Gene drive in species complexes: defining target organisms.

Engineered gene drives, which bias their own inheritance to increase in frequency in target populations, are being developed to control mosquito malaria vectors. Such mosquitoes can belong to complexes of both vector and nonvector species that can produce fertile interspecific hybrids, making vertical gene drive transfer (VGDT) to sibling species biologically plausible. While VGDT to other vectors could positively impact human health protection goals, VGDT to nonvectors might challenge biodiversity ones. Therefore, environmental risk assessment of gene drive use in species complexes invites more nuanced considerations of target organisms and nontarget organisms than for transgenes not intended to increase in frequency in target populations. Incorporating the concept of target species complexes offers more flexibility when assessing potential impacts from VGDT.

Recommendations for environmental risk assessment of gene drive applications for malaria vector control.

Building on an exercise that identified potential harms from simulated investigational releases of a population suppression gene drive for malaria vector control, a series of online workshops identified nine recommendations to advance future environmental risk assessment of gene drive applications.

Potential use of gene drive modified insects against disease vectors, agricultural pests and invasive species poses new challenges for risk assessment.

Potential future application of engineered gene drives (GDs), which bias their own inheritance and can spread genetic modifications in wild target populations, has sparked both enthusiasm and concern. Engineered GDs in insects could potentially be used to address long-standing challenges in control of disease vectors, agricultural pests and invasive species, or help to rescue endangered species, and thus provide important public benefits. However, there are concerns that the deliberate environmental release of GD modified insects may pose different or new harms to animal and human health and the wider environment, and raise novel challenges for risk assessment. Risk assessors, risk managers, developers, potential applicants and other stakeholders at many levels are currently discussing whether there is a need to develop new or additional risk assessment guidance for the environmental release of GD modified organisms, including insects. Developing new or additional guidance that is useful and practical is a challenge, especially at an international level, as risk assessors, risk managers and many other stakeholders have different, often contrasting, opinions and perspectives toward the environmental release of GD modified organisms, and on the adequacy of current risk assessment frameworks for such organisms. Here, we offer recommendations to overcome some of the challenges associated with the potential future development of new or additional risk assessment guidance for GD modified insects and provide considerations on areas where further risk assessment guidance may be required.

Risk management recommendations for environmental releases of gene drive modified insects.

The ability to engineer gene drives (genetic elements that bias their own inheritance) has sparked enthusiasm and concerns. Engineered gene drives could potentially be used to address long-standing challenges in the control of insect disease vectors, agricultural pests and invasive species, or help to rescue endangered species. However, risk concerns and uncertainty associated with potential environmental release of gene drive modified insects (GDMIs) have led some stakeholders to call for a global moratorium on such releases or the application of other strict precautionary measures to mitigate perceived risk assessment and risk management challenges. Instead, we provide recommendations that may help to improve the relevance of risk assessment and risk management frameworks for environmental releases of GDMIs. These recommendations include: (1) developing additional and more practical risk assessment guidance to ensure appropriate levels of safety; (2) making policy goals and regulatory decision-making criteria operational for use in risk assessment so that what constitutes harm is clearly defined; (3) ensuring a more dynamic interplay between risk assessment and risk management to manage uncertainty through closely interlinked pre-release modelling and post-release monitoring; (4) considering potential risks against potential benefits, and comparing them with those of alternative actions to account for a wider (management) context; and (5) implementing a modular, phased approach to authorisations for incremental acceptance and management of risks and uncertainty. Along with providing stakeholder engagement opportunities in the risk analysis process, the recommendations proposed may enable risk managers to make choices that are more proportionate and adaptive to potential risks, uncertainty and benefits of GDMI applications, and socially robust.

The use of statistical tools in field testing of putative effects of genetically modified plants on nontarget organisms.

To fulfill existing guidelines, applicants that aim to place their genetically modified (GM) insect-resistant crop plants on the market are required to provide data from field experiments that address the potential impacts of the GM plants on nontarget organisms (NTO's). Such data may be based on varied experimental designs. The recent EFSA guidance document for environmental risk assessment (2010) does not provide clear and structured suggestions that address the statistics of field trials on effects on NTO's. This review examines existing practices in GM plant field testing such as the way of randomization, replication, and pseudoreplication. Emphasis is placed on the importance of design features used for the field trials in which effects on NTO's are assessed. The importance of statistical power and the positive and negative aspects of various statistical models are discussed. Equivalence and difference testing are compared, and the importance of checking the distribution of experimental data is stressed to decide on the selection of the proper statistical model. While for continuous data (e.g., pH and temperature) classical statistical approaches - for example, analysis of variance (ANOVA) - are appropriate, for discontinuous data (counts) only generalized linear models (GLM) are shown to be efficient. There is no golden rule as to which statistical test is the most appropriate for any experimental situation. In particular, in experiments in which block designs are used and covariates play a role GLMs should be used. Generic advice is offered that will help in both the setting up of field testing and the interpretation and data analysis of the data obtained in this testing. The combination of decision trees and a checklist for field trials, which are provided, will help in the interpretation of the statistical analyses of field trials and to assess whether such analyses were correctly applied. We offer generic advice to risk assessors and applicants that will help in both the setting up of field testing and the interpretation and data analysis of the data obtained in field testing.

Isolation, characterization, and sensitivity to 2,4-diacetylphloroglucinol of isolates of Phialophora spp. from Washington wheat fields.

Dark pigmented fungi of the Gaeumannomyces-Phialophora complex were isolated from the roots of wheat grown in fields in eastern Washington State. These fungi were identified as Phialophora spp. on the basis of morphological and genetic characteristics. The isolates produced lobed hyphopodia on wheat coleoptiles, phialides, and hyaline phialospores. Sequence comparison of internal transcribed spacer regions indicated that the Phialophora isolates were clearly separated from other Gaeumannomyces spp. Primers AV1 and AV3 amplified 1.3-kb portions of an avenacinase-like gene in the Phialophora isolates. Phylogenetic trees of the avenacinase-like gene in the Phialophora spp. also clearly separated them from other Gaeumannomyces spp. The Phialophora isolates were moderately virulent on wheat and barley and produced confined black lesions on the roots of wild oat and two oat cultivars. Among isolates tested for their sensitivity to 2,4-diacetylphloroglucinol (2,4-DAPG), the 90% effective dose values were 11.9 to 48.2 microg ml(-1). A representative Phialophora isolate reduced the severity of take-all on wheat caused by two different isolates of Gaeumannomyces graminis var. tritici. To our knowledge, this study provides the first report of an avenacinase-like gene in Phialophora spp. and demonstrated that the fungus is significantly less sensitive to 2,4-DAPG than G. graminis var. tritici.

Diversity, virulence, and 2,4-diacetylphloroglucinol sensitivity of Gaeumannomyces graminis var. tritici isolates from Washington state.

We determined whether isolates of the take-all pathogen Gaeumannomyces graminis var. tritici become less sensitive to 2,4-diacetylphloroglucinol (2,4-DAPG) during wheat monoculture as a result of exposure to the antibiotic over multiple growing seasons. Isolates of G. graminis var. tritici were baited from roots of native grasses collected from noncropped fields and from roots of wheat from fields with different cropping histories near Lind, Ritzville, Pullman, and Almota, WA. Isolates were characterized by using morphological traits, G. graminis variety-specific polymerase chain reaction and pathogenicity tests. The sensitivity of G. graminis var. tritici isolates to 2,4-DAPG was determined by measuring radial growth of each isolate. The 90% effective dose value was 3.1 to 4.4 microg ml(-1) for 2,4-DAPG-sensitive isolates, 4.5 to 6.1 microg ml(-1) for moderately sensitive isolates, and 6.2 to 11.1 microg ml(-1) for less sensitive isolates. Sensitivity of G. graminis var. tritici isolates to 2,4-DAPG was normally distributed in all fields and was not correlated with geographic origin or cropping history of the field. There was no correlation between virulence on wheat and geographical origin, or virulence and sensitivity to 2,4-DAPG. These results indicate that G. graminis var. tritici does not become less sensitive to 2,4-DAPG during extended wheat monoculture.

Effects of Pseudomonas putida modified to produce phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol on the microflora of field grown wheat.

Pseudomonas putida WCS358r, genetically modified to have improved activity against soil-borne pathogens, was released into the rhizosphere of wheat. Two genetically modified derivatives carried the phz or the phl biosynthetic gene loci and constitutively produced either the antifungal compound phenazine-1-carboxylic acid (PCA) or the antifungal and antibacterial compound 2,4-diacetylphloroglucinol (DAPG). In 1997 and 1998, effects of single introductions of PCA producing derivatives on the indigenous microflora were studied. A transient shift in the composition of the total fungal microflora, determined by amplified ribosomal DNA restiction analysis (ARDRA), was detected. Starting in 1999, effects of repeated introduction of genetically modified microorganisms (GMMs) were studied. Wheat seeds coated with the PCA producer, the DAPG producer, a mixture of the PCA and DAPG producers, or WCS358r, were sown and the densities, composition and activities of the rhizosphere microbial populations were measured. All introduced strains decreased from 10(7) CFU per gram of rhizosphere sample to below the detection limit after harvest of the wheat plants. The phz genes were stably maintained in the PCA producers, and PCA was detected in rhizosphere extracts of plants treated with this strain or with the mixture of the PCA and DAPG producers. The phl genes were also stably maintained in the DAPG producing derivative of WCS358r. Effects of the genetically modified bacteria on the rhizosphere fungi and bacteria were analyzed by using amplified ribosomal DNA restriction analysis. Introduction of the genetically modified bacterial strains caused a transient change in the composition of the rhizosphere microflora. However, introduction of the GMMs did not affect the several soil microbial activities that were investigated in this study.