Slavomír Rakouský University of South Bohemia, Fac. Health & Soc. Studies and Fac. Science, České Budějovice, Czech Republic Workshop on Biosafety AGR 46779, Kiev, th April 2012 Assessment of the Impacts of Genetically Modified Plants in Czech Republic

Mandatory GMO legislative  National (CZ) (Act No 78/2004 Coll., am. by Act No 346/ transpose Directive 2001/18/EC and Directive 98/81/EC; Ordinance No 209/2004 Coll. and 29/2010 Coll. - details of handling) - fully harmonized to EU legislative  European Union Regulations (EC) Nos 1829/2003, 1830/2003, No 1946/ directly apllied  International Cartagena Protocol on Biosafety Convention on Biological Diversity

3 levels of handling: Contained use of GMOs (laboratory, glasshouse… 1 st -2 nd categories only) Release into environment ″B″ (field experiments, since 1996, annually ca ) Release into environment ″C″(import, processing, market, growing; commercial cultivation Bt-maize MON 810 since 2005, potato cv. Amflora in 2010)

Safety to environment Environmental Risk Assessment - ERA, (event, conditions of release), seed samples, regular field evaluations, annual reports, inspections & sampling CEI, GMO Committee, CISTA, SPA accredited laboratories for GMOs analysis

GMOs Research & Monitoring supported by grant agencies of Czech ministries (agri., env., edu.), AS CR, GA CR, EU projects - (e.g. SIGMEA, MOBITAG), other internat. projects (UNEP/GEF), biotech companies

Examples of GMOs’ Impact Monitoring

SIGMEA - 6th FP project, (2005-7) PRIORITY [FP SSP1] CZ participants: University of South Bohemia (USB) – oilseed rape (volunteer population dynamics, seed persistence, gene transfer to related species) Czech University of Agriculture (CUA), Prague – sugar beet (wild beet population persistence & weed managenent)

Aims of the study Long-term monitoring of GM winter oilseed rape (GM-OSR) volunteers incidence, its possible impact on experimental field areas in the Czech Republic (CZ) where this crop was cultivated during the seasons 2000/01, Influence of various environmental conditions and agricultural practices on dynamics of GM- OSR survival/ incidence, its possible spreading around the fields, Possible transfer of transgene coding for herbicide-tolerance (HT) to OSR wild relatives.

USB: 8 Locations – oilseed rape

Approach Fields monitored in regular intervals for 4-6 seasons for the presence of OSR survivors and volunteers, all farming practices registered over the whole period of time, Polymerase-chain reaction (PCR) used to confirm/ exlude the HT transgene presence, Limited number of volunteers was selfed and analyzed for HT gene (herbicide appl., PCR) to estimate their origin and possible degree of outcrossing.

Herbicide tolerance test before treatment 4 weeks following herbicide application

Survey of sampling dates, locations & analysed plant samples since 2002 Sampling date Location [No.] OSR volunt. fr. [No./m 2 ] Samples total [No.] OSR samples [No.] Other species samples [No.] 09/05/ /06/02 1, 2, 3, 4, , (33*) 18 (16*) YC, 65* YF 20/05/ /06/03 1, 2, 3, 5, , * YC, WR 06/05/ /06/04 1, 3, 5, 6, 7, , BM, 4 TR 19/04/ /06/05 1, 2, 3, 5, 6, , YC, 11* YR, 7 SP, 15 FP, 1 TC 12/05/ /06/06 1, 3, 5, 6, , FP, 41 (15)* SP, 8* TM, 8* WR, * - field borders or its near surrounding BM - Black mustard (B. nigra (L.) Koch), TR - Turnip rape (B. rapa L.) - both volunteers FP - Field penny cress, SP - Shepherd's purse, TC - Thale cress, TM – Treacle mustard, YC - Yellow charlock, YF - Yellow fieldcress, YR - Yellow-rocket, WR - Wild radish - all wild related species

Data obtained Survey of agricultural practices used Survey of agricultural practices used following the winter oilseed rape (OSR) cultivation on 8 fields in the Czech Republic (CZ) since 2001 to next-used crops harvest in 2006 Volunteer densities decline medium No s. of OSR plants/m 2 found at terms of monitoring (1-6/year)

Results of molecular analyses Results of molecular analyses OSR ( GM/non-GM plants per location, year) and related species Implications for safe GM-OSR cultivation Implications for safe GM-OSR cultivation recommendations to Ministry of Agriculture

Overview of farming practices

Volunteer frequencies following the harvest

Results Indicate that GM-OSR plants coming from the soil seed bank could be still present at experimental fields after five years since the GM rape cultivation, Volunteer frequency drops down dramatically during the first year following the crop harvest. Later on its (low) number could persist/vary for years depending on agricultural practices used, Proper agricultural practices can suppress efficiently its frequency to minimize possible risks of cross-contamination of non-GM rape production or gene transfer to related species,

Quite often the found ratio of GM : non-GM rape did not corespond to the expected one, Up to now our studies performed with limited numbers of either related cultural- or wild species did not indicate the HT transgene transfer.

Implications Recommended period of at least 6 years for repeated conventional OSR cultivation on the same field should be kept also in the case of GM-OSR. One of the most effective steps in controlling GM-OSR volunteers is to allow seed germination directly on a field immediately after the harvest, skimming(s) and/or herbicide treatment (if used) should be postponed to the treatment-sensitive OSR plant developmental stage.

This should be an imperative of farmers in seasons with climatic or other disturbances, which do not allow for the complete GM-OSR harvest in a given field. The cultivation of high-density- or competitive crops (e.g. cereals, alfalfa) in the following years helps to further suppress volunteers.

CUA: Geneflow in a beet complex (Beta sp.) CUA: Gene flow in a beet complex (Beta sp.) Ouside the agroecosystem In agroecosystem Beta maritimaRuderal types Weed beet Sugarbeet Photo: Stevanato, Soukup

Experiments of the Institute of Entomology BC AS CR (IE) with maize MON 810 ( ) Impact of the protection strategy against European corn borer (ECB) - Ostrinia nubilalis on arthropod communities

Aphids and their parasitoids, thrips, larval and adult lady birds, white flies and Orius bugs, lacewings (spidermites)

Selected groups of arthropods were collected 3 x during season on 10 randomly chosen plants per plot Sampling of arthropods on the plants

Sampling of epigeic arthropods 5 x during season in 5 pitfall traps per plot Dipteran larvae were analyzed in soil samples taken on Soil samples for the screening of entomopatogenic nematodes were taken 3 x per season Pitfall trap

Rove beetles (Staphylinidae), ground beetles (Carabidae), and spiders (Aranea) are predators associated with soil

Eggs and larvae of the European corn borer (ECB), aphids, thrips, and aphid predators per 100 plants of the GM Bt maize MON 810 and control non-Bt maize

Abundance of sucking insects in relation to Bt expression, date and year

Species numbers of ground beetles, rove beetles, and spiders in the Bt and non-Bt plots

Numbers of ground beetles, rove beetles, and spiders in the Bt and non-Bt plots (2.5 ha total)

Dominant ground beetle species

Representation of common spider species

Maize MON project ( ) Diabrotica v.virgifera (corn western rootworm, CWR) was brought to Europe from USA and spreads rapidly. Adults consume maize pollen, larvae feed on roots. Cry3Bb1 transgene Cry3Bb1 from Bacillus thuringiensis partial resistance to Diabrotica virgifera virgifera) CP4 EPSPS transgene CP4 EPSPS from Agrobacterium tolerance to glyphosate herbicides

It will soon reach the region of IE experiments CWR spreading in Europe The International Working Group on Ostrinia and other Maize Pests

Experimental set-up in Scale 1:5200, taken from www. maps.google.cz C C: MON N N: „isogenic“ cultivar DK 315 I I: „isogenic“ cultivar DK 315 treated with the Dursban 10 insecticide A A: reference cultivar KIPOUS B B: reference cultivar PR38N86 14 ha (48°59´ N, 14°20´ E) 25 plots, 0.5 ha each pre- and post- emergence application of herbicides and fertilizers plants of waxy stage were shred and all biomass was ploughed into soil or used in a biogas station

Quantification of transgene product The content was measured in plant tissues with Double Antibody Sandwich ELISA using a commercial kit Values averaged from samples taken at the 6- leaf, flowering, and waxy ripening stage Plant organµg Cry3Bb1 per g of fresh plant tissue Roots30.8 ± 0.7 Aerial roots18.3 ± 0.6 Stem11.8 ± 0.2 Leaves36.5 ± 0.1 Male flowers 28.9 ± 0.5 Female flowers27.5 ± 0.9

Entomopathogenic nematodes Plots where S. affine occurred – in yellow Single species found: Steinernema affine Nematodes occurred only in plots with lighter soil and adjacent to the forest. Differences between plots are due to their position, not to the type of treatment.

General conclusion: Neither biodiversity nor the abundance of examined arthropods were affected by GM maize

Acknowledgement University of South Bohemia – Marek Hraška, Jindřich Bříza, Daniela Pavingerová, Hana Niedermaierová Institute of Entomology BC AS CR – Oxana Habuštová, František Sehnal, Zdeňka Svobodová, Hany M. Hussein, Radka Fabiánová, Michal Holec, Vladimír Půža, Lukáš Spitzer, Moataz Mostafa, Jiří Zelený, Jaroslav Boháč Czech University of Agriculture – Josef Soukup, Josef Holec Staff of Co-operating institutions AGR Workshop organizers