Survival and dispersion of genetically modified rhizobia in the field and genetic interactions with native strains

Abstract A genetically modified strain of the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum biovar viciae was used to inoculate a typical host, pea, and a control non-host cereal crop in the field. The inoculant was monitored for survival and spread from the site of application, and for genetic interactions with the native population. It could be identified by chromosomally located antibiotic resistance markers and additional markers conferred by the transposon Tn 5 inserted on its conjugative symbiotic plasmid. These markers facilitated enumeration of the strain on selective agar, enabling survival and spread to be monitored over a six year period. Although culturable cell numbers dropped two to three orders of magnitude after the first year, subsequently they remained around 10² viable cells per g soil, even in subplots where only the non-host cereals had been grown. However, peas did give the inoculant a small survival advantage compared with non-hosts. Soil cultivation appeared to play a major role in inoculant dissemination from the site of application. Transfer of the Tn 5 marker to other rhizobia could be monitored by screening for isolates with Tn 5 -encoded antibiotic resistance in the absence of the inoculant chromosomal markers. Over three years, more than 4000 pea root nodules were screened for indigenous rhizobia that had acquired the Tn 5 -marked symbiotic plasmid from the inoculant. None were detected, although overall about 2% of nodules contained the inoculant strain, and transfer of the Tn 5 -marked symbiotic plasmid to three out of four R. leguminosarum biovar viciae isolates from the field site could be demonstrated under laboratory conditions.     

中华根瘤菌自体诱导物合成酶基因的筛选及其在大肠杆菌中的表达

通过携带有mariner转座子的质粒pJZ290随机插入诱变中华根瘤菌(Sinorhizobium meliloti)建立突变子文库,并从中筛选到自体诱导物(autoinducer,AI)部分缺失突变株YW1。Arbitrary PCR扩增、DNA测序得到YW1基因组DNA中mariner转座子两端侧翼序列,经DNA序列拼接在GenBank上进行同源性分析后获得一个621bp的完整的开放阅读框(ORF),该ORF编码的酶具有206个氨基酸,与草木樨中华根瘤菌(Sinorhizobium medicae)WSM419的LuxI类自体诱导物合成酶(autoinducer synthase)TraI的同源性高达99%。因此,也将该基因命名为traⅠ。将该基因克隆到广宿主范围表达载体pYC12并在大肠杆菌Escherichia coli DH5α中成功表达,C18反相薄层层析(TLC)在阳性重组子培养上清中检测到四种自体诱导物分子,其中的两种正是AI缺失突变株YW1所缺失的AI,这些结果表明该traⅠ基因在苜蓿中华根瘤菌负责合成两种自体诱导物分子,为进一步研究其群体感应系统奠定了理论基础。     

Elevated atmospheric CO2 alters microbial population structure in a pasture ecosystem

An increase in concentration of atmospheric CO₂ is one major factor influencing global climate change. Among the consequences of such an increase is the stimulation of plant growth and productivity. Below‐ground microbial processes are also likely to be affected indirectly by rising atmospheric CO₂ levels, through increased root growth and rhizodeposition rates. Because changes in microbial community composition might have an impact on symbiotic interactions with plants, the response of root nodule symbionts to elevated atmospheric CO₂ was investigated. In this study we determined the genetic structure of 120 Rhizobium leguminosarum bv. trifolii isolates from white clover plants exposed to ambient (350 μmol mol^?1) or elevated (600 μmol mol^?1) atmospheric CO₂ concentrations in the Swiss FACE (Free‐Air‐Carbon‐Dioxide‐Enrichment) facility. Polymerase Chain Reaction (PCR) fingerprinting of genomic DNA showed that the isolates from plants grown under elevated CO₂ were genetically different from those isolates obtained from plants grown under ambient conditions. Moreover, there was a 17% increase in nodule occupancy under conditions of elevated atmospheric CO₂ when strains of R. leguminosarum bv. trifolii isolated from plots exposed to CO₂ enrichment were evaluated for their ability to compete for nodulation with those strains isolated from ambient conditions. These results indicate that a shift in the community composition of R. leguminosarum bv. trifolii occurred as a result of an increased atmospheric CO₂ concentration, and that elevated atmospheric CO₂ affects the competitive ability of root nodule symbionts, most likely leading to a selection of these particular strains to nodulate white clover.     

Development of a cultivation-independent approach for the study of genetic diversity of Sinorhizobium meliloti populations

The development of a species-specific marker for the analysis of the genetic polymorphism of the nitrogen-fixing symbiotic bacterium Sinorhizobium meliloti directly from environmental DNA is reported. The marker is based on terminal-restriction fragment length polymorphism (T-RFLP) methodology targeting specifically the 16S-23S Ribosomal Intergenic Spacer of S. meliloti. Species-specificity and polymorphism of the marker were tested on DNA extracted from soil samples and from a collection of 130 S. meliloti bacterial isolates. These primers and the T-RFLP approach proved useful for the detection and analysis of polymorphism of S. meliloti populations.     

Monitoring genetically modified rhizobia in field soils using the polymerase chain reaction

Monitoring genetically modified (GM) bacterial inoculants after field release using conventional culture methods can be difficult. An alternative is the detection of marker genes in DNA extracted directly from soil, using specific oligonucleotide primers with the polymerase chain reaction (PCR). The PCR was used to monitor survival of two GM Rhizobium leguminosarum bv. viciae inoculants after release in the field at Rothamsted. One strain, RSM2004, is marked by insertion of transposon Tn 5 ; the second strain, CT0370, released at the same site, is modified by chromosomal integration of a single copy of the gene from E. coli conferring GUS activity. Both GM strains provide a realistic case study for the development of PCR-based detection techniques. Specific primers were developed to amplify regions of the Tn 5 and GUS genetic markers using PCR and conditions optimized for each primer set to routinely detect a signal from 10 fg of purified template DNA, the equivalent of one cell per reaction. Procedures to improve the sensitivity of detection are described, to detect fewer than 50 cells g⁻¹ soil in soil-extracted DNA.     

新疆濒危野生樱桃李的种群结构与动态

为了解野生樱桃李种群结构和种群生态现状,采用“空间序列代替时间变化”的方法,对新疆天然分布区内的野生樱桃李不同种群的径级结构、冠幅结构及株高结构进行了分析,运用静态生命表描述了野生樱桃李种群年龄结构的动态变化规律.结果表明,樱桃李天然种群的株高和冠幅结构呈现为一定的衰退趋势,冠幅在总体呈现为近乎对数型的衰减趋势;种群的径级结构趋于稳定型,但种群结构表现出较强的波动性,种群衰退风险较高;同时,不同样地种群因所处微环境等的差异,在径级、株高和冠幅结构上表现出一定差异.野生樱桃李种群的存活曲线接近于B1型(台阶型),说明幼龄的死亡率较高,且不同生活史时期的死亡率差异较大.新疆的野生樱桃李天然种群表现出一定的不稳定性,亟待补充低龄级的个体,并减少对成年树的砍伐破坏,从而为种群的保育和恢复提供保障.     

Population dynamics of ticks on indigenous cattle in a pastoral dry to semi-arid rangeland zone of Uganda

Studies on seasonality and population dynamics of ticks on indigenous cattle and their crosses (calves) were carried out in Buruli Ranching Scheme, Nabiswera, Luwero district of Uganda on three treatment groups of animals: group 1 (twice a week dipping), group 2 (once a month dipping) and group 3 (no tick control). During this study, four major species of ticks of economic importance were recorded in decreasing order of abundance: Rhipicephalus appendiculatus, Rhipicephalus evertsi evertsi, Amblyomma variegatum and Boophilus decoloratus. Of these ticks, a seasonal pattern of activity was only observed in R. appendiculatus ticks, with peak activities occurring during rainy seasons. Greater numbers of ticks were recorded on cows than calves in the three treatment groups, with the exception of A. variegatum where the reverse occurred. The mean numbers of ticks per animal were highly significantly different (p < 0.01) when group 1 animals were compared with group 2 and 3 animals. However, no significant differences (p > 0.05) were observed in mean tick numbers between group 2 and 3 animals. Highly significant differences (p < 0.01) were observed in mean tick numbers on cows and calves (more than 12 months old) in different calving seasons. The state of lactation only affected tick counts on cows in group 1; significantly more ticks (p < 0.01) were observed in lactating than non-lactating cows. Furthermore, significantly greater (p < 0.05) numbers of ticks (with the exception of B. decoloratus) were recorded during the second year of study (March 1992-May 1993) than the first year (January 1991-February 1992), despite lower rainfall during the former period.     

黄山松种群结构与动态研究

以“空间代替时间”的方法,以种群径级结构代替年龄结构,采用静态生命表和生存分析的方法,探讨了天柱山国家森林公园黄山松种群结构和动态规律。结果表明,随海拔升高,黄山松种群密度增大,径级分布范围减小,种群存活曲线由Deevey-Ⅱ型之间过渡到Deevey—Ⅰ型,种群由稳定型过渡为增长型。表明由低海拔向高海拔,黄山松种群处于不同的演替和发展阶段。     

Population dynamics: Limits of predictability

Problems related to the complex pattern of ecosystem dynamics are discussed. Examples of studies on the complex population dynamics are considered, including those of plankton populations in a spatially heterogeneous environment and of an agroecosystem invaded by pests resistant to Bt toxins produced by genetically modified insecticidal crops.     

The dynamics of Rhizobium leguminosarum biovar trifolii introduced into soil as determined by immunofluorescence and selective plating techniques

Abstract After the introduction of Rhizobium leguminosarum biovar trifolii into a loamy sand and a silt loam, high recovery percentages were determined using quantitative immunofluorescence. Soil type, but not inoculum density between 10⁴ and 10⁸ cells per gram of soil, significantly influenced the recovery percentage of the immunofluorescence technique. Recovery percentages determined using selective plating were independent of either soil type or inoculum density and exceeded those determined by immunofluorescence.
The serological and genetic markers used for detection were stable during 55 days of incubation in phosphate-buffered saline and soil extract solution. After the introduction of R. leguminosarum biovar trifolii into both sterilized soil types, the population increased to 0.5–1×10⁹ cells per gram of soil, but a decline was demonstrated in non-sterile loamy sand and silt loam during incubation of 90 days at 15°C. Starvation of rhizobial cells in the phosphate-buffered saline and soil extract solution, as well as incubation in both soil types, resulted in a significant decrease in mean cell size.     

The construction of recA–deficient Rhizobium meliloti and R. leguminosarum strains marked with gusA or luc cassettes for use in risk–assessment studies

A vector system was developed employing the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar. viciae as target sequences for the stable genomic integration of foreign DNA. The plasmid vectors can be used either as integration vectors (single cross–over), or as gene replacement vectors (double cross–over). Gene replacement results in the antibiotic–marker–free integration of cloned DNA into the recA genes of R. meliloti and R. leguminosarum bv. viciae. Consequently, the recombinant strains become recombination deficient (RecA^-). The expression of integrated genes is under the control of the neomycin phosphotransferase II (nptll) promoter of transposon Tn5. The system was used to construct recA mutant strains of R. meliloti and R. leguminosarum by. viciae, carrying the Escherichia coli gusA gene encoding β–glucuronidase as well as the firefly (Photinus pyralis) luc gene encoding luciferase as marker genes. The GUS activity in the constructed strains was found to be absolutely stable over more than 100 generations of non–selective growth in liquid culture. The stability was also confirmed in root–nodule passages. In addition, the potential use of the luc gene as a stable genetic marker in the unequivocal identification of tagged strains among indigenous microbes in non–sterile soil was demonstrated. It is proposed to use bioluminescent recA mutants as model organisms in risk assessment studies with genetically engineered Rhizobium strains.     

子午沙鼠种群数量动态及预测

子千沙鼠（Ｍｅｒｉｏｎｅｓ ｍｅｒｉｄｉａｎｕｓ）是荒漠和半荒漠地区常见的鼠种之一,作者于１９９１～、９９５年４～１０月每月中旬,在内蒙古达拉特旗中国农业守草原研究所鄂尔多斯沙地草场改良试验站,利用直线夹日法在站内的流动消逝天,半流动消逝一,固定沙地,丘间滩地,林地和农田中开展了种群数量调查。６ａ共布放１２４２４５闪日,捕获鼠７４９８只,其中子午沙鼠１４０８只,在各种鼠中占１８．７７％,居第二位。     

湖南珍稀濒危植物——珙桐种群数量动态

在湖南省珙桐(Davidia involucrata)主要分布区八大公山和壶瓶山自然保护区设置了7个具有代表性的样地,采用样方法对珙桐群落的组成进行调查,研究珙桐种群的年龄结构、静态生命表和生存规律.结果表明:(1)两个保护区珙桐种群的径级结构和高度结构都近似于倒金字塔型,并且珙桐种群幼树死亡率高,珙桐种群有衰退迹象.(2)珙桐种群结构存在波动性,幼年阶段的个体数量少,并且多为萌生苗,实生苗很少;成年个体数量相对也较少,种群趋于Deevey的Ⅱ型和Ⅲ型曲线之间.(3)珙桐种群死亡率和消失率曲线变化趋势基本一致,均在第5龄级阶段出现一个高峰;珙桐种群的生存率单调上升,累计死亡率单调下降,生存率上升趋势前期低于后期,累计死亡率则相反.(4)珙桐种群具有前期稳定、中期锐减和后期衰退的特点.     

Population dynamics of hypolimnetic rotifers in the Pluss-see (North Germany)

The hypolimnetic rotifer populations of the Pluss-see (Keratella hiemalis, Filinia terminalis, Filinia hofmanni) show similar patterns of changing population parameters indicating that they respond to particular environmental signals in the same way.     

异迟眼蕈蚊在不同植物上的生长发育及种群参数

【目的】异迟眼蕈蚊Bradysia difformis Frey的幼虫取食为害作物的地下部分,影响作物的品质,为了明确韭菜、蚕豆、生菜、白菜和甘蓝5种植物对异迟眼蕈蚊生长发育以及繁殖的影响。【方法】本试验采用室内人工饲养测定的方法,研究了5种不同植物对异迟眼蕈蚊生长发育,繁殖力和存活率的影响,并统计了其对异迟眼蕈蚊种群参数的影响。【结果】结果表明:卵到蛹的发育历期依次为甘蓝、白菜、韭菜、生菜、蚕豆;5种植物对雌雄虫寿命影响不显著,对雌虫产卵量以及蛹重均有影响,其中在韭菜上的产卵量最大,甘蓝最少,在韭菜上蛹最重,生菜上蛹最轻;异迟眼蕈蚊的存活率随着生长发育降低,总体在韭菜上的存活率高于其他寄主植物,在生菜上的存活率均最低。统计分析不同植物对异迟眼蕈蚊种群参数的影响,净增殖率和内禀增长率在韭菜上最大而在甘蓝上最小;平均世代周期在蚕豆上最短,甘蓝上最长;种群加倍时间在韭菜上最短,而在甘蓝上最长。【结论】由此可知,异迟眼蕈蚊均可以在韭菜,蚕豆,生菜,白菜和甘蓝上完成生长发育及繁殖,其对5种植物的适应性依次为:韭菜、蚕豆、白菜、甘蓝和生菜。