Katherine K. Donegan

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.

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).

Bracken (Pteridium aquilinum L.) frond biomass and rhizosphere microbial community characteristics are correlated to edaphic factors

Bracken is a broadly distributed weedy fern common in disturbed habitats. Frond and rhizosphere soil samples were obtained from bracken growing in three clearcut locations in the Willamette National Forest in western Oregon. The highest frond biomass was correlated with soil having the highest total %N, lowest Fe content and oldest geological age. Based on analysis of variance of principal component scores for patterns of utilization of substrates on Biolog GN plates, metabolic profiles of rhizosphere microbial communities of bracken differed significantly between locations. Utilization of carbohydrates and phosphorylated compounds was positively correlated with organic matter (OM) and total N and negatively correlated with extractable Fe and Mn content of soil. Carboxylic acid utilization was positively correlated with pH and OM and negatively correlated with extractable Mn and P content of soils. Pseudomonas rDNA fingerprints of bracken rhizosphere samples suggested that the diversity of pseudomonads at the location with the most acidic (pH 5.5) soil (Burnside Road) differed from those at less acidic (pH 6.2 and 6.1) locations (Falls Creek and Toad Road). Mycorrhizal infection of bracken was lowest at Falls Creek, the location with the highest %N soil content. Our results suggest that bracken frond biomass and rhizosphere microbial community characteristics are correlated with local edaphic factors such as soil chemistry and geological age.