This report is the outcome of an EFSA procurement (OC/EFSA/GMO/2015/02) which aims at reviewing relevant scientific information on RNA interference (RNAi) that could serve as baseline information for the environmental risk assessment of RNAi‐based genetically modified (GM) plants. The report is based on a systematic literature search on the use of RNAi molecules in arthropods, nematodes, annelids and molluscs with dsRNA, siRNA and miRNA applied primarily through feeding and soaking (oral ingestion). The numbers of retrieved publications covering these areas are reported, along with the species name, life stages tested, the target gene and its function, details of the test substances and their concentrations used, methods of delivery and effects. Separate sections discuss the available information on: (1) the uptake and systematic spread of RNAi activity, including a description of the various components involved in this process; (2) the mechanisms of dsRNA‐, siRNA‐ and miRNA‐elicited gene silencing and the different factors involved in RNAi efficiency; (3) routes of exposure of the biotic and abiotic environment to dsRNA, siRNA and miRNA from GM plants; (4) the environmental fate of dsRNA, siRNA and miRNA; and (5) the various factors that may limit non‐target effects including exposure, factors influencing the silencing efficiency of dsRNA, siRNA and miRNA, possible unintended and off‐target effects, and their mechanisms. Finally, an overview of the species of arthropods, nematodes, annelids and molluscs for which genomic data are available is also presented. The report identifies some of the challenges involved in developing plants with RNAi systems which affect invertebrate gene expression. The report also concludes that, currently, knowledge on issues such as exposure, specificity, offtarget effects, sequence similarities and bioinformatics is very limited, as only a few RNAi expressing plants which specifically target invertebrate species have been developed and comprehensively studied.

RNA interference (RNAi) is being developed and exploited to improve plants by modifying endogenous gene expression as well as to target pest and pathogen genes both within plants (i.e. host-induced gene silencing) and/or as topical applications (e.g. spray-induced gene silencing). RNAi is a natural mechanism which can be exploited to make a major contribution towards integrated pest management and sustainable agricultural strategies needed worldwide to secure current and future food production. RNAi plants are being assessed and regulated using existing regulatory frameworks for GMO. However, there is an urgent need to develop appropriate science-based risk assessment procedures for topical RNAi applications within existing plant protection products legislation. Keywords RNAi • dsRNA • Biosafety • Agriculture • Regulations • HIGS • SIGS Key message • RNAi is a natural mechanism found in most eukaryotic organisms in nature and can be exploited to improve plant health. • RNAi-based technology is already being exploited, and the realized examples confirm its great potential in a range of areas of crop production and protection.

The spatial distribution and niche differentiation of three closely related species (Erysiphe alphitoides, E. quercicola and E. hypophylla) causing oak powdery mildew was studied at scales ranging from the European continent, where they are invasive, to a single leaf. While E. alphitoides was dominant at all scales, E. quercicola and E. hypophylla had restricted geographic, stand and leaf distributions. The large-scale distributions were likely explained by climatic factors and species environmental tolerances, with E. quercicola being more frequent in warmer climates and E. hypophylla in colder climates. The extensive sampling and molecular analyses revealed the cryptic invasion of E. quercicola in nine countries from which it was not yet recorded. The presence of the three species was also strongly affected by host factors, such as oak species and developmental stage. Segregation patterns between Erysiphe species were finally observed at the leaf scale, between and within leaf surfaces, suggesting competitive effects.

The genus Phytophthora comprises many economically and ecologically important plant pathogens. Hybrid species have previously been identified in at least six of the 12 phylogenetic clades. These hybrids can potentially infect a wider host range and display enhanced vigour compared to their progenitors. Phytophthora hybrids therefore pose a serious threat to agriculture as well as to natural ecosystems. Early and correct identification of hybrids is therefore essential for adequate plant protection but this is hampered by the limitations of morphological and traditional molecular methods. Identification of hybrids is also important in evolutionary studies as the positioning of hybrids in a phylogenetic tree can lead to suboptimal topologies. To improve the identification of hybrids we have combined genotyping-by-sequencing (GBS) and genome size estimation on a genus-wide collection of 614 Phytophthora isolates. Analyses based on locus- and allele counts and especially on the combination of species-specific loci and genome size estimations allowed us to confirm and characterize 27 previously described hybrid species and discover 16 new hybrid species. Our method was also valuable for species identification at an unprecedented resolution and further allowed correct naming of misidentified isolates. We used both a concatenation- and a coalescent-based phylogenomic method to construct a reliable phylogeny using the GBS data of 140 non-hybrid Phytophthora isolates. Hybrid species were subsequently connected to their progenitors in this phylogenetic tree. In this study we demonstrate the application of two validated techniques (GBS and flow cytometry) for relatively low cost but high resolution identification of hybrids and their phylogenetic relations.