Lidia S. Watrud

Bt Crop Effects on Functional Guilds of Non-Target Arthropods: A Meta-Analysis

Background Uncertainty persists over the environmental effects of genetically-engineered crops that produce the insecticidal Cry proteins of Bacillus thuringiensis (Bt). We performed meta-analyses on a modified public database to synthesize current knowledge about the effects of Bt cotton, maize and potato on the abundance and interactions of arthropod non-target functional guilds. Methodology/Principal Findings We compared the abundance of predators, parasitoids, omnivores, detritivores and herbivores under scenarios in which neither, only the non-Bt crops, or both Bt and non-Bt crops received insecticide treatments. Predators were less abundant in Bt cotton compared to unsprayed non-Bt controls. As expected, fewer specialist parasitoids of the target pest occurred in Bt maize fields compared to unsprayed non-Bt controls, but no significant reduction was detected for other parasitoids. Numbers of predators and herbivores were higher in Bt crops compared to sprayed non-Bt controls, and type of insecticide influenced the magnitude of the difference. Omnivores and detritivores were more abundant in insecticide-treated controls and for the latter guild this was associated with reductions of their predators in sprayed non-Bt maize. No differences in abundance were found when both Bt and non-Bt crops were sprayed. Predator-to-prey ratios were unchanged by either Bt crops or the use of insecticides; ratios were higher in Bt maize relative to the sprayed non-Bt control. Conclusions/Significance Overall, we find no uniform effects of Bt cotton, maize and potato on the functional guilds of non-target arthropods. Use of and type of insecticides influenced the magnitude and direction of effects; insecticde effects were much larger than those of Bt crops. These meta-analyses underscore the importance of using controls not only to isolate the effects of a Bt crop per se but also to reflect the replacement of existing agricultural practices. Results will provide researchers with information to design more robust experiments and will inform the decisions of diverse stakeholders regarding the safety of transgenic insecticidal crops.

Influence of adverse soil conditions on the formation and function of Arbuscular mycorrhizas

Abstract The majority of plants have mycorrhizal fungi associated with them. Mycorrhizal fungi are ecologically significant because they form relationships in and on the roots of a host plant in a symbiotic association. The host plant provides the fungus with soluble carbon sources, and the fungus provides the host plant with an increased capacity to absorb water and nutrients from the soil. Adverse conditions are a pervasive feature in both natural and agronomic soils. The soil environment is constantly changing with regard to moisture, temperature and nutrient availability. In addition, soil properties are often manipulated to improve crop yields. In many cases, soils may be contaminated through disposal of chemicals that are toxic to plants and microorganisms. The formation and function of mycorrhizal relationships are affected by edaphic conditions such as soil composition, moisture, temperature, pH, cation exchange capacity, and also by anthropogenic stressors including soil compaction, metals and pesticides. Arbuscular mycorrhizal fungi are of interest for their reported roles in alleviation of diverse soil-associated plant stressors, including those induced by metals and polychlorinated aliphatic and phenolic pollutants. Much mycorrhizal research has investigated the impact of extremes in water, temperature, pH and inorganic nutrient availability on mycorrhizal formation and nutrient acquisition. Evaluation of the efficacy of plant–mycorrhizal associations to remediate soils contaminated with toxic materials deserves increased attention. Before the full potential benefits of arbuscular mycorrhizal fungi to reclaim contaminated soils can be realized, research advances are needed to improve our understanding of the physiology of mycorrhizae subjected to adverse physical and chemical conditions. This paper will review literature and discuss the implications of soil contamination on formation and function of arbuscular mycorrhizal associations.

An improved method for purifying DNA from soil for polymerase chain reaction amplification and molecular ecology applications

analysis for studying microbial diversity has encouraged the development of direct soil DNA extraction methods. The advantage of the direct method is that it yields higher amounts of DNA from cells that are lysed directly rather than cells extracted from the soil and then lysed (Steffan et al. 1988). However, DNA extracted by the direct method is heavily contaminated with humic substances (Zhou et al. 1996), and various techniques have been used with varying degrees of success to remove those substances: caesium chloride, glassmilk and spearmine HCl (Smalla et al. 1993); Elutip-dTM and Sephadex G-200 columns (Tsai & Olson 1992); polyvinyl polypyrrolidone (PVPP) columns and Microcon-100TM (Amicon, Beverly, MA, USA) microconcentrators (Widmer et al. 1996); phenol, a chemical toxic to humans (Tsai & Olson 1991) and labour-intensive agarose gel electrophoresis (Zhou et al. 1996). Researchers have also attempted to increase the amount of DNA recovered from soil microbes. One approach is to use a rapid treatment such as bead beating that may shear the DNA (Picard et al. 1992). This DNA may not be usable for PCR analysis because small fragments may form chimeric hybrids during amplification (Liesack et al. 1991). The other approach is to use a longer gentle treatment generally yielding higher molecular weight DNA that may utilize sodium dodecyl sulphate (SDS), enzymes, heat, or freeze–thaw processes (Zhou et al. 1996). Our objective was to develop a method for obtaining purer, high-molecular-weight DNA to increase sensitivity with PCR and to limit the formation of mismatched hybrid DNA molecules during amplification (Liesack et al. 1991; Romanowski et al. 1993). Triplicate samples of two soil types were processed by the new method and subjected to PCR and restriction enzyme analysis to determine the extent of formation of chimeric hybrids during PCR. A direct soil DNA extraction and agarose gel purification method developed in our laboratory (Porteous et al. 1994) that isolated DNA from a wide variety of bacteria, fungi, plants and soil was modified to increase purity of PCR-ready high-molecular-weight DNA, about 20–25 kb in size (Fig. 1). The previously reported method recovered from 1 to 50 μg DNA g–1 of soil after purification. The new method has a similar recovery efficiency. The old agarose gel method was limited to yielding less than 1 μg of usable PCR-ready DNA from 100 mg initial sample (we routinely filled each well with 15 μL of the crude extract containing the DNA from 15 mg of soil). The new method (Fig. 1) yields more usable PCR-ready DNA from 500 mg initial sample (we routinely process 300 μL of the crude extract containing the DNA from 300 mg of soil). In this study 2.8 and 4.6 μg DNA g–1 (wet weight) from two soil types were recovered after purification. The previous agarose gel purification method yielded less than 0.1 μg usable PCRready DNA for both soils. However, using the new method (Fig. 1) 0.8 and 1.4 μg usable pure PCR-ready DNA were obtained. Results from similar methods indicated that 1–20 μg usable PCR-ready DNA were obtained from seven soils (Zhou et al. 1996) and 2 μg usable PCRready DNA was obtained from one soil (Tsai & Olson 1992). We suggest the differences are due primarily to differences in soil compositions and microbial populations. In the new method (Fig. 1) cells are lysed by a longer gentle treatment and all reactions are carried out in microfuge tubes. Sample weights of 500 mg were used in this study; however, soils with low biomass or significant microbial heterogeneity may require larger sample weights (Holben 1994). Additional or modified treatments T E C H N I C A L N O T E

Sensitive detection of transgenic plant marker gene persistence in soil microcosms

Genetic engineering offers the opportunity to generate plants with useful new traits conferred by genes originating from a variety of organisms. The objectives of this study were to establish methods for investigating persistence of recombinant plant marker DNA after introduction into soil and to collect data from controlled laboratory test systems. As a model system, we studied the stability of DNA encoding recombinant neomycin phosphotransferase II (rNPT‐II), a neomycin/kanamycin resistance marker, used in plant genetic engineering. The recombinant nature of the target (i.e. fusion of nopaline synthase promoter and NPT‐II coding region) allowed us to design a rNPT‐II‐specific PCR primer pair. DNA preparation and quantitative PCR protocols were established. Effects of temperature and moisture, on DNA persistence in soil were determined in two laboratory test systems. In the first system, purified plasmid DNA was added to soil and incubated under controlled conditions. Up to 0.08% of the rNPT‐II target sequences were detectable after 40 days. In the second system, fresh leaf tissue of transgenic tobacco was ground, added to soil, and incubated under controlled conditions. After 120 days, up to 0.14% of leaf tissue‐derived genomic rNPT‐II sequences were detectable. Under most experimental conditions, leaf tissue‐derived and plasmid DNA were initially degraded at a high rate. A small proportion of the added DNA resisted degradation and was detectable for several months. We hypothesize that this DNA may have been adsorbed to soil particles and was protected from complete degradation.

Establishment of transgenic herbicide‐resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats

Concerns about genetically modified (GM) crops include transgene flow to compatible wild species and unintended ecological consequences of potential transgene introgression. However, there has been little empirical documentation of establishment and distribution of transgenic plants in wild populations. We present herein the first evidence for escape of transgenes into wild plant populations within the USA; glyphosate‐resistant creeping bentgrass (Agrostis stolonifera L.) plants expressing CP4 EPSPS transgenes were found outside of cultivation area in central Oregon. Resident populations of three compatible Agrostis species were sampled in nonagronomic habitats outside the Oregon Department of Agriculture control area designated for test production of glyphosate‐resistant creeping bentgrass. CP4 EPSPS protein and the corresponding transgene were found in nine A. stolonifera plants screened from 20 400 samples (0.04 ± 0.01% SE). CP4 EPSPS‐positive plants were located predominantly in mesic habitats downwind and up to 3.8 km beyond the control area perimeter; two plants were found within the USDA Crooked River National Grassland. Spatial distribution and parentage of transgenic plants (as confirmed by analyses of nuclear ITS and chloroplast matK gene trees) suggest that establishment resulted from both pollen‐mediated intraspecific hybridizations and from crop seed dispersal. These results demonstrate that transgene flow from short‐term production can result in establishment of transgenic plants at multi‐kilometre distances from GM source fields or plants. Selective pressure from direct application or drift of glyphosate herbicide could enhance introgression of CP4 EPSPS transgenes and additional establishment. Obligatory outcrossing and vegetative spread could further contribute to persistence of CP4 EPSPS transgenes in wild Agrostis populations, both in the presence or absence of herbicide selection.

Comparison of parental and transgenic alfalfa rhizosphere bacterial communities using biolog GN metabolic fingerprinting and enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR)

A bstractRhizosphere bacterial communities of parental and two transgenic alfalfa (Medicago sativa L.) of isogenic background were compared based on metabolic fingerprinting using Biolog GN microplates and DNA fingerprinting of bacterial communities present in Biolog GN substrate wells by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR). The two transgenic alfalfa expressed either bacterial (Bacillus licheniformis) genes for alpha-amylase or fungal (Phanerochaete chrysosporium) genes for Mn-dependent lignin peroxidase (Austin S, Bingham ET, Matthews DE, Shahan MN, Will J, Burgess RR, Euphytica 85:381–393). Cluster analysis and principal components analysis (PCA) of the Biolog GN metabolic fingerprints indicated consistent differences in substrate utilization between the parental and lignin peroxidase transgenic alfalfa rhizosphere bacterial communities. Cluster analysis of ERIC-PCR fingerprints of the bacterial communities in Biolog GN substrate wells revealed consistent differences in the types of bacteria (substrate-specific populations) enriched from the rhizospheres of each alfalfa genotype. Comparison of ERIC-PCR fingerprints of bacterial strains obtained from substrate wells to substrate community ERIC-PCR fingerprints suggested that a limited number of populations were responsible for substrate oxidation in these wells. Results of this study suggest that transgenic plant genotype may affect rhizosphere microorganisms and that the methodology used in this study may prove a useful approach for the comparison of bacterial communities.

Phytoremediation of soil contaminated with low concentrations of radionuclides

Ecosystems throughout the world have been contaminated with radionuclides by above-ground nuclear testing, nuclear reactor accidents and nuclear power generation. Radioisotopes characteristic of nuclear fission, such as 137Cs and 90Sr, that are released into the environment can become more concentrated as they move up the food chain often becoming human health hazards. Natural environmental processes will redistribute long lived radionuclides that are released into the environment among soil, plants and wildlife. Numerous studies have shown that 137 Cs and 90Sr are not removed from the top 0.4 meters of soil even under high rainfall, and migration rate from the top few centimeters of soil is slow. The top 0.4 meters of the soil is where plant roots actively accumulate elements. Since plants are known to take up and accumulate 137 Cs and 90Sr removal of these radionuclides from contaminated soils by plants could provide a reliable and economical method of remediation. One approach is to use fast growing plants inoculated with mycorrhizal fungi combined with soil organic amendments to maximize the plant accumulation and removal of radionuclides from contaminated soils, followed by harvest of above-ground portion of the plants. High temperature combustion would be used to oxidize plant material concentrating 137 Cs and 90Sr, in ash for disposal. When areas of land have been contaminated with radionuclides are large, using energy intensive engineering solutions to remediate huge volumes of soil is not feasible or economical. Plants are proposed as a viable and cost effective method to remove radionuclides from the soils that have been contaminated by nuclear testing and nuclear reactor accidents.

Accumulation of 137Cs and 90Sr from contaminated soil by three grass species inoculated with mycorrhizal fungi

Abstract The use of plants to accumulate low level radioactive waste from soil, followed by incineration of plant material to concentrate radionuclides may prove to be a viable and economical method of remediating contaminated areas. We tested the influence of arbuscular mycorrhizae on 137Cs and 90Sr uptake by bahia grass (Paspalum notatum), johnson grass (Sorghum halpense) and switchgrass (Panicum virginatum) for the effectiveness on three different contaminated soil types. Exposure to 137Cs or 90Sr over the course of the experiment did not affect above ground biomass of the three grasses. The above ground biomass of bahia, johnson and switchgrass plants accumulated from 26.3 to 71.7% of the total amount of the 137Cs and from 23.8 to 88.7% of the total amount of the 90Sr added to the soil after three harvests. In each of the three grass species tested, plants inoculated with Glomus mosseae or Glomus intraradices had greater aboveground plant biomass, higher concentrations of 137Cs or 90Sr in plant tissue, % accumulation of 137Cs or 90Sr from soil and plant bioconcentration ratios at each harvest than those that did not receive mycorrhizal inoculation. Johnson grass had greater aboveground plant biomass, greater accumulation of 137Cs or 90Sr from soil and plant higher bioconcentration ratios with arbuscular mycorrhizal fungi than bahia grass and switchgrass. The greatest accumulation of 137Cs and 90Sr was observed in johnson grass inoculated with G. mosseae. Grasses can grow in wide geographical ranges that include a broad variety of edaphic conditions. The highly efficient removal of these radionuclides by these grass species after inoculation with arbuscular mycorrhizae supports the concept that remediation of radionuclide contaminated soils using mycorrhizal plants may present a viable strategy to remediate and reclaim sites contaminated with radionuclides. ©

An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonucleasePalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi includingGossypium hirsucum (cotton),Pseudomonas, Bacillus, Streptomyces, andColletotrichum. Up to 100 μg DNA/g (wet weight) of soil and 400 μg DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonucleasePalI, contained 12–20 DNA fragments, appearing as sample “fingerprints.” Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.

Transgenic perennial biofuel feedstocks and strategies for bioconfinement

The use of transgenic tools for the improvement of plant feedstocks will be required to realize the full economic and environmental benefits of cellulosic and other biofuels, particularly from perennial plants. Traits that are targets for improvement of biofuels crops include herbicide resistance, pest, drought, cold and salt tolerance, nutrient use efficiency, altered cell wall composition and improved processing and end-use characteristics. However, controlling gene flow is a major issue and there is no regulatory experience with perennial plants as dedicated biofuels feedstocks. Bioconfinement of transgenes is thus an obvious regulatory and biosafety objective to the release and commercialization of transgenic bioenergy feedstocks. In this article, we review bioconfinement strategies that target pollen or seeds that can be applied to perennial plants used as biofuels. These include male sterility, integration of transgenes into plastid genomes, removal of transgenes in pollen and seeds, transgene expression in vegetative organs for harvest before appearance of reproductive structures or gene use restriction technologies.