34 research outputs found
Environmental fate of double-stranded RNA in agricultural soils.
A laboratory soil degradation study was conducted to determine the biodegradation potential of a DvSnf7 dsRNA transcript derived from a Monsanto genetically modified (GM) maize product that confers resistance to corn rootworm (CRW; Diabrotica spp.). This study provides new information to improve the environmental assessment of dsRNAs that become pesticidal through an RNAi process. Three agricultural soils differing in their physicochemical characteristics were obtained from the U.S., Illinois (IL; silt loam), Missouri (MO; loamy sand) and North Dakota (ND; clay loam), and exposed to the target dsRNA by incorporating insect-protected maize biomass and purified (in vitro-transcribed) DvSnf7 RNA into soil. The GM and control (non-GM maize) materials were added to each soil and incubated at ca. 22 °C for 48 hours (h). Samples were collected at 12 time intervals during the incubation period, extracted, and analyzed using QuantiGene molecular analysis and insect bioassay methods. The DT50 (half-life) values for DvSnf7 RNA in IL, MO, and ND soils were 19, 28, and 15 h based on QuantiGene, and 18, 29, and 14 h based on insect bioassay, respectively. Furthermore, the DT90 (time to 90% degradation) values for DvSnf7 RNA in all three soils were <35 h. These results indicate that DvSnf7 RNA was degraded and biological activity was undetectable within approximately 2 days after application to soil, regardless of texture, pH, clay content and other soil differences. Furthermore, soil-incorporated DvSnf7 RNA was non-detectable in soil after 48 h, as measured by QuantiGene, at levels ranging more than two orders of magnitude (0.3, 1.5, 7.5 and 37.5 µg RNA/g soil). Results from this study indicate that the DvSnf7 dsRNA is unlikely to persist or accumulate in the environment. Furthermore, the rapid degradation of DvSnf7 dsRNA provides a basis to define relevant exposure scenarios for future RNA-based agricultural products
Independent action between DvSnf7 RNA and Cry3Bb1 protein in southern corn rootworm, Diabrotica undecimpunctata howardi and Colorado potato beetle, Leptinotarsa decemlineata.
In recent years, corn rootworm (CRW)-resistant maize events producing two or more CRW-active Bt proteins have been commercialized to enhance efficacy against the target pest(s) by providing multiple modes of action (MoA). The maize hybrid MON 87411 has been developed that produces the CRW-active Cry3Bb1 Bt protein (hereafter Cry3Bb1) and expresses a RNAi-mediated MoA that also targets CRW. As part of an environmental risk assessment for MON 87411, the potential for an interaction between the CRW-active DvSnf7 RNA (hereafter DvSnf7) and Cry3Bb1 was assessed in 12-day diet incorporation bioassays with the southern corn rootworm (SCR, Diabrotica undecimpunctata howardi). The potential for an interaction between DvSnf7 and Cry3Bb1 was evaluated with two established experimental approaches. The first approach evaluated each substance alone and in combination over three different response levels. For all three response levels, observed responses were shown to be additive and not significantly different from predicted responses under the assumption of independent action. The second approach evaluated the potential for a fixed sub-lethal concentration of Cry3Bb1 to decrease the median lethal concentration (LC50) of DvSnf7 and vice-versa. With this approach, the LC50 value of DvSnf7 was not altered by a sub-lethal concentration of Cry3Bb1 and vice-versa. In addition, the potential for an interaction between the Cry3Bb1 and DvSnf7 was tested with Colorado potato beetle (CPB, Leptinotarsa decemlineata), which is sensitive to Cry3Bb1 but not DvSnf7. CPB assays also demonstrated that DvSnf7 does not alter the activity of Cry3Bb1. The results from this study provide multiple lines of evidence that DvSnf7 and Cry3Bb1 produced in MON 87411 have independent action
DvSnf7 RNA degrades rapidly when applied to soil.
<p>Soil samples were amended with lyophilized maize tissue and fortified with 7.5 µg DvSnf7 RNA per gram of soil. DvSnf7 RNA concentration in soil was measured with a QuantiGene assay. Biological activity against Southern Corn Rootworm was determined by assessing insect mortality in a 12-day bioassay. Error bars represent one standard error of the mean (n = 4).</p
DvSnf7 RNA degrades in soil alone.
<p>Soil samples were fortified with 7.5 µg DvSnf7 RNA per gram of soil in the absence of maize tissue. DvSnf7 RNA concentration was determined with a QuantiGene assay. Error bars represent one standard error of the mean (n = 2).</p
Degradation kinetics for DvSnf7 RNA are independent of initial dsRNA concentration.
<p>Soil samples were amended with lyophilized maize tissue and fortified with the indicated amounts of DvSnf7 RNA (µg RNA/g soil). Soil concentration of DvSnf7 RNA was determined with a QuantiGene assay. Numbers in parenthesis indicate the relative DvSnf7 RNA treatment. Error bars represent one standard error of the mean (n = 3).</p
Physicochemical characteristics of three soils used to determine the environmental fate of DvSnf7 RNA.
<p>Physicochemical characteristics of three soils used to determine the environmental fate of DvSnf7 RNA.</p
QuantiGene and insect bioassay methods provide comparable degradation rate estimates.
<p>The degradation rate estimates (DT<sub>50</sub> and DT<sub>90</sub>), in hours, were calculated for DvSnf7 RNA in each soil type as measured by QuantiGene and insect bioassay. Numbers in parenthesis represent the standard error of the mean (SE) in hours. LCI and UCI represent the lower and upper 95% confidence intervals respectively, in hours.</p
Concentration response curves of CPB mortality for Cry3Bb1 alone (Δ) and Cry3Bb1 in the presence of DvSnf7 RNA (○) at a fixed concentration of 1000 ng/ml diet in 12-day diet bioassays.
<p>The responses were plotted based on the tested Cry3Bb1 concentrations, rather than the mixture concentrations. Data points represent mean mortality with standard errors of three replicated bioassays. LC<sub>50</sub> values and associated 95% confidence intervals are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118622#pone.0118622.t002" target="_blank">Table 2</a>.</p
12-day LC<sub>50</sub> values for the Cry3Bb1 protein and the mixture with DvSnf7 for the Colorado potato beetle, <i>Leptinotarsa decemlineata</i>.
<p>* A fixed concentration of DvSnf7 at 1000 ng/ml diet was added in each effective concentration level of Cry3Bb1 in the mixture. Concentration responses are illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118622#pone.0118622.g005" target="_blank">Fig. 5</a>.</p><p>12-day LC<sub>50</sub> values for the Cry3Bb1 protein and the mixture with DvSnf7 for the Colorado potato beetle, <i>Leptinotarsa decemlineata</i>.</p