaligenes, Campylobacter, Erwinia, Escherichia, Helicobacter, Pseudomonas, Legionella, and Shigella in agar lower, liquid media and cells attached to a surface. Helicobacter pylori was studied employing both actively growing and viable but nonculturable (VBNC) cells. The majority of the bacterial strains tested were found to be susceptible to Bdellovibrio. A significant observation was that Bdellovibrio attacked both actively growing and VBNC H. pylori, that phenomenon has never been reported. The results indicate that bdellovibrios have potential as biocontrol agents.

Fate of a genetically modified bacterium in foregut of glassy-winged sharpshooter (Hemiptera: Cicadellidae).

Symbiotic control is a new strategy being investigated to prevent the spread of insect-transmitted pathogens by reducing vector competence. We are developing this strategy to reduce the spread of Xylella fastidiosa by Homalodisca vitripennis (Germar) [formerly Homalodisca coagulata (Say)] (Hemiptera: Cicadellidae), the glassy-winged sharpshooter. In this study, the fate of a transformed symbiotic bacterium, Alcaligenes xylosoxidans variety denitriicans (S1Axd), in the foregut of glassy-winged sharpshooter when fed on citrus (Citrus spp.) and grape (Vitris spp.) was assessed. TaqMan-based quantitative real-time polymerase chain reaction (PCR) was used to detect and quantify bacterial cells remaining in the foregut at 0, 2, 4, 9, and 12 d after acquisition. S1Axd titer dropped rapidly by 2 d after acquisition, but in spite of this, at end of the 12-d experimental period, 45 and 38% of the glassy-winged sharpshooters retained the transformed bacteria, when fed on grape and citrus, respectively.

[Isolation and characterization of a new glyphosate-resistant strain from extremely polluted environment].

OBJECTIVE: To isolate and characterize a glyphosate-resistant strain from extremely polluted environment. METHODS: A glyphosate-resistant strain was isolated from extremely polluted soil taking glyphosate as the selection pressure. Its glyphosate resistance, growth optimal pH and antibiotic sensitivity were detected. Its morphology, cultural characteristics, physiological and biochemical properties, chemotaxonomy and 16S rDNA sequences were studied. Based on these results, the strain was identified according to the ninth edition of Bergey's manual of determinative bacteriology. RESULTS: The isolate was named SL06500. It could grow in M9 minimal medium containing up to 500 mmol/L glyphosate. The cell growth optimal pH of SL06500 was 4.0. It was resistant to ampicillin, kanamycin, tetracycline and chloromycetin. The 16S rDNA of SL06500 was amplified by PCR and sequenced. Compared with the published nucleotide sequence of 16S rDNA in NCBI (National Center for Biotechnology Information), SL06500 showed high identity with Achromobacter and Alcaligenes. Based on morphological, physiological and biochemical characteristics, the strain was identified as Alcaligenes xylosoxidans subsp.xylosoxidans SL06500 according to the ninth edition of Bergey's manual of determinative bacteriology. CONCLUSION: Strain SL06500 is worthy to be studied because of its high glyphosate resistance.

Synthesis of PHAs from waster under various C:N ratios.

Polyhydroxyalkanoates (PHAs) production was carried out under various C:N ratios. A ratio of 100 resulted best polymer yield. C-source was an important factor in synthesis. For example, as the ratio of valeric acid (C5) to butyric acid (C4) in N-free medium was increased, the mole fraction of HV in the copolymer increased. When soy waste was used as a C-source a copolymer, a high HV mole fraction (HB:HV, 75:25) was produced while when malt waste was used, a much lower HV mole fraction (HB:HV, 90:10) was generated. It was concluded that activated sludge bacteria could be induced to produce PHAs using food wastes as C-sources and this could be the basis for production of biodegradable plastics.

Reclassification of Alcaligenes latus strains IAM 12599T and IAM 12664 and Pseudomonas saccharophila as Azohydromonas lata gen. nov., comb. nov., Azohydromonas australica sp. nov. and Pelomonas saccharophila gen. nov., comb. nov., respectively.

The aim of this study was to clarify the taxonomic position of the nitrogen-fixing and hydrogen-oxidizing bacteria Alcaligenes latus strains IAM 12599T, IAM 12664 and IAM 12665 and Pseudomonas saccharophila IAM 14368T. It was found that the type strain of Alcaligenes latus, IAM 12599T, showed 99 x 9 and 96 x 1 % 16S rRNA gene sequence similarity to strains IAM 12665 and IAM 12664, respectively. A comparison using DNA-DNA hybridization suggested that strains IAM 12599T and IAM 12665 belong to a single species (89 x 7 %) and that strain IAM 12664 (35 x 1 %) forms a separate species. The phenotypic characteristics also support the conclusion that these bacteria should be identified as two species of a new genus: Azohydromonas lata gen. nov., comb. nov. (type strain IAM 12599T=DSM 1122T=LMG 3321T=ATCC 29712T; reference strain IAM 12665=DSM 1123=LMG 3325=ATCC 29714) and Azohydromonas australica sp. nov. (type strain IAM 12664T=DSM 1124T=LMG 3324T=ATCC 29713T). Pseudomonas saccharophila IAM 14368T was found to be closely related to the phototrophic bacterium Roseateles depolymerans, with 96 x 8 % 16S rRNA gene sequence similarity, but the two bacteria are quite different with respect to their metabolism and some significant phenotypic characteristics, suggesting that they cannot be included in a single genus. Further studies on their nifH gene sequences, G+C content of the DNA and cellular fatty acid composition confirm that Pseudomonas saccharophila should be reclassified: the name Pelomonas saccharophila gen. nov., comb. nov. is proposed, with the type strain IAM 14368T (=LMG 2256T=ATCC 15946T).

Surface active properties of bacterial strains isolated from petroleum hydrocarbon-bioremediated soil.

Two bacterial strains identified as Ralstonia picketti (BP 20) and Alcaligenes piechaudii (CZOR L-1B) were isolated from petroleum hydrocarbon-contaminated soil following bioremediation treatment. The surface active properties, e.g. surface tension, emulsification and foamability of their culture filtrates were evaluated. Bacterial cell-surface hydrophobicity (BAH) as measured by analyzing cell affinity towards aliphatic and aromatic compounds was also determinated. The bacteria grew in liquid cultures containing 1% (v/v) of crude oil as carbon and energy source at 30 degrees C under aerobic conditions. The surface tensions were reduced to 61 mN/m and 55 mN/m by Ralstonia picketti and Alcaligenes piechaudii, respectively. The emulsification index (EI24) was almost 100% for all tested compounds except diesel oil. The stability of the emulsions was deteminated at 4 degrees C, 45 degrees C and 65 degrees C. The emulsions were stable at 4 degrees C. Ralstonia picketti was better foam inducer (FV = 50 ml) compared to Alcaligenes piechaudii (FV = 10 ml). The BAH measurements revealed higher adhesion of Alcaligenes piechaudii cells towards different hydrocarbons compared to Ralstonia picketti cells. The strains were found to have a surface hydrophobicity in the following order: aliphatic hydrocarbons, BTEX, and PAHs. The ability to adhere to bulk hydrocarbon is mostly a characteristic of hydrocarbon-degrading bacteria. The strains were found to be better emulsifiers than surface tension reducers. They produce water-soluble extracellular bioemulsifiers. Both bacterial isolates have good properties to use them, mainly in the petroleum industry, e.g. in enhanced oil recovery and in bioremediation processes-primarily due to their emulsification property, i.e. emulsion forming and stabilizing capacity.

Effect of Alcaligenes faecalis on nitrous oxide emission and nitrogen removal in three phase fluidized bed process.

Nitrous oxide (N2O), one of the greenhouse effect gases, has not been known that how much N2O is produced from municipal wastewater treatment and what its management should be. In this study, for controlling nitrous oxide emission and removing nitrogen from municipal wastewater, we experimented the three phase fluidized bed process equipped with draft tube along with immobilized Alcaligenes faecalis, a typical heterotrophic nitrifer and a predominant genus. Also we evaluated the optimum treatment condition of the three phase fluidized bed process for emitting nitrous oxide. The results of this study showed that the three phase fluidized bed process was more effective than the activated sludge process for controlling nitrous oxide emission and removing nitrogen. Increasing amount of A. faecalis in reactor should be encouraged for controlling nitrous oxide emission and removing nitrogen. In addition, the activated sludge process using immobilized A. faecalis as a carrier had more nitrogen removal efficiency than conventional activated sludge process. The accumulation of NO2-N, NO3-N resulted in high N2O emission. Therefore, we suggested that it is necessary to reduce NO2-N and NO3-N for both reducing N2O emission and improving nitrogen removal.

Extraction of exopolymers from biofilms: the protective effect of glutaraldehyde.

The extraction of the exopolymeric matrix is a prerequisite to properly assessing the composition of the biofilm. Several extraction methods have already been developed, however, no universal method has yet been adopted because the compromise between high yields of extraction and minimum cell lysis is difficult to establish. In fact, most of the extraction methods promote leakage of intracellular material. The most common extraction methods, Dowex resin and sonication, were assayed in biofilms of Pseudomonas fluorescens and Alcaligenes denitrificans submitted to a pre-treatment with glutaraldehyde (GTA). The assessment of ATP released after extraction was used as a criterion of cell lysis. The results showed that GTA is a protective agent against cell lysis. The pre-treatment with GTA is particularly useful combined with sonication.

Biological control of Fusarium wilt of pigeonpea Cajanus cajan (L.) Millsp with chitinolytic Alcaligenes xylosoxydans.

Alcaligenes xylosoxydans protected pigeonpea from Fusarium wilt in a pot experiment and field trials. When seeds of pigeonpea (C. cajan) were treated with A. xylosoxydans and sown in soil infested with Fusarium, the incidence of wilt was reduced by 43.5% and resulted in 58% higher grain yield. The antifungal activity of A. xylosoxydans was based on chitinase production and was comparable in efficacy to commercial antifungal agents such as benlate, monitor WP, thiram and bavistin.