Pyrosequencing Reveals Bacterial Diversity in the Rhizosphere of Three Phragmites australis Ecotypes

Here we present the use of high-throughput DNA pyrosequencing to assess bacterial diversity in the rhizosphere of three Phragmites australis ecotypes from the Hexi Corridor, China. In total, 43404 sequences were obtained for the three ecotypes, representing 31 phyla and a small amount of unclassified bacteria. The predominant bacterial groups in the rhizosphere of P. australis were Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes and Planctomycetes. The bacterial community structure varied with the different degrees of wetland degradation, which were exhibited by the three P. australis ecotypes in the study area. At the phylum level, the Caldiserica, Chlamydiae, Deferribacteres, Lentisphaerae, and candidate division WS3 were only detected in the swamp reed (SR) sample. Then, δ-proteobacteria, Acidobacteria, Cyanobacteria and Fusobacteria decreased, the Actinobacteria increased with the degree of degradation from SR through salt meadow reed (SMR) to dune reed (DR). The functional bacterial genera also varied with wetland degradation. The sulfur and sulfate-reducing, nitrifying and nitrogen-fixing bacteria were more abundant in the rhizosphere of the SR sample. Methane-oxidizing bacteria were abundant in the SR and DR samples but less so in the SMR. In our study, pyrosequencing of different P. australis ecotypes provided insight into the structural variation of the rhizosphere bacterial community. This study gave a database for the use of bacteria in the protection and ecological restoration of wetland.     

Isolation and Characterization of a Novel Antialgal Allelochemical from Phragmites communis

Antialgal allelochemicals were isolated from Phragmites communis Tris. The isolated allelopathic fraction showed strong inhibition activity on the growth of Chlorella pyrenoidosa and Microcystis aeruginosa but had no inhibition on Chlorella vulgaris. The 50% effective concentrations (EC₅₀) of the allelopathic fractions on C. pyrenoidosa and M. aeruginosa were 0.49 and 0.79 mg/liter, respectively. The allelopathic activity of the fraction was species-specific. The isolated allelopathic fraction caused metal ion leakage from algal cells. The fraction decreased the activities of antioxidant enzymes, such as superoxide dismutase and peroxidase. The addition of the isolated fraction increased the concentration of unsaturated lipid fatty acids in cell membrane of C. pyrenoidosa and M. aeruginosa. This caused a change in plasma membrane integrity and the leakage of ions in the protoplast. The allelopathic compound was identified by nuclear magnetic resonance and gas chromatography-mass spectrometry as ethyl 2-methylacetoacetate. Synthesized ethyl 2-methylacetoacetate also showed allelopathic activity on C. pyrenoidosa and M. aeruginosa. The EC₅₀ of synthesized ethyl 2-methylacetoacetate on C. pyrenoidosa and M. aeruginosa were 0.49 and 0.65 mg/liter, respectively.     

棉花根际亲和性高效促生细菌的分离筛选

为了从棉花根际土壤筛选能与棉花凝集素具有亲和作用的高效促生细菌,以选择性培养基从棉花根部初步筛选具有固氮能力、解磷能力及解钾能力的促生细菌,再以异硫氰酸磺(FITC)标记的棉花凝集素为复筛工具,从棉花根际促生细菌中筛选能与棉花凝集素结合的亲和性菌株,分别挑选2株固氮菌、2株解磷细菌和2株解钾细菌作为微生物肥料接种到棉花根部进行盆栽试验.观察其在根部定殖情况.结果是在选择性平板上有20%～30%的菌株具有凝集素染色阳性.盆栽试验显示,接种的6株亲和性菌株能在棉花根部成功定殖,根际细菌数量约是灭活对照的`0倍.通过初步鉴定,固氮菌株N1111为固氮菌属(Azotobacter),N2121属于德克斯氏菌属(Derxia);解磷菌株P2126属于黄单胞菌属(Xanthomonas),P1108菌株为假单胞菌属(Pseudomonas);解钾菌株K2204和K2116属于芽孢杆菌属(Bacillus).     

Detoxification of herbicides in Phragmites australis

Unintentional loss of herbicides into drainage ditches, shores or other waterbodies may cause large problems in farmland. Therefore strategies for the phytoremediation of agrochemicals and especially herbicides have become a topic of great interest in many agricultural areas. However, in order to establish effective biological pollution control, information on the detoxification capacity of riparian plants and aquatic macrophytes (e.g., Phragmites australis) is important to build up effective buffer stripes. We determined the detoxification capacity of Phragmites australis roots and leaves for the conjugation of agrochemicals to glutathione by assaying the model substrate CDNB as well as the herbicides fenoxaprop-P, propachlor, pethoxamid and terbuthylazine. Specific GST activities were always higher in the rhizomes (6.78 +/- 0.88 microkat/mg protein for CDNB) than in leaves (1.08 +/- 0.21 microkat/mg protein). The detoxification capacity is distributed across an array of GST isoforms. In summary, Phragmites australis seems to be efficient in herbicide detoxification and a good candidate for phytoremediation of effluents from agricultural sites.     

Diel methane emission patterns from Scirpus lacustris and Phragmites australis

In mature Phragmites australis and Scirpus lacustris vegetated sediment methane was emitted almost exclusively by plant-mediated transport, whereas in unvegetated, but otherwise identical sediment, methane was emitted almost exclusively by ebullition. Diel variations in methane emission, with highest emission rates at daytime and emission peaks following sunrise, were demonstrated for Phragmites and Scirpus. The diel difference and magnitude of the emission peaks were much smaller for Scirpus than for Phragmites. In contrast to Phragmites, methane concentrations within Scirpus stems did not change significantly over the diel period. These patterns are consistent with a two-way transport mechanism for Phragmites (convective at daytime and diffusive at night-time) and an all day diffusive mechanism for Scirpus. The patterns could not be accounted for by diel variation in air and sediment temperature, plant transpiration, or photosynthetically coupled methane production. Comparison of the experimentally derived ratio of methane emission in helium and nitrogen under light and dark conditions with the theoretical derived ratio (calculated according to the kinetic theory of gases) confirmed the exploitation of the different transport mechanism for Phragmites and Scirpus. Methane emission from Phragmites correlated significantly with incident light, which probably drove the pressure differential associated with thermally induced convection. Decrease of the radial resistance of Scirpus stems for methane transport under light compared to dark conditions, in combination with morphological characteristics of the plant species, suggested that stomatal aperture, regulated by light, controls methane emission from Scirpus. Diel variation in bubble emission from the non-vegetated sediment coincided with sediment temperature changes. The results have important implications for sampling and scaling strategies for estimating methane emission from wetlands.     

Isolation and Characterization of Acetate-Utilizing Anaerobes from a Freshwater Sediment

Acetate-degrading anaerobic microorganisms in freshwater sediment were quantified by the most probable number technique. From the highest dilutions a methanogenic, a sulfate-reducing, and a nitrate-reducing microorganism were isolated with acetate as substrate. The methanogen (culture AMPB-Zg) was non-motile and rod-shaped with blunted ends (0.5–1 μm × 3–4 μm long). Doubling times with acetate at 30–35°C were 5.6–8.1 days. The methanogen grew only on acetate. Analysis of the 16S rRNA sequence showed that AMPB-Zg is closely related toMethanosaeta concilii. The isolated sulfate-reducing bacterium (strain ASRB-Zg) was rod-shaped with pointed ends (0.5–0.7 μm × 1.5–3.5 μm long), weakly motile, spore forming, and gram positive. At the optimum growth temperature of 30°C the doubling times with acetate were 3.9–5.3 days. The bacterium grew on a range of organic acids, such as acetate, butyrate, fumarate, and benzoate, but did not grow autotrophically with H₂, CO₂, and sulfate. The closest relative of strain ASRB-Zg isDesulfotomaculum acetoxidans. The nitrate-reducing bacterium (strain ANRB-Zg) was rod-shaped (0.5–0.7 μm × 0.7–1 μm long), weakly motile, and gram negative. Optimum growth with acetate occurred at 20–25°C. The bacterium grew on a range of organic substrates, such as acetate, butyrate, lactate, and glucose, and did grow autotrophically with H₂, CO₂, and oxygen but not with nitrate. In the presence of acetate and nitrate, thiosulfate was oxidized to sulfate. Phylogenetically, the closest relative of strain ANRB-Zg isVariovorax paradoxus.     

芦苇生态型研究进展

芦苇生态幅极广,适生于多种生境类型。不同的环境选择压力如水深、盐度、养分、气候等交互影响致使芦苇个体及种群间发生不同程度的分化和变异,形成了形态、生理或遗传上互有差异、异地性的不同生态型。尽管基于芦苇表型变异以及遗传变异进行生态型划分的研究已开展很多,但针对芦苇生态型变异规律及其可能的形成机制的认知仍存在较多分歧。在总结近年来有关芦苇生态型研究文献的基础上,通过对影响芦苇生态型变异的主要因素——环境因素和遗传因素的分析,以期为芦苇生态型的划分及其可能的形成机制提供新的研究思路。(1)空间尺度的选择应成为研究者分析、划分芦苇生态型的首要定位。在较大的地理空间尺度上,高度异质性的生境导致某些性状的变异式样具有相对的不连续性,可作为不同芦苇生态型鉴别与描述的主要依据;(2)在合理的尺度定位、取样设计和统计分析的基础上开展的表型变异研究,及进一步基于种群水平的分子标记研究(分子指纹特征或特有等位基因),可为芦苇生态型的鉴定、划分提供更为可靠的参考数据,并且可以甄别生境差异(环境响应)和遗传变异对芦苇不同生态型分化的贡献;(3)应同时进行不同生态型的特定性状与功能(株高、茎粗、生物量、生理抗逆性、水体污染物净化能力等)的定位,推动优良基因型的选育与扩繁。     

Isolation and Characterization of Thymineless Mutants from Ultraviolet-Sensitive Bacillus subtilis Strains

Thymineless mutants of Bacillus subtilis 168 ind, hcr-9 were isolated by using trimethoprim. These and other Thy⁻ strains differed drastically from Thy⁺ ones in their patterns of [³H]thymidine uptake and growth in trimethoprim-containing medium. Transformation was negligible between most mutants derived from the ultraviolet-sensitive strain 168 ind, hcr-9 but significant between 168 ind, thy and these mutants. The latter and these new mutants all grow in the presence of trimethoprim plus thymidine or thymine and fail to grow if thymine or thymidine is omitted.     

Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme,Sphingobium fuliginis ATCC 27551

The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.     

Phragmites australis and silica cycling in tidal wetlands

Tidal marshes have recently been shown to be important biogenic Si recycling surfaces at the land–sea interface. The role of vegetation in this recycling process has not yet been quantified. In situ and ex situ decomposition experiments were conducted with Phragmites australis stems. In a freshwater tidal marsh, litterbags were incubated at different elevations and during both winter and summer. Biogenic Si (BSi) dissolution followed a double exponential decay model in the litterbags (from ca. 60 to 15 mg g⁻¹ after 133 days), irrespective of season. Si was removed much faster from the incubated plant material compared to N and C, resulting in steadily decreasing Si/N and Si/C ratios. Ex situ, decomposition experiments were conducted in estuarine water, treated with a broad-spectrum antibiotic, and compared to results from untreated incubations. The bacterial influence on the dissolution of dissolved Si (DSi) from P. australis stems was negligible. Although the rate constant for dissolved Si dissolution decreased from 0.004 to 0.003 h⁻¹, the eventual amount of BSi dissolved and saturation concentration in the incubation environment were similar in both treatments. P. australis contributes to and enhances dissolved Si recycling capacity of tidal marshes: in a reed-dominated small freshwater tidal marsh, more than 40% of DSi export was attributable to reed decomposition. As the relation between tidal marsh surface and secondary production in estuaries has been linked to marsh Si cycling capacity, this provides new insight in the ecological value of the common reed.     

Rhizome dynamics and resource storage in Phragmites australis

Seasonal changes in rhizome concentrations of total nonstructural carbohydrates (TNC), water soluble carbohydrates (WSC), and mineral nutrients (N, P and K) were monitored in two Phragmites australis stands in southern Sweden. Rhizome biomass, rhizome length per unit ground area, and specific weight (weight/ length ratio) of the rhizomes were monitored in one of the stands.Rhizome biomass decreased during spring, increased during summer and decreased during winter. However, changes in spring and summer were small (< 500 g DW m-2) compared to the mean rhizome biomass (approximately 3000 g DW m^–2). Winter losses were larger, approximately 1000 g DW m-2, and to a substantial extent involved structural biomass, indicating rhizome mortality. Seasonal changes in rhizome length per unit ground area revealed a rhizome mortality of about 30% during the winter period, and also indicated that an intensive period of formation of new rhizomes occurred in June.Rhizome concentrations of TNC and WSC decreased during the spring, when carbohydrates were translocated to support shoot growth. However, rhizome standing stock of TNC remained large (> 1000 g m^–2). Concentrations and standing stocks of mineral nutrients decreased during spring/ early summer and increased during summer/ fall. Only N, however, showed a pattern consistent with a spring depletion caused by translocation to shoots. This pattern indicates sufficient root uptake of P and K to support spring growth, and supports other evidence that N is generally the limiting mineral nutrient for Phragmites.The biomass data, as well as increased rhizome specific weight and TNC concentrations, clearly suggests that reloading of rhizomes with energy reserves starts in June, not towards the end of the growing season as has been suggested previously. This resource allocation strategy of Phragmites has consequences for vegetation management.Our data indicate that carbohydrate reserves are much larger than needed to support spring growth. We propose that large stores are needed to ensure establishment of spring shoots when deep water or stochastic environmental events, such as high rhizome mortality in winter or loss of spring shoots due to late season frost, increase the demand for reserves.     

Isolation and Characterization of Methicillin-Resistant Staphylococcus aureus Strains from Louisiana Retail Meats

We investigated the prevalence of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in 120 retail meat samples from 30 grocery stores in Baton Rouge, LA. S. aureus strains were recovered from 45.6% of pork samples and 20% of beef samples, whereas MRSA strains were isolated from six meat samples (five pork samples and one beef sample). The MRSA isolates were of two strain types (clones), one harboring Panton-Valentine leucocidin and belonging to pulsed-field gel electrophoresis type USA300 and the other one belonging to USA100.     

Isolation and Characterization of Bacillus thuringiensis Strains from Aquatic Environments in Spain

Samples collected from aquatic environments from Spain were analyzed for the occurrence and dipteran toxicity of Bacillus thuringiensis. From a total of 41 samples, 122 isolates were obtained, yielding a B. thuringiensis index of 0.22. Isolates were assigned to 13 different serovars, with serovar thuringiensis (serotype H1) the most frequently found. Toxicity tests carried out revealed that eight isolates (6.6% out of the total) were active against Tipula oleracea larvae. Serological tests assigned these toxic isolates to serovar thuringiensis. The toxicity found in these isolates against the tipulid was approximately seven times lower than that shown by the standard strain B. thuringiensis ser. israelensis IPS-82. Implication of Cry2A protein in toxic activity is hypothesized. Received: 3 December 1999 / Accepted: 5 January 2000     

Isolation and Characterization of Circular Deoxyribonucleic Acid Obtained from Lactose-Fermenting Salmonella Strains

Six lac elements originally contained in Salmonella strains were transferred to Escherichia coli WR3026. All of the six E. coli strains that received one of the lac elements were observed to contain supercoiled, circular deoxyribonucleic acid (DNA) when examined by the dye-buoyant density method. Segregants of each of these E. coli WR3026 strains that had lost the ability to utilize lactose, when examined in the same manner as the lactose-fermenting strains, were not observed to contain these supercoiled, circular DNA molecules. Thus the DNA of the lac elements is maintained in E. coli WR3026 in the supercoiled, circular form. Molecular weights of the supercoiled, circular molecules isolated from strains carrying the lac elements were determined by sucrose density gradient centrifugation to be 30 million to 56 million. The calculated number of copies per chromosome of the lac elements varied from 1.4 to 3.7, depending upon the particular lac element examined. Each of the elements was determined to have a guanine plus cytosine composition of 50%. All six of the E. coli WR3026 strains containing a transmissible lac element were tested with the E. coli male-specific phage, R-17, and the E. coli female-specific phage, II, and did not respond to either of these phages as do F-containing derivatives of E. coli K-12.     

北京地区芦苇资源状态及其多样性

为探明北京地区芦苇(Phragmites australis)的资源状态和多样性, 实地考察北京主要河流、湿地和水库, 发现北京地区芦苇总生长面积已超过600 hm²。芦苇染色体倍性以八倍体为主, 四倍体次之。在面积较大的湿地内, 八倍体单一芦苇群落占据优势地位; 而在城市的浅河内有形态和遗传性多样的混合种群。研究表明, 植物性状和倍性水平之间无显著相关性。在小清河发现了6种形态各异的芦苇克隆, 均属于叶绿体DNA片段的P单倍型; 其单倍体基因组大小为(0.499±0.019) pg, 变异系数为3.8%。这表明表型与单倍型之间也不具相关性。此外, 发现1个具有变叶特性的芦苇, 将其命名为金条芦苇。北京地区芦苇形态和遗传多样性为研究芦苇基因型与环境适应性之间的关系提供了珍贵的资源。     

Characterization of Two Compatible Small Plasmids from Sphingobium yanoikuyae

We isolated and characterized two small cryptic indigenous plasmids, pYAN-1 (4,896 bp) and pYAN-2 (4,687 bp), from Sphingobium yanoikuyae, and developed a versatile system that permitted genetic manipulation of the genus Sphingomonas. Nucleotide sequencing of both plasmids revealed that they contained mobA, mobs, and repA genes, which are predicted to encode proteins associated with mobilization and replication, in common. Transformation with each plasmid harboring the antibiotic resistance gene by electroporation was fully successful, using Novosphingobium capsulatum as a host.     

Characterization of two compatible small plasmids from Sphingobium yanoikuyae

We isolated and characterized two small cryptic indigenous plasmids, pYAN-1 (4,896 bp) and pYAN-2 (4,687 bp), from Sphingobium yanoikuyae, and developed a versatile system that permitted genetic manipulation of the genus Sphingomonas. Nucleotide sequencing of both plasmids revealed that they contained mobA, mobs, and repA genes, which are predicted to encode proteins associated with mobilization and replication, in common. Transformation with each plasmid harboring the antibiotic resistance gene by electroporation was fully successful, using Novosphingobium capsulatum as a host.     

Morphology and Genetic Diversity of Phragmites australis in Beijing

为探明北京地区芦苇(Phragmites australis)的资源状态和多样性, 实地考察北京主要河流、湿地和水库, 发现北京地区芦苇总生长面积已超过600 hm²。芦苇染色体倍性以八倍体为主, 四倍体次之。在面积较大的湿地内, 八倍体单一芦苇群落占据优势地位; 而在城市的浅河内有形态和遗传性多样的混合种群。研究表明, 植物性状和倍性水平之间无显著相关性。在小清河发现了6种形态各异的芦苇克隆, 均属于叶绿体DNA片段的P单倍型; 其单倍体基因组大小为(0.499±0.019) pg, 变异系数为3.8%。这表明表型与单倍型之间也不具相关性。此外, 发现1个具有变叶特性的芦苇, 将其命名为金条芦苇。北京地区芦苇形态和遗传多样性为研究芦苇基因型与环境适应性之间的关系提供了珍贵的资源。     

Characterization of nutrient elements at different leaf positions in Phragmites australis in Songnen degraded grassland

对松嫩草地不同退化程度样地的芦苇(Phragmites australis)各叶位叶片的生长及营养元素代谢特征进行分析, 以探讨土壤盐碱化对芦苇叶片营养元素代谢的影响及其适应机制。结果表明: 松嫩草地土壤中Na ⁺含量、全盐含量、pH值是衡量土壤盐碱化程度的主要决定因子, 从典型草地到重度退化草地, 土壤盐碱化程度逐级加剧。芦苇具有一定程度的耐盐碱性, 植株高度和地上部分生物量随土壤盐分增加而降低。检测出10种营养元素: K、Na、Ca、Mg、Fe、Cu、Zn、Mn、P、B, 主成分分析结果显示全部样本均处于95%的置信区间内。方差分析结果表明, 芦苇不同叶位叶片对营养元素的富集能力有所差异。K、P含量随叶位降低而减少; 而Na、Ca、Mg呈现相反分布规律。Fe、Cu集中分布在功能叶和老叶中; Mn只大量聚集在老叶中; 而Zn集中分布在幼叶中。表明土壤盐碱化对老叶营养元素的影响大于幼叶, Na在老叶中的大量积累保护了幼叶免于或者减轻离子的毒害。功能叶和老叶中Ca、Mg、Fe、Cu的积累有利于保障芦苇正常的光合作用。盐碱胁迫下幼叶仍维持较高K、P含量, 这不仅为幼叶的生长提供所需营养, 同时提高了其抗逆性, 这可能是芦苇的生理响应策略。     

松嫩退化草地芦苇不同叶位叶片营养元素代谢特征

对松嫩草地不同退化程度样地的芦苇(Phragmites australis)各叶位叶片的生长及营养元素代谢特征进行分析, 以探讨土壤盐碱化对芦苇叶片营养元素代谢的影响及其适应机制。结果表明: 松嫩草地土壤中Na ⁺含量、全盐含量、pH值是衡量土壤盐碱化程度的主要决定因子, 从典型草地到重度退化草地, 土壤盐碱化程度逐级加剧。芦苇具有一定程度的耐盐碱性, 植株高度和地上部分生物量随土壤盐分增加而降低。检测出10种营养元素: K、Na、Ca、Mg、Fe、Cu、Zn、Mn、P、B, 主成分分析结果显示全部样本均处于95%的置信区间内。方差分析结果表明, 芦苇不同叶位叶片对营养元素的富集能力有所差异。K、P含量随叶位降低而减少; 而Na、Ca、Mg呈现相反分布规律。Fe、Cu集中分布在功能叶和老叶中; Mn只大量聚集在老叶中; 而Zn集中分布在幼叶中。表明土壤盐碱化对老叶营养元素的影响大于幼叶, Na在老叶中的大量积累保护了幼叶免于或者减轻离子的毒害。功能叶和老叶中Ca、Mg、Fe、Cu的积累有利于保障芦苇正常的光合作用。盐碱胁迫下幼叶仍维持较高K、P含量, 这不仅为幼叶的生长提供所需营养, 同时提高了其抗逆性, 这可能是芦苇的生理响应策略。