Halotolerant Aerobic Heterotrophic Bacteria from the Great Salt Plains of Oklahoma

The Salt Plains National Wildlife Refuge (SPNWR) near Cherokee, Oklahoma, contains a barren salt flat where Permian brine rises to the surface and evaporates under dry conditions to leave a crust of white salt. Rainfall events dissolve the salt crust and create ephemeral streams and ponds. The rapidly changing salinity and high surface temperatures, salinity, and UV exposure make this an extreme environment. The Salt Plains Microbial Observatory (SPMO) examined the soil microbial community of this habitat using classic enrichment and isolation techniques and phylogenetic rDNA studies. Rich growth media have been emphasized that differ in total salt concentration and composition. Aerobic heterotrophic enrichments were performed under a variety of conditions. Heterotrophic enrichments and dilution plates have generated 105 bacterial isolates, representing 46 phylotypes. The bacterial isolates have been characterized phenotypically and subjected to rDNA sequencing and phylogenetic analyses. Fast-growing isolates obtained from enrichments with 10% salt are predominantly from the gamma subgroup of the Proteobacteria and from the low GC Gram-positive cluster. Several different areas on the salt flats have yielded a variety of isolates from the Gram-negative genera Halomonas, Idiomarina, Salinivibrio, and Bacteroidetes. Gram-positive bacteria are well represented in the culture collection including members of the Bacillus, Salibacillus, Oceanobacillus, and Halobacillus.     

Methanotrophic Bacteria in the Rhizosphere of Trichloroethylene-Degrading Plants

The presence and density of methanotrophic bacteria has been shown to play an important role in the bioremediation of trichloroethylene (TCE). This article describes the methanotrophic bacterial densities in rhizosphere soils from two areas of the Department of Energy's Savannah River Site in Aiken, South Carolina. A direct fluorescent antibody (DFA) technique was evaluated to determine the presence of methanotrophic bacteria in roots and rhizospheres from vascular plants. The first site, the Miscellaneous Chemical Basin (MCB), was contaminated with a mixture of chemicals, including chlorinated solvents. The second site will be potentially affected by outcropping of TCE-contaminated groundwater. Significantly higher numbers of methanotrophic bacteria were observed with DFA in rhizosphere soils and on roots of Lespedeza cuneata and Pinus taeda (that previously showed higher rates of ¹⁴C-TCE mineralization) compared with nonvegetated soils. In addition, viable and heterotrophic microbial counts were consistently higher in rhizosphere soils and on plant roots compared with nonvegetated soils. Therefore, the presence of these plant species may enhance ¹⁴C-TCE mineralization by selectively increasing the microbial population in the root zone. Methanotrophic bacteria were directly observed by DFA in soils, on the surface of root hairs, within plant roots, and in higher densities associated with mycorrhizal fungi. These experiments provide further evidence that specific types of bacterial interactions with vegetation in the rhizosphere may play an important role in remediation of TCE-contaminated soils and groundwater.     

Secondary Metabolite- and Endochitinase-Dependent Antagonism toward Plant-Pathogenic Microfungi of Pseudomonas fluorescens Isolates from Sugar Beet Rhizosphere

Forty-seven isolates representing all biovars of Pseudomonas fluorescens (biovars I to VI) were collected from the rhizosphere of field-grown sugar beet plants to select candidate strains for biological control of preemergence damping-off disease. The isolates were tested for in vitro antagonism toward the plant-pathogenic microfungi Pythium ultimum and Rhizoctonia solani in three different plate test media. Mechanisms of fungal inhibition were elucidated by tracing secondary-metabolite production and cell wall-degrading enzyme activity in the same media. Most biovars expressed a specific mechanism of antagonism, as represented by a unique antibiotic or enzyme production in the media. A lipopeptide antibiotic, viscosinamide, was produced independently of medium composition by P. fluorescens bv. I, whereas the antibiotic 2,4-diacetylphloroglucinol was observed only in glucose-rich medium and only in P. fluorescens bv. II/IV. Both pathogens were inhibited by the two antibiotics. Finally, in low-glucose medium, a cell wall-degrading endochitinase activity in P. fluorescens bv. I, III, and VI was the apparent mechanism of antagonism toward R. solani. The viscosinamide-producing DR54 isolate (bv. I) was shown to be an effective candidate for biological control, as tested in a pot experiment with sugar beet seedlings infested with Pythium ultimum. The assignment of different patterns of fungal antagonism to the biovars of P. fluorescens is discussed in relation to an improved selection protocol for candidate strains to be used in biological control.     

Antibiotic and Biosurfactant Properties of Cyclic Lipopeptides Produced by Fluorescent Pseudomonas spp. from the Sugar Beet Rhizosphere

Cyclic lipopeptides (CLPs) with antibiotic and biosurfactant properties are produced by a number of soil bacteria, including fluorescent Pseudomonas spp. To provide new and efficient strains for the biological control of root-pathogenic fungi in agricultural crops, we isolated approximately 600 fluorescent Pseudomonas spp. from two different agricultural soils by using three different growth media. CLP production was observed in a large proportion of the strains (approximately 60%) inhabiting the sandy soil, compared to a low proportion (approximately 6%) in the loamy soil. Chemical structure analysis revealed that all CLPs could be clustered into two major groups, each consisting of four subgroups. The two major groups varied primarily in the number of amino acids in the cyclic peptide moiety, while each of the subgroups could be differentiated by substitutions of specific amino acids in the peptide moiety. Production of specific CLPs could be affiliated with Pseudomonas fluorescens strain groups belonging to biotype I, V, or VI. In vitro analysis using both purified CLPs and whole-cell P. fluorescens preparations demonstrated that all CLPs exhibited strong biosurfactant properties and that some also had antibiotic properties towards root-pathogenic microfungi. The CLP-producing P. fluorescens strains provide a useful resource for selection of biological control agents, whether a single strain or a consortium of strains was used to maximize the synergistic effect of multiple antagonistic traits in the inoculum.     

Population of Aerobic Heterotrophic Nitrogen-Fixing Bacteria Associated with Wetland and Dryland Rice

Nitrogen-fixing activity and populations of nitrogen-fixing bacteria associated with two varieties of rice grown in dryland and wetland conditions were measured at various growth stages during the dry season. Acetylene reduction activities were measured both in the field and for the hydroponically grown rice, which was transferred from the field to water culture 1 day before assay. The activities measured by both methods were higher in wetland than in dryland rice. The population of nitrogen-fixing heterotrophic bacteria associated with rhizosphere soil, root, and basal shoots was determined by the most probable number method with semisolid glucose-yeast extract and semisolid malate-yeast extract media. The number of nitrogen-fixing bacteria was higher in wetland conditions than in dryland conditions. The difference between two conditions was most pronounced in the population associated with the basal shoot. The glucose medium gave higher counts than did the malate medium. Colonies were picked from tryptic soy agar plates, and their nitrogen-fixing activity was tested on a semisolid glucose-yeast extract medium. The incidence of nitrogen-fixing bacteria among aerobic heterotrophic bacteria in association with rhizosphere soil, root, and basal shoots was much lower in dryland rice than in wetland rice.     

Rehydration of Sugar Beet Plants after Water Stress: Effect of Cytokinins

The possibility to improve the recovery of sugar beet plants after water stress by application of synthetic cytokinins N⁶-benzyladenine (BA) or N⁶-(m-hydroxybenzyl)adenosine (HBA) was tested. Relative water content (RWC), net photosynthetic rate (P_N), transpiration rate (E), stomatal conductance (g_s), chlorophyll (Chl) a and Chl b contents, and photosystem 2 efficiency characterized by variable to maximal fluorescence ratio (F_v/F_m) were measured in control plants, in water-stressed plants, and after rehydration (4, 8, 24, and 48 h). Water stress markedly decreased parameters of gas exchange, but they started to recover soon after irrigation. Application of BA or HBA to the substrate or sprayed on leaves only slightly stimulated recovery of P_N, E, and g_s in rehydrated plants, especially during the first phases of recovery. Chl contents decreased only under severe water stress and F_v/F_m ratio was not significantly affected by water stress applied. Positive effects of BA or HBA application on Chl content and F_v/F_m ratio were mostly not observed.     

Production of Cyclic Lipopeptides by Pseudomonas fluorescens Strains in Bulk Soil and in the Sugar Beet Rhizosphere

The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 μg g⁻¹) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ~2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.     

Interaction of Rhizospheric Bacteria and Nonrhizobial Endophytic Bacteria Moving from the Roots to the Rhizosphere of Pea Plants (Pisum sativum)

Applied Biochemistry and Microbiology - Six of 11 strains of endophytic bacteria from pea (Pisum sativum L.) seeds were found in an aqueous medium of seedling-root growth under hydroculture...     

Exopolysaccharide-Producing Bacteria from Sugar Beets

Six hundred microorganisms were isolated from sugar beets collected from different parts of Finland to study their slime production. A total of 170 of them produced exopolysaccharides, of which 35% were heteropolysaccharides. The yield of heteropolysaccharides from sucrose was lower than that of dextrans. Five isolates, which were chosen for closer study, were identified as Leuconostoc mesenteroides (two species), Rahnella aquatilis (two species), and Enterobacter amnigenus.     

Sucrose Leakage from Isolated Parenchyma of Sugar Beet Roots

The kinetics of sugar efflux from slices of sugar beet rootswas investigated using washing solutions of different osmoticpressure and calcium concentration. The leakage of sucrose isstrongly reduced in solutions of high osmotic pressure (>0·8MPa) or high calcium concentration (10 mM). Turgor-dependentnecrosis of parenchyma cells (plasmoptysis) is the main causeof sucrose efflux from the tissue in hypotonic media with lowcalcium activity. This was shown by good correlation betweenthe percentage of leaked sucrose and the percentage of tissuewater, which was in the free space after the washing procedure.The kinetics of sugar leakage from beet root parenchyma is nobasis for the estimation of the sugar contents of the free spaceor the cytoplasm in situ.     

Populations of Aerobic Bacteria Associated with the Roots of Apple and Cherry Plants

S ummary . The incidence of bacteria on the surface of apple and cherry roots was compared over a period of 20 months with those in rhizosphere and root-free soils. Seasonal variation in total and in Gram negative bacterial populations in soils and on roots was essentially similar. The highest numbers occurred between November and March and the lowest in September. Little general stimulation of bacteria near roots was detected and, on occasion, negative values were recorded for rhizosphere effect. The results indicated that edaphic factors rather than root activity had the major influence on population levels. Fluorescent pseudomonads were low in number and this suggests either that fruit tree roots were unable to support large numbers of these bacteria or that colonization from the soil populations was irregular. However, in contrast to other bacteria, there was a marked stimulatory effect on fluorescent pseudomonads, particularly in apple rhizo-spheres where a maximum rhizosphere/soil ratio of 131 was recorded.     

On the Relationship Between Soil Moisture and Osmotic Potential in Maize and Sugar Beet Plants

Investigations on the relationship between soil moisture and maize and sugar beet plant osmotic potential were carried out during three years under field trial conditions. It was established that between soil moisture and osmotic potential there is an inverse ratio dependence, expressed in significant correlation coefficients (r =?0.84** for maize and r =?0.88** for sugar beet on an average). The correlation appears more striking in the lower third of the active moisture range, where slight variations in soil moisture determine important changes in osmotic potential. The possibility of using the osmotic potential as an index in estimating plant water requirement is asserted and the limit values indicating a good water supply in maize and sugar beet plants are established according to growth stages.     

甜菜(Beta vulgaris L.)叶绿体转化体系建立及抗虫和抗除草剂植株的获得

为建立外源基因甜菜叶绿体转化体系,利用分子生物学方法构建了包含有编码苏云金芽孢杆菌晶体蛋白基因By crylAc 和编码膦丝菌素乙酰转移酶基因bar 的甜菜叶绿体转化载体pSKARBt/bar,以甜菜叶绿体基因组中atpB/rbcL做同源片段,以甜菜叶绿体16S启动子和终止子为调控基因,以bar矿基因为筛选标记基因.基因枪法转化甜菜叶柄,经筛选获得抗性转基因植株.对转基因植株进行外源基因 Bt crylAc和bar的PCR检测、DNA印迹分析,结果表明:外源基因Bt crylAc和bar确已导入到甜菜叶绿体基因组中.转基因植株除草剂抗性鉴定及其离体叶片虫试鉴定结果表明:转基因植株具有较强的杀虫活性和抗除草剂特性,表达了相应的蛋白质.研究结果还表明:bar基因在植物叶绿体转化中,既可以用作抗性基因,又可用作转化体筛选的标记基因.建立了甜菜叶绿体转化体系.     

Natural Hot Spots for Gain of Multiple Resistances: Arsenic and Antibiotic Resistances in Heterotrophic,Aerobic Bacteria from Marine Hydrothermal Vent Fields

Microorganisms are responsible for multiple antibiotic resistances that have been associated with resistance/tolerance to heavy metals, with consequences to public health. Many genes conferring these resistances are located on mobile genetic elements, easily exchanged among phylogenetically distant bacteria. The objective of the present work was to isolate arsenic-, antimonite-, and antibiotic-resistant strains and to determine the existence of plasmids harboring antibiotic/arsenic/antimonite resistance traits in phenotypically resistant strains, in a nonanthropogenically impacted environment. The hydrothermal Lucky Strike field in the Azores archipelago (North Atlantic, between 11°N and 38°N), at the Mid-Atlantic Ridge, protected under the OSPAR Convention, was sampled as a metal-rich pristine environment. A total of 35 strains from 8 different species were isolated in the presence of arsenate, arsenite, and antimonite. ACR3 and arsB genes were amplified from the sediment''s total DNA, and 4 isolates also carried ACR3 genes. Phenotypic multiple resistances were found in all strains, and 7 strains had recoverable plasmids. Purified plasmids were sequenced by Illumina and assembled by EDENA V3, and contig annotation was performed using the “Rapid Annotation using the Subsystems Technology” server. Determinants of resistance to copper, zinc, cadmium, cobalt, and chromium as well as to the antibiotics β-lactams and fluoroquinolones were found in the 3 sequenced plasmids. Genes coding for heavy metal resistance and antibiotic resistance in the same mobile element were found, suggesting the possibility of horizontal gene transfer and distribution of theses resistances in the bacterial population.     

Aerobic Heterotrophic Bacteria Indigenous to pH 2.8 Acid Mine Water: Microscopic Examination of Acid Streamers

"Acid streamers" found in acid coal mine drainage consist of bacteria trapped within an extracellular fibrillar polymer network. Inorganic compounds also precipitate within the polymer network. Several bacteria which appear to be different and are presumed to be different species are associated in the slimy mass of the "acid streamers." The "streamers" contain individual microcolonies or microcosms that can be recognized by a selective polysaccharide stain, which suggests that the slime streamer is a conglomeration of polymers produced by more than one species.     

Production of Regenerants in Sugar Beet

The experimental results are presented on production of plants-regenerants of the sugar beet (Beta vulgaris L.) via callus formation or direct organogenesis from the leaf tissues. The method of aseptic treatment was developed for the seeds with strong bacterial and fungal invasion. The regenerant plants were obtained in the presence of various concentrations of synthetic hormones, such as cytokinin (6-benzaminopurine) and auxin (naphthylacetic acid), and inhibitor of auxin transport in plants (2,3,5-triiodobenzoic acid). This combination of growth regulators made it possible to avoid callus formation. The genotype of initial plants affected the capacity for callus formation and regeneration. The temperature influenced rhizogenesis in regenerants.     

Lipids from Bean, Barley and Sugar Beet in Relation to Salt Resistance

A comparison was made between the lipid and fatty acid composition of the salt-sensitive bean (Phaseolus vulgaris L. cv. Saxa), the less salt-sensitive barley (Hordeum vulgaris L. cv. Wisa) and the salt-tolerant sugar beet (Beta vulgaris L. cv. Kawemono). Sugar beet roots showed a higher content of sterol components and sulfolipid as compared with bean and barley roots. The lipids of sugar beet roots contained more linoleic acid and less linolenic acid than those of bean and barley roots. For barley and sugar beet roots a higher amount of extra-long chain fatty acids was observed than for bean roots. It was concluded that differences in membrane structure are correlated with differences in membrane permeability to sodium and chloride and in salt-resistance of the studied species.