Microbial diversity in soil: effect of releasing genetically engineered micro-organisms

This review examines the potential for change in microbial diversity, with the emphasis on bacteria, in soil resulting from the introduction of genetically engineered microorganisms (GEMs). With the advent of GEMs came the impetus for new technologies to recover these micro-organisms from soil and to assess their effects on microbial diversity. This review also presents general aspects of and genetic approaches to accessing bacterial diversity in the environment.     

Release of genetically engineered insects: a framework to identify potential ecological effects

Genetically engineered (GE) insects have the potential to radically change pest management worldwide. With recent approvals of GE insect releases, there is a need for a synthesized framework to evaluate their potential ecological and evolutionary effects. The effects may occur in two phases: a transitory phase when the focal population changes in density, and a steady state phase when it reaches a new, constant density. We review potential effects of a rapid change in insect density related to population outbreaks, biological control, invasive species, and other GE organisms to identify a comprehensive list of potential ecological and evolutionary effects of GE insect releases. We apply this framework to the Anopheles gambiae mosquito – a malaria vector being engineered to suppress the wild mosquito population – to identify effects that may occur during the transitory and steady state phases after release. Our methodology reveals many potential effects in each phase, perhaps most notably those dealing with immunity in the transitory phase, and with pathogen and vector evolution in the steady state phase. Importantly, this framework identifies knowledge gaps in mosquito ecology. Identifying effects in the transitory and steady state phases allows more rigorous identification of the potential ecological effects of GE insect release.     

Mice fed on a diet enriched with genetically engineered multivitamin corn show no sub‐acute toxic effects and no sub‐chronic toxicity

Multivitamin corn is a novel genetically engineered variety that simultaneously produces high levels of β‐carotene, ascorbate and folate, and therefore has the potential to address simultaneously multiple micronutrient deficiencies caused by the lack of vitamins A, B9 and C in developing country populations. As part of the development process for genetically engineered crops and following European Food Safety Authority (EFSA) recommendations, multivitamin corn must be tested in whole food/feed sub‐chronic animal feeding studies to ensure there are no adverse effects, and potential allergens must be identified. We carried out a 28‐day toxicity assessment in mice, which showed no short‐term sub‐acute evidence of diet‐related adverse health effects and no difference in clinical markers (food consumption, body weight, organ/tissue weight, haematological and biochemical blood parameters and histopathology) compared to mice fed on a control diet. A subsequent 90‐day sub‐chronic feeding study again showed no indications of toxicity compared to mice fed on control diets. Our data confirm that diets enriched with multivitamin corn have no adverse effects on mice, do not induce any clinical signs of toxicity and do not contain known allergens.     

Carbon source utilization of soil extracted microorganisms as a tool to detect the effects of soil supplemented with genetically engineered and non-engineered Corynebacterium glutamicum and a recombinant peptide at the community level

Abstract: Substrate utilization of microbial cells extracted from soil with a 0.85% aqueous sodium chloride solution, was determined to estimate effects on soil microorganisms at the community level with microtiter plates (Biolog GN®) containing 95 different sources of organic carbon. A consistent pattern of utilized substrates was obtained after 24 h of microtiter plate incubation at 28°C. The absorbance values (OD₅₉₀) obtained from a microtiter plate reader after background correction were transformed by using the average absorbance values of oxidized substrates as a threshold to distinguish between well utilized and poorly or non-utilized substrates and thereby reduce variances between replicates. Doubling times of the extracted soil microorganisms in the microtiter plates were tested with 12 substrates and ranged from 1.96 h to 3.23 h, depending on the carbon source. The carbon source utilization assay was used to assess the effects of soil inoculation with Corynebacterium glutamicum with and without a genetically engineered plasmid (pUN1; 6.3 kb), which encoded for the synthesis of the mammalian protease inhibiting peptide, aprotinin. Additionally, aprotinin itself was added at two concentrations to soil samples. An identical decrease in the number of carbon sources utilized, especially carbohydrates, occurred upon soil inoculation with both C. glutamicum strains after inoculation with 10⁶ cells g⁻¹ soil. This effect was only detectable during the first three weeks of incubation, as long as cell numbers of C. glutamicum (pUN1) were above 10⁵ cfu g⁻¹. Soil amendment with aprotinin resulted in utilization of additional substrates, most of them carbohydrates. With 0.1 mg aprotinin g⁻¹ soil this stimulation lasted 2 days and with 10 mg g⁻¹ it lasted for 7 days.     

Rapid method for the detection of genetically engineered microorganisms by polymerase chain reaction from soil and sediments

A rapid and sensitive method for the detection of genetically engineered microorganisms in soil and sediments has been devised by in vitro amplification of the target DNAs by a polymerase chain reaction. A cloned catechol 2,3-dioxygenase gene located on the recombinant plasmid pOH101 was transferred to Pseudomonas putida MMB2442 by triparental crossing and used as a target organism. For the polymerase chain reaction from soil and sediment samples, the template DNA was released from a 100-mg soil sample. Bacterial seeded soil samples were washed with Tris-EDTA buffer (pH 8.0) and treated with a detergent lysis solution at 100°C. After addition of 1% polyvinylpolypyrrolidine solution, the samples were boiled for 5 min. Supernatant containing nucleic acid was purified with a PCR purification kit. The purified DNA was subjected to polymerase chain reaction, using two specific primers designed for the amplification of catechol 2,3-dioxygenase gene sequences. The detection limit was 10² cells per gram of soil. This method is rapid and obviates the need for lengthy DNA purification from soil samples. Received 28 February 1997/ Accepted in revised form 23 November 1997     

转基因抗虫作物对捕食性瓢虫的安全性研究进展

转基因抗虫作物的商业化种植带来了显著的经济、生态和社会效益,对转基因抗虫作物的安全性评价也一直是国内外学者研究的热点。对生物非靶标效应的研究是转基因抗虫作物安全性评价的重要组成部分,农田天敌类生物是其中的重点内容之一。瓢虫是农田生态系统重要的捕食性天敌,评价其在转基因抗虫作物田间是否能通过捕食猎物或取食花粉而接触到杀虫蛋白并富集于体内,进而对自身产生一定的非靶标效应,这对捕食性瓢虫的安全性研究具有重要的意义。本文从捕食性天敌瓢虫的生命表参数、行为功能参数、田间群落参数及体内微环境指标等方面综述了转基因抗虫作物对瓢虫的安全性研究进展,并对后期转基因抗虫作物对田间捕食性天敌的研究方向提出了建议,以期为转基因抗虫作物的环境安全性研究提供理论指导和为进一步完善转基因抗虫作物对瓢虫安全性评价的技术体系提供系统的数据资料。     

Evaluation of the potential exposure of butterflies to genetically modified maize pollen in protected areas in Italy

Environmental impacts of genetically modified crops are mandatorily assessed during their premarket phase. One of the areas of concern is the possible impact on nontarget organisms. Crops expressing Cry toxins might affect Lepidoptera larvae living outside cultivated fields, through pollen deposition on wild plants, which constitute their food source. While pollen toxicity varies among different events, possible exposure of nontarget species depends on the agro‐environmental conditions. This study was conducted in two protected areas in Italy, characterized by different climatic conditions, where many Lepidoptera species thrive in proximity to maize cultivations. To estimate the possible exposure in absence of the actual stressor (e.g., Cry1‐expressing maize plants), we conducted a two‐year field survey of butterflies and weeds. Indicator species were selected—Aglais (Inachis) io in the Northern site and Vanessa cardui in the Southern site—and their phenology was investigated. Pollen dispersal from maize fields was measured by collection in Petri dishes. Duration and frequency of exposure was defined by the overlap between pollen emission and presence of larvae on host plants. Different risk scenarios are expected in the two regions: highest exposure is foreseen for A. io in the Northern site, while minimal exposure is estimated for V. cardui in the Southern site. In the latter case, locally grown maize cultivars flower in mid‐summer in coincidence with an aestivation period for several butterfly species due to hot and dry conditions. Moreover, host plants of V. cardui are at the end of their life cycle thus limiting food availability.     

Influence of introducing the genetically modified strain <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> ACH-5 on the structure of the soil microbial community

To study the influence of genetically modified microorganisms (GMM) on the number and structure of the soil microbial community, we introduced the genetically modified strain of Sinorhizobium meliloti into soil under controlled laboratory conditions. The analysis of the dynamics of soil microorganisms of all the main groups (archaea, bacteria, fungi) using the PCR with real-time detection and the analysis of the species structure of all the indicated components using T-RFLP were carried out for a month. The results of the quantitative PCR demonstrated that none of the components of the soil microbial community was appreciably influenced by the GMM introduced. The number of GMM decreased over a month more than 300-fold. The analysis of the dynamics of the eubacterial, archaeal, and fungal communities using T-RFLP did not detect fundamental changes in their structure.     

The influence of moisture on microbial transport, survival and 2,4-D biodegradation with a genetically marked Burkholderia cepacia in unsaturated soil columns

The influence of moisture on the survival, movement anddegradation activity of a 2,4-D degrading bacterium,Burkholderia cepacia strain BRI6001L, geneticallyengineered to contain bioluminescent and lactoseutilization genes, was studied in unsaturated soil columns.The distance traveled by BRI6001L was dependent on theclay content of the soil, higher clay contents beingresponsible for higher filtration coefficients. Long termsurvival, in excess of one year, was attributed to strainBRI6001L's ability to survive dry conditions. Changes inthe 2,4-D biodegradation rate showed a better correlationwith the BRI6001L population density than with the totalviable bacterial population. At moisture levels betweenfield capacity and 40% moisture (– 33 kPa to –100 kPa)2,4-D degradation was attributed mainly to BRI6001L. Atmoisture levels between 6 and 15%, 2,4-D disappearancewas attributed to the indigenous microbial population,with no degradation occurring at moisture levels below6%. Returning the moisture to above 40% led to anincrease of 4 orders of magnitude in the BRI6001Lpopulation density and to a 10-fold increase in the 2,4-Ddegradation rate. The ability to monitor a specificmicrobial population using reporter genes hasdemonstrated the importance of controlling moisturelevels for maximizing biodegradation rates in unsaturatedsoil environments.     

Bt水稻秸秆还田对赤子爱胜蚓生长发育和生殖的影响

Bt蛋白能通过转Bt基因作物的秸秆还田进入土壤,进而可能会对土壤动物如蚯蚓的生长发育和生殖造成影响.为评估Bt水稻对赤子爱胜蚓的影响,本文模拟秸秆还田,在土壤中添加2.5%、5%、7.5%和10% Bt水稻(b2B138)及其同源水稻(安丰A)秸秆,分别在饲养赤子爱胜蚓7、15、30、45、60、75和90 d后观测蚯蚓的存活率、相对生长率和生殖情况,以及秸秆土壤混合物和蚯蚓体内的Cry1Ab蛋白含量.结果表明:较高还田量(7.5%和10%)Bt水稻秸秆处理对赤子爱胜蚓存活率有抑制作用;Bt水稻秸秆还田对赤子爱胜蚓的相对生长率没有不利影响;还田量为5%、7.5%和10%时,Bt水稻秸秆还田能促进蚯蚓的生殖.酶联免疫吸附测定法(ELISA)结果表明: 在Bt水稻土壤混合物中,蚯蚓体内均能检测到Cry1Ab蛋白,且前者随着时间延长而显著减少.因此,还田量为2.5%和5%时,Bt水稻秸秆还田释放的Cry1Ab蛋白对赤子爱胜蚓的生长发育和生殖没有不利影响.     

Bt水稻秸秆还田对赤子爱胜蚓生长发育和生殖的影响

Bt蛋白能通过转Bt基因作物的秸秆还田进入土壤,进而可能会对土壤动物如蚯蚓的生长发育和生殖造成影响.为评估Bt水稻对赤子爱胜蚓的影响,本文模拟秸秆还田,在土壤中添加2.5%、5%、7.5%和10% Bt水稻(b2B138)及其同源水稻(安丰A)秸秆,分别在饲养赤子爱胜蚓7、15、30、45、60、75和90 d后观测蚯蚓的存活率、相对生长率和生殖情况,以及秸秆土壤混合物和蚯蚓体内的Cry1Ab蛋白含量.结果表明:较高还田量(7.5%和10%)Bt水稻秸秆处理对赤子爱胜蚓存活率有抑制作用;Bt水稻秸秆还田对赤子爱胜蚓的相对生长率没有不利影响;还田量为5%、7.5%和10%时,Bt水稻秸秆还田能促进蚯蚓的生殖.酶联免疫吸附测定法(ELISA)结果表明: 在Bt水稻土壤混合物中,蚯蚓体内均能检测到Cry1Ab蛋白,且前者随着时间延长而显著减少.因此,还田量为2.5%和5%时,Bt水稻秸秆还田释放的Cry1Ab蛋白对赤子爱胜蚓的生长发育和生殖没有不利影响.     

旅游干扰对土壤生态系统的影响研究进展

为了解旅游对土壤生态环境的影响,综述了近40年来有关旅游干扰对土壤环境因子、植物、土壤动物和土壤微生物影响的研究进展。旅游干扰致使土壤紧实度、容重、pH、重金属含量升高,氮(N)、磷(P)、水分、有机质含量降低;旅游干扰导致地表植物的高度、盖度、多样性、丰富度、均匀度下降,伴人种植物增多;游客活动区土壤动物的种类、数量、密度均小于缓冲区和背景区,土壤线虫、节肢动物对旅游干扰比较敏感;旅游干扰使土壤酶活性、微生物丰度与多样性显著下降。旅游干扰对土壤生态系统的上述影响与游道、步道等游客活动中心地带的距离呈负相关。将来可构建旅游地生态数据库,从个体、细胞、分子等微观水平深入探讨旅游干扰对土壤生态系统的胁迫机理,发展旅游与环境的和谐共生关系,实现旅游业的可持续发展。     

Evaluation of the field impacts of simulated Bacillus thuringiensis‐transgenic Pinus radiata on nontarget native Lepidoptera and their natural enemies in a New Zealand plantation forest

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Prevalence of pSmeSM11a-like plasmids in indigenous Sinorhizobium meliloti strains isolated in the course of a field release experiment with genetically modified S. meliloti strains

Plasmid pSmeSM11a, residing in the indigenous Sinorhizobium meliloti strain SM11 originating from a field in Strassmoos (Bavaria, Germany), was analysed previously at the genomic level. Thirty-seven indigenous S. meliloti strains, originating from two different locations in Germany, were screened for genes identified previously on pSmeSM11a. Seven of these strains harbour accessory plasmids that are very similar to pSmeSM11a. The identified pSmeSM11a-like plasmids are c. 130-150 kb in size and possess nearly identical restriction profiles. Up to 30 genes identified previously on pSmeSM11a could be detected on these plasmids by hybridisation experiments, e.g., the nodulation genes nodP and nodQ, the ethylene level modulation gene acdS and the taurine metabolism gene tauD. A few pSmeSM11a genes were also detected on other plasmids. The reference plasmid pSmeSM11a contains a region that is similar to a segment of S. meliloti strain Rm1021 pSymA. Regions with similarity to pSymA were also detected on the aforementioned seven pSmeSM11a-like plasmids. The specifications of these regions are nearly identical to the one on pSmeSM11a and differ from Rm1021 pSymA as determined by nucleotide sequence analysis. Two further plasmids similar to pSmeSM11a completely lack the pSymA-region. Those strains carrying accessory plasmids that contain the acdS gene encoding 1-aminocyclopropane-1-carboxylate deaminase are able to grow on 1-aminocyclopropane-1-carboxylate as the sole source of nitrogen, demonstrating functionality of the acdS gene product. About 36% of the analysed plasmids, including three pSmeSM11a-like plasmids, could be transferred to another S. meliloti recipient strain, allowing for their dissemination in S. meliloti populations.     

Optimising the capacity of field trials to detect the effect of genetically modified maize on non‐target organisms through longitudinal sampling

To assess risks of cultivation of genetically modified crops (GMCs) on non‐target arthropods (NTAs), field tests are necessary to verify laboratory results and in situations where exposure pathways are very complex and cannot be reproduced in the laboratory. A central concern in the design of field trials for this purpose is whether the tests are capable of detecting differences in the abundance or activity of NTAs in a treated crop in comparison with a non‐treated comparator plot. The detection capacity of a trial depends on the abundance and variability of the taxon, the values assumed for type I (α) and II (β) errors, and the characteristics of the trial and statistical design. To determine the optimal trial layout and statistical analysis, 20 field trials carried out in Spain from 2000 to 2009 to assess risks of GMCs on NTAs were examined with α and β set at 0.05 and 0.20, respectively. In this article we aim to determine the optimal number of sampling dates during a season, or longitudinal samples, in the design of field trials for assessing effects of GM maize on NTAs, and the ones that contribute most to achieving detectable treatment effects (d_c) less than 50% of the mean of the control. Detection capacities are a function of the number of individual samples taken during the season but a high number of samples is rarely justified because gains of repeated sampling can be relatively low. These gains depend primarily on field tests relative experimental variability in individual samplings (i.e. experimental variability relative to the mean of the control in each sampling date) which in turn depends on the sampling method (visual counts, pitfall traps or yellow sticky traps) and the density (or abundance) of the taxon in question. Taxa showing more density (or abundance) have less relative experimental variability. The smaller the experimental variability, the lower the profit of increasing the number of sampling dates. Sticky traps have a good effect detection capacity and need very few sampling dates, whereas visual counts and pitfall traps have a poorer effect detection capacity and need more individual samples to achieve d_c values lower than 50%. In maize field trials, it is recommended to concentrate sampling efforts in certain growth stages; the optimal ones for achieving an acceptable detection capacity are variable but, in general, samples in the first half of the season render better detection capacity than samples in the second half.     

生源要素有效性及生物因子对湿地土壤碳矿化的影响

湿地土壤是全球碳存储的重要场所,湿地生态系统的碳循环过程对全球变化有重要指示作用。土壤碳矿化是湿地生态系统碳循环的重要环节,对于认知湿地生态系统生物地球化学循环过程具有重要的意义。综述了生源要素及生物因素对湿地土壤碳矿化的内在作用机制。土壤活性有机碳库通过调节土壤能源物质和微生物活性影响土壤碳库的有效性,是表征土壤碳矿化的敏感指标。湿地其它养分如N、P、S等元素的有效性也是影响土壤碳矿化的关键要素。电子受体(NO₃^-、SO₄^2-、Fe³⁺、Mn⁴⁺等)对湿地土壤碳矿化和有机碳转变的影响主要通过电子受体的还原过程完成,在厌氧分解过程中,湿地土壤利用难溶性电子受体可能是土壤C矿化的更重要途径。动物、植物、微生物群落和区系等则是土壤碳矿化的主要驱动因子。土壤动物区系在有机态养分矿化为无机态养分的过程有着独特的功能,能显著增加土壤碳矿化。土壤微生物的活性,决定着土壤中有机碎屑的降解速率,是土壤有机碳分解周转的主要诱导因素。湿地植物则通过影响根系、微生物呼吸底物的供应以及对小气候和土壤因子的调节而影响土壤有机质的分解。湿地生源要素和生物因子还极易与土壤理化性质如温度、水分、pH值和质地等环境因素形成交互和制约,共同影响土壤碳矿化。最后,提出了进一步研究生源要素和生物因素与湿地土壤碳矿化关系需要解决的一些重要问题。     

Evaluation of PAHs concentration and cancer risk assessment on human health in a roadside soil: A case study

Abstract

An explanatory study was carried out to divulge the sources, contamination level of different classes of Polycyclic Aromatic Hydrocarbons (PAHs) distribution and the impact of vehicular traffic on the roadside soil by assessing incremental lifetime cancer risk at each site to understand the potential health risk of nearby residents along the National Highway-2 Delhi–Kolkata India. Comparison of the cancer risk assessment was performed using Monte Carlo simulation for the entire study area. The results revealed 90% cancer risk value of 6.40?×?10^?5 and 6.5?×?10^?5 for children and adults, respectively, whereas, without simulation the Total Cancer Risk (TCR) for adults was 6.925?×?10^?5 and 6.220?×?10^?5 for children, observed maximum at the location (S5). The dilemma of risk assessment indicating profoundly contaminated soil. Comparison of PAHs concentration with the background values of PAHs ranged from 1.478 to 27.493?mg kg^?1. The (IP/BgP) ratio specified that the PAHs content of the highway roadside sample is preponderate by diesel vehicle emission, biomass combustion and coal combustion. The study clearly revealed and advocated the influence of organic and inorganic pollution, which aggravates and causes health issues to the nearby inhabitants. This study could also be advantageous to similar consequences seen elsewhere in the world.