EU project LIFE13 ENV IT 000461 / “EVERGREEN” “Environmentally friendly biomolecules from

1. EU project LIFE13 ENV IT 000461 / “EVERGREEN” “Environmentally friendly biomolecules from agricultural wastes as substitutes of pesticides for plant diseases control” BENEFICCIARY; CSIC (CEBAS-CSIC) Carlos García Izquierdo. CEBAS-CSIC

2. EVERGREEN (LIFE) CEBAS Project LIFE13 ENV/IT/000461 CEBAS-CSIC (EVERGREEN) Dr. Carlos Garcia Dra. Teresa Hernández Dr. J. Moreno Lda. Mª Dolores Coll Lda. Blanca D. Manuel Campoy Dña. C. Montesinos

3. CEBAS-CSIC GROUP: EVERGREEN Project LIFE13 ENV/IT/000461 WHAT CEBAS-CSIC GROUP WILL DO IN THE EVERGREEN PROJECT?? EVERGREEN:Thisprojectwilldemostratethesuccessful use of biomoleculesrecoveredfromagricultural vegetable wastes as substitutes of pesticidesfortheenvironmentalyfriendly and sustainable control of plantdiseases ACTIONS B1, B3, B4, (B6) B1) Demostration of the performances of traditionalpesticidesforthe control of bacterial and nematodesdiseases of platsimportantforthe EU. B4) Demostration of thebiologicalactivity of thehighqualitypolyphenolsextractsrecoveredfromnotediblebiomass and wastes, againstplantpathogenic bacteria and nematode, in plant. B3) Polyphenolsactivity?? ACTIONS C1, C2 and C4: C1: Monitoringontheenvironmentalimpact of cooper and nematicidesforthecropdefenceagainstphytopathogenic bacteria and nematodes C2 (B6?).Monitoring of theabsences of sideeffectsforthehighqualitypolyfhenolicpreparationsoncommon targets of the living organisms at laboratorylevel. C4. Monitoring of the short-termenvironmentalbenefictsfromthe use of thehighqualitypolyphenolspreparations in plantdisease control at pilotscalelevel in fieldscreening ACTION D…:Communication and disseminationactions

4. EVERGREEN (LIFE) Project LIFE13 ENV/IT/000461 B1) and C1Demostration and monitoring of the performances of traditionalpesticidesforthe control of bacterial and nematodesdiseases of platsimportantforthe EU. (FromOctober 2014 to March 2015: DONE) AGRICULTURE: WE NEED TO PRODUCE FOOD (9000 MILL IN 2050) 1) Use of agrochemical to combatplantdiseases; isituseful? YES 2) Are goodtheagrochemicalsfortheenvironment?? Whataboutsoil quality?? (Cu and nematicides) (OUR ACTYVITY B1 and C1) SUSTAINABLE PRODUCTION: WE MUST PRODUCE WITH NO RISK FOR THE ENVIRONMENT

5. EVERGREEN Project LIFE13 ENV/IT/000461 C2-(B6) ACTIVITY (FROM APRIL 2015) Monitoring of theabsences of sideeffectsforthehighqualitypoly- phenolicpreparationsoncommon targets of the living organisms at laboratorylevel(EFFECT ON SOIL MICROORGANISMS). CEBAS EXPERIMENT (FINISHED)) CONTROL SOIL SOIL + POLYPHENOLS SOIL + Pathogen (bacteria and nematicide) SOIL + POLYPHENOLS + Pathogen

6. EVERGREEN (LIFE) Project LIFE13 ENV/IT/000461 EFFECT OF POLYPHENOLS ON SOIL MICROORGANISMS Photo 1. Microcosm containers with different treatments al the beginning of the assay, MICROCOSM TIMES: 0,1 AND 2 MONTHS

7. EVERGREEN (LIFE) Project LIFE13 ENV/IT/000461 More labile structures of N and C are affected by polyphenols addition. However, soil enzymes are not affected with this addition (no effect on specific microbial population)

8. Table 18. Bacterial, fungal, Gram+, Gram-, satured and monosatured PLFAs concentration in soils T0. At the beginning of this experiment, soil biodiversity microorganisms (bacterial and fungi) were affected by polyphenols addition. But two months latter, soils with polyphenols showed a similar soil bio- diversity that control soil. Only fungi could be affected Table 20. Bacterial, fungal, Gram+, Gram-, satured and monosatured PLFAs concentration in soils T30. Polyphenols added to the soil have not influence on soil bio-biodiversity and soil population microorganisms Table 22. Bacterial, fungal, Gram+, Gram-, satured and monosatured PLFAs concentration in soils T60.

9. EVERGREEN (LIFE) Project LIFE13 ENV/IT/000461 A possible effect was observed on fungi/bacteria ratio: when polyphenols are added to the soil, this ratio decreased. It could be due to biopesticide effect from polyphenols against fungi.

10. EVERGREEN Project LIFE13 ENV/IT/000461 POLYPHENOLS “ACTIVITY” B3 ACTIVITY??? (fromApril 2015) CEBAS EXPERIMENT a) POLYPHENOLS ANALYSIS b) PHYTOTOXICITY EFFECT c) ECOTOXICOLOGICAL ASPECTS d) CHEMICAL STABILITY

11. a) EVERGREEN-ANALYSIS Project LIFE13 ENV/IT/000461 GALLOTANINOS mg/g dry matter ELLAGITANINOS mg/g dry matter mg/g dry matter TOTAL GALLOTANINOS mg/g dry matter mg/g dry matter POLYPHENOLS from chestnuts showed a good quantity of gallotannins and ellagitannins. TCC showed the highest value.

12. a) EVERGREEN-ANALYSIS Project LIFE13 ENV/IT/000461 mg/g dry matter The ellagic acid was found in polyphenols from chestnuts used in EVERGREEN.

13. a) EVERGREEN-ANALYSIS Project LIFE13 ENV/IT/000461 mg/g dry matter mg/g dry matter Polyphenols extracted by grapefruits showed some compounds such as catequin, nDP; some polyphenols showed also epicatequin and epigallocatequin. POLYPHENOLS EXTRACTED BY CHESTNUT AND GRAPEFRUIT ARE NOT SIMILAR.

14. b) EVERGREEN-PHYTOTOXICITY Project LIFE13 ENV/IT/000461 Germination tests on filter paper: In Petri dishes with filter paper and 15 seeds of rye-grass (Lolliumperenne) or 8 seeds of barley (Hordeum vulgaris), 2ml of different digestate dilutions in distilled water (1/100; 5/100; 10/100; 15/100; 25/100, and 50/100) will be added and dishes were put in a germination chamber at 28 ºC, 75% relative humidity and darkness. After germination, the number of germinated seeds was recorded and the length of the seedling roots and shoots was measured. All treatments were carried out by quadruplicate, and Petri dishes with 2 ml of distilled water instead of digestate will be used as control. Germination index (GI) will be calculated according to the following equation (Zucconi and De Bertoldi 1987): GI = % GS (LR/LRC); where GI = Germination index in percentage; %GS = Percentage of germinated seed with respect to the control; LR = Average root length in the treated seedling; and LRC = Average root length in the control seedling.

15. b) EVERGREEN-PHYTOTOXICITY PLANT TEST WITH POLYPHENOLS (DIRECTLY) Project LIFE13 ENV/IT/000461 Plants test were carried out using directly polyphenols. Some inhibition was showed with some polyphenols. A bio-stimulant effect was also showed by TCO polyphenol

16. b) EVERGREEN (LIFE) PLANT TEST WITH EXTRACTS OF SOILS + POLYFENOLS (MICROCOSM) Project LIFE13 ENV/IT/000461 Plants test were carried out using extracts of microcosms Experiment at the beginning of the microcosm: -- No inhibition is showed for any polyphenols, -- A possible bio-stimulant effect was appreciated by all studied polyphenols

17. b) EVERGREEN (LIFE) Project LIFE13 ENV/IT/000461 Best results both parameters (IG (%) and Lmr (cm), Table 1-2 and figures 1-4)) were obtained with TCO polyphenol solution at 0,1 g/l, following by PFV and EPV polyphenols vs control. In polyphenols 0,01 g/l solutions TCO at the concentrated solution (0, 1 g/l) continues obtaining the best results in IG and Lmr vs control. Maybe some organic compound contained in these organic products could have some phytohormonal effect on germination seed. For some polyphenols, inhibition on seed germination has been observed; it is expected because some polyphenols (particularly, derived of tannins), have a biopesticide effect on plants. When the extracts of soil are used, no inhibitory effect was showed, And a possible bio-stimulator effect was appreciated by all polyphenols.

18. c) EVERGREEN-ECOTX Project LIFE13 ENV/IT/000461 ECOTOXICITY ASSAY A toxicity test was carried out using luminescent bacteria (Microtox), in which the inhibition of the luminescence of Photobacteriumphosphoreum was measured using a luminometer (Kapanen and Itävaara, 2001) after adding extracts of the samples. This assay uses a suspension of luminescent bacteria (Photobacteriumphosphoreum) as bioassay organism for measuring acute toxicity in aqueous extracts (Bulich, 1979; Matthews and Hastings, 1987). Lyophilized bacteria were used after rehydration in the commercial solution. All assays were carried out at 15 ºC with 15 min and 30 min contact periods between 0.5 ml of bacterial suspension and compost suspension. Compost suspension was prepared by mixing 1g sample with 10ml of 2 % NaCl (w/w) solution DATA RELEVANCE Ecotoxicity assays were carried out with all polyphenols at two different concentrations (1g/L and 0.1 g/L). Ecotoxicity assay were done with the seven Evergreen polyphenols at two concentrations 1 g/l and 0,1 g/l (polyphenol concentration used in plants assays) (Table 3). According to our results and with Spanish low (BOE 10 de noviembre de 1989, número 270/1989) it is considered that a substance is toxic when its leached has an EC50 (15 minutes, 15ºC) less or equal than 3000 mg/l. The EC50 obtained in polyphenols samples demonstrated that none of them are toxic for soil organisms.

19. d) EVERGREEN (LIFE) Project LIFE13 ENV/IT/000461 CHEMICAL STABILITY: To know the chemical stability of polyphenols, were prepared solutions at work concentration (0,1 g/l), and these solutions were subjected to : FACTORS ASSAYED: pH changes (5-8) and Temperature (Heat) (25,30 and 35ºC) Polyphenols are very stables with the temperature and pH; Water soluble N and C showed only a minor changes with the Tª and pH. It is indicative of the chemical stability of polyphenols.

20. EVERGREEN (LIFE) B4 Project LIFE13 ENV/IT/000461 Effects ON PLANTS: SOME INITIAL EXPERIMENTS B4 ACTIVITY KIWI, OLIVE AND TOBACCO

21. CEBAS-CSIC: PLANTS EXPERIMENT WITH OLIVE AND KIWI (BACTERIA INOCULATION) CEBAS Project LIFE13 ENV/IT/000461

22. EVERGREEN (LIFE) B4 Project LIFE13 ENV/IT/000461 PLANT KIWI AND OLIVE

23. EVERGREEN (LIFE) B4 Project LIFE13 ENV/IT/000461 PLANT KIWI AND OLIVE (PREPARATION)

24. EVERGREEN (LIFE) B4 Firstresultswith kiwi and polyphenols (Thisexperimentwill be done another time; summerisnotgoodforthis culture) Control SOIL Bacteria Polyphenols TC

25. EVERGREEN (LIFE) B4 Olive plants inoculate with bacteria and treated with polyphenols No symptoms at this moment were showed.

26. EVERGREEN (LIFE) B4 B4 ACTIVITY FOR EVERGREEN a) TOBACCO PLANTS: plantspreparation b) NEMATODE: plantsinoculation c) POLYPHENOLS USED: TC and TCO

27. EVERGREEN (LIFE) B4 a) TOBACCO PLANTS (PREPARATION) Project LIFE13 ENV/IT/000461

28. EVERGREEN (LIFE) B4 Project LIFE13 ENV/IT/000461 b). Nematodeexperimentt at greenhouselevelontobaccoplants

29. EVERGREEN (LIFE) B4 c). Nematode (M. incognita) inoculationontobaccoplants. Project LIFE13 ENV/IT/000461 d)

30. EVERGREEN (LIFE) B4 Project LIFE13 ENV/IT/000461 PLANTS ROOT WITH NEMATODE (TOBACCO ASSAY)

31. EVERGREEN (LIFE) B4 Project LIFE13 ENV/IT/000461 PLANTS ROOT WITH NEMATICIDES (TOBACCO ASSAY)

32. EVERGREEN (LIFE) B4 Project LIFE13 ENV/IT/000461 PLANTS ROOT WITH POLYPHENOLS (TOBACCO ASSAY)

33. EVERGREEN (LIFE) Project LIFE13 ENV/IT/000461 • ACTIVITY D (DISSEMINATION) • MATERIALS FOR DISSEMINATION • Notebooks for EVERGREEN (Done) • POSTER (Done) • NOTICE BOARD (Done) • Cap of coffeefor EVERGREEN (Done) • EVENTS (Future): • Meeting: agriculturalstakeholder (January, 2016) • Sometechnical meeting from CSIC

34. EVERGREEN (LIFE). D-ACT. Project LIFE13 ENV/IT/000461 NOTICE BOARD: EVERGREEN FROM CEBAS

35. CEBAS CEBAS WORKING GROUP THANK YOU FOR YOUR ATTENTION