Ramon J. Seidler

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.

Quantification in soil of Bacillus thuringiensis var. kurstakiδ-endotoxin from transgenic plants

Transgenic plants that produce pesticidal proteins have the potential to release these products into the environment when the plants are incorporated into soil. This could result in novel exposure of soil organisms to these pesticidal proteins. There is a lack of knowledge about the fate and persistence of transgenic pesticidal products in the soil. A model system of transgenic cotton, which produces Bacillus thuringiensis kurstakiδ‐endotoxin (Bt toxin), was used to address this issue. Methods were developed to quantify Btk toxin in soil and soil/plant litter by extraction of the Btk toxin with an aqueous buffer and quantification by ELISA. The highest recovery of Btk toxin from soil was obtained with a high salt, high pH buffer. In addition, for certain soil types, addition of a non‐ionic detergent, Tween‐20, was needed for optimal recovery. Recovery of Btk toxin from soil ranged from 60% for a low clay content, low organic matter soil to 27% for a high clay content, high organic matter soil. The limit of detection of this method is 0.5 ng of extractable toxin per g dry weight soil. The method was shown to be useful in tracking over time the persistence of both purified and transgenic Btk toxin in laboratory experiments.

Quantification of transgenic plant marker gene persistence in the field

Methods were developed to monitor persistence of genomic DNA in decaying plants in the field. As a model, we used recombinant neomycin phosphotransferase II (rNPT‐II) marker genes present in genetically engineered plants. Polymerase chain reaction (PCR) primers were designed, complementary to 20‐bp sequences of the nopaline synthase promoter in a transgenic tobacco and the cauliflower mosaic virus 35S promoter in a transgenic potato. The PCR reverse primer was complementary to a 20‐bp sequence of the N‐terminal NPT‐II coding region. The PCR protocol allowed for quantification of as few as 10 rNPT‐II genes per reaction. We analysed rNPT‐II marker gene amounts in samples obtained from two field experiments performed at different locations in Oregon. In transgenic tobacco leaves, buried at 10 cm depth in a field plot in Corvallis, marker DNA amount dropped to 0.36% during the first 14 days and was detectable for 77 days at a final level of 0.06% of the initial amount. Monitoring of residual potato plant litter, from the soil surface of a test field in Hermiston, was performed for 137 days. After 84 days marker gene amounts dropped to 2.74% (leaf and stem) and 0.50% (tuber) of the initially detected amount. At the final sample date 1.98% (leaf and stem) and 0.19% (tuber) were detectable. These results represent the first quantitative analysis of plant DNA stability under field conditions and indicate that a proportion of the plant genomic DNA may persist in the field for several months.

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.

Microbial populations, fungal species diversity and plant pathogen levels in field plots of potato plants expressing theBacillus thuringiensis var.tenebrionis endotoxin

The environmental release of genetically engineered (transgenic) plants may be accompanied by ecological effects including changes in the plant-associated microflora. A field release of transgnic potato plants that produce the insecticidal endotoxin ofBacillus thuringiensis var.tenebrionis (Btt) was monitored for changes in total bacterial and fungal populations, fungal species diversity and abundance, and plant pathogen levels. The microflora on three phenological stages of leaves (green, yellow and brown) were compared over the growing season (sample days 0, 21, 42, 63 and 98) for transgenic potato plants, commercial Russet Burbank potato plants treated with systemic insecticide (Di-Syston) and commercial Russet Burbank potato plants treated with microbialBtt (M-Trak). In addition, plant and soil assays were performed to assess disease incidence ofFusarium spp.,Pythium spp.,Verticillium dahliae, potato leaf roll virus (PLRV) and potato virus Y (PVY). Few significant differences in phylloplane microflora among the plant types were observed and none of the differences were persisent. Total bacterial populations on brown leaves on sample day 21 and on green leaves on sample day 42 were significantly higher on the transgenic potato plants. Total fungal populations on gree leaves on sample day 63 were significantly different among the three plant types; lowest levels were on the commerical potato plants treated with systemic insecticide and highest levels were on the commercial potato plants treated with microbialBtt. Differences in fungal species assemblages and diversity were correlated with sampling dates, but relatively consistent among treatments.Alternaria alternata, a common saprophyte on leaves and in soil and leaf litter, was the most commonly isolated fungus species for all the plant treatments. Rhizosphere populations of the soilborne pathogensPythium spp.,Fusarium spp. andV. dahliae did not differ between the transgenic potato plants and the commercial potato plants treated with systemic insecticide. The incidence of tuber infection at the end of the growing season by the plant pathogenV. dahliae was highest for the transgenic potato plants but this difference was related to longer viability of the transgenic potato plants. This difference in longevity between the transgenic potato plants and the commercial + systemic insecticide potato plants also made comparison of the incidence of PVY and PLRV problematic. Our results indicate that under field conditions the microflora of transgenicBtt-producing potato plants differed minimally from that of chemically and microbially treated commerical potato plants.

Decomposition of genetically engineered tobacco under field conditions : persistence of the proteinase inhibitor I product and effects on soil microbial respiration and protozoa, nematode and microarthropod populations

To evaluate the potential effects of genetically engineered (transgenic) plants on soil ecosystems, litterbags containing leaves of non-engineered (parental) and transgenic tobacco plants were buried in field plots. The transgenic tobacco plants were genetically engineered to constitutively produce proteinase inhibitor I, a protein with insecticidal activity. The litterbag contents and surrounding soil, as well as soil from control plots without litterbags, were sampled over a 5-month period at 2- to 4-week intervals and assayed for proteinase inhibitor concentration, litter decomposition rates, carbon and nitrogen content, microbial respiration rates and population levels of nematodes, protozoa and microarthropods. The proteinase inhibitor concentration in the transgenic plant litter after 57 days was 0.05% of the sample day 0-value and was not detectable on subsequent sample days. Although the carbon content of the transgenic plant litter was comparable to that of the parental plant litter on sample day 0, it became significantly lower over the course of the experiment. Nematode populations in the soil surrounding the transgenic plant litterbags were greater than those in the soil surrounding parental plant litterbags and had a different trophic group composition, including a significantly higher ratio of fungal feeding nematodes to bacterial feeding nematodes on sample day 57. In contrast, Collembola populations in the soil surrounding the transgenic plant litterbags were significantly lower than in the soil surrounding parental plant litterbags. Our results demonstrated that under field conditions proteinase inhibitor remained immunologically active in buried transgenic plant litter for at least 57 days and that decomposing parental and transgenic plant litter differed in quality (carbon content) and in the response of exposed soil organisms (Collembola and nematodes).

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.

Temporal and Spatial Distribution of the nifH Gene of N2 Fixing Bacteria in Forests and Clearcuts in Western Oregon

A bstractDecomposition of plant litter is a primary mechanism of nutrient recycling and redistribution in most terrestrial ecosystems. Previously we demonstrated by a nested PCR protocol that 20 distinctive nifH (the gene encoding nitrogenase reductase) HaeIII restriction fragment length polymorphism (RFLP) patterns were derived from bulk DNA associated with samples of plant litter and soil collected at one Douglas Fir (DF) forest [33]. Five of the nifH DNA patterns (II–VI) were dominant types in DF litter with characteristic fragments of 237–303 bp length, whereas samples from soil contained primarily seven other patterns 131–188 bp length (IX–XV). Here we report that the 237–303 bp fragments characteristic for forest litter could generally not be detected in plant litter or soil samples collected in clearcuts that adjoin the forest sites. The same fragments (237–303 bp) were also found in the litter at this DF forest site over 16 months and were consistently found in litter at 12 other DF forest or recent (<2 yrs) clearcut sites. However, trace to none of these fragments were detected in 6 clearcut (5–10 yrs) or different forest types (oak, alder) collected over a 200 km east–west direction in western Oregon, USA. Data suggest that the logging practice in DF forests that creates a clearcut removes a unique gene pool of nitrogen-fixing microorganisms. These organisms could potentially contribute more to nitrogen fixation in forest litter than litter from natural or invasive plants that grow in clearcuts [26].

Microcosm method to assess survival of recombinant bacteria associated with plants and herbivorous insects

A microcosm method was developed to investigate survial and fate of genetically engineered bacteria associated with plant surfaces and a plant-feeding insect, the variegated cutworm,Peridroma saucia. Larvae on radish plants in microcosms were sprayed with nonrecombinantPseudomonas cepacia and a recombinant strain ofP. cepacia carrying the transmissible plasmid R388::Tn1721. Leaf, whole insect, foregut, and frass samples were periodically assayed over a 48-h period to enumerate total bacteria andP. cepacia strains. Immediately after spraying,P. cepacia comprised about 20%–30% of the total population on leaves, which was 2×107 cfu/g of leaf. Approximately 4×107 total cfu were recovered from each gram of whole insect, when theP. cepacia strains averaged about 3×105 cfu/g. After 2 days, the total epiphytic population had increased approximately fourfold, while theP. cepacia strains had decreased to 2%–30% of their initial numbers. After 24 and 48 h, eachP. cepacia strain numbered between 104 and 105 cfu/g of insect in foregut samples, whereas none was detectable in frass. Plasmid transfer fromP. cepacia R388::Tn1721 to the nonrecombinant recipientP. cepacia strain was not observed.