ACTIVITY OF CONSORTIUM INSTM Oct. 1, 2014 – March 31, 2015 EU project LIFE13 ENV IT 000461 / “EVERGREEN” “Environmentally friendly biomolecules from agricultural wastes as substitutes of pesticides for plant diseases control” ACTIVITY OF CONSORTIUM INSTM Oct. 1, 2014 – March 31, 2015 Monitoring Meeting 28-04-2015 - Sergio Miele, Enrica Bargiacchi –

SWEET CHESTNUT TANNIN EXTRACT The source: Sweet chestnut (Castanea sativa Mill.), a typical tree of Southern Europe. Bark & wood water extraction It is a traced, solvent-free, high in hydrolysable tannins (+ non-tannins) extract, with consistent characteristics, due to concentration by reverse osmosis and membrane-nanotechnology GA=Gallic Acid; GGlu=Galloyl Glucose; G-HHDP-Glu=Galloyl HHDP-Glucose; EA=Ellagic Acid; ET m/z=Ellagic Tannins m/z fractions-CAST/VESC=Castalagin/Vescalagin; PEDUNC-ISO= Peduncolagin isomer TETRA-GALLOYL-GLUCOSE GALLIC ACID ELLAGIC ACID

THE PROCESS (B2-B3-B5) STANDARDIZED EXTRACTS 1 TRACED SOURCE 8 2 STANDARDIZED EXTRACTS 9 REVERSE OSMOSIS & NANO-FILTRATION FEED FIBER (1% TANNINS) MULCHING PRE-TREATMENTS Forest Stewardship Council 5 7 4 6 3 WATER EXTRACTION

PREVIOUS ACTIVITY Since 1999 several uses in agriculture were studied Chestnut Tannins were identified for their favorable action both on crop nutrition and “well-being” (plant health). Actually Chestnut tannins affect both some soil-plant nutritional aspects (water, N-P & micronutrient availability), and act as a biostimulant, enhancing plant resistance to abiotic-biotic stresses (higher tolerance to salinity, and some seed-borne diseases, enhanced early plant growth and rooting, higher tolerance to soil nematodes, etc.) Some patents were applied, e.g. for uses such as “starter fertilizer” at sowing or transplanting, and seed treatment

THE SITES OF INSTM 2014-2015 ACTIVITY

ACTIVITY FOR THE PROJECT Month 1-6 B1/C1 – Demonstration of the performances of traditional pesticides for the control of bacterial and nematode diseases of plants important for the EU (1st CYCLE EXPERIMENT-beginning date: May 2014) B3 – Demonstration of the biological and of the chemical stability of the crude polyphenolic extracts and of their fractions, recovered from not edible vegetable biomass and waste, at laboratory scale B4 - Demonstration of the biological activity of the high quality polyphenolic extracts recovered from not edible biomass and waste, against plant pathogenic bacteria and nematode, in planta B7 - Demonstration of the in vivo performances of the high quality polyphenolic bioactive preparations, recovered from vegetable not edible biomass and waste, at pilot scale level in field screenings (PRELIMINARY ACTIVITY: FORMULATIONS FOR DELIVERY) D7 - Articles and press releases D10 - International conferences and fairs

B1 – Demonstration of the performances of traditional pesticides for the control of bacterial and nematode diseases of plants important for the EU (1st CYCLE EXPERIMENT) Tobacco Virginia Bright, field grown, single plot 500 m2, May 2014 Treatments Conclusions Agrochemicals: the early application related to necessity of respecting the 60-day pre-harvest interval is not sufficient to protect plants in a rainy season (2014) CHT: act as a biostimulant, with minimal effects on nematode count; however low evidence of nematode feeding on tobacco roots was found. Better yield results for the botanicals/biocides than agrochemicals

B3 – Demonstration of the biological and of the chemical stability of the crude polyphenolic extracts and of their fractions, recovered from not edible vegetable biomass and waste, at laboratory scale Initially activity on Sweet Chestnut tannins was carried out using the liquid 13% tannin extract. However previous experimental activity indicated poor consistency among results obtained with liquid extracts, especially after some storage period Apparently, there was no microbial spoiling during storage, but some molecular reassembling happened, which resulted in a reduced biological action on treated plants Therefore, the more recent experimental activity has been entirely based on the use of a spray-dried tannin extract formulation, as later indicated (see B7).

B4 - Demonstration of the biological activity of the high quality polyphenolic extracts recovered from not edible biomass and waste, against plant pathogenic bacteria and nematode, in planta Tobacco Virginia Bright, greenhouse pot test, Oct-Nov 2014 Treatments CHT Non-infested control Etoprofos Infested control Azadirachtine Conclusions Etoprofos and Azadirachtine acted as nematicides, with marked reductions in nematode counts at the end of the test, while CHT showed a significant reduction vs. the infested control, but to a much less extent. Both Etoprofos and Azadirachtine had a depressive effet on root mass density, and the latter on fine root development too. Biostimulant effect of CHT on fine root emission

B7 - Demonstration of the in vivo performances of the high quality poly-phenolic bioactive preparations, recovered from vegetable not edible biomass and waste, at pilot scale level in field screenings (preliminary activity: January-March 2015) Preliminary activity consisted in preparing at industrial pilot level* formulations for field application of Chestnut tannins, to be used in next 2015 growing season: A microgranulated fertilizer for application at sowing/transplanting along the row (pilot plant) A fully and easily, non-foamy, wettable tannin extract powder for application in microirrigation (pilot plant) * 2014 B1 action used a lab-prepared micro granulated fertilizer and a spray-dried wettable powder

MAJOR MICROGRANULATED FERTILIZERS WITH TANNIN EXTRACTS % MICRO TAN MICRO NP Tannin extract 18 12 Nitrogen (N) 7 Phosphate (P2O5) 30 Total Iron (Fe) 3 2 Complex Tannin-Fe (Fe) 2.4 1.2 Granule Size mm (> 80%) 100% 0.6-1.2 mm 0.8 Authorization: Italian D.Lgs. 75/2010 as Iron-Tannin complex fertilizer and Nitrogen-Phosphate Fertilizer w/ Iron-Tannin complex

CORRECTIVES-VARIOUS CORRECTIVES * DRY TANNINS EXTRACT WP pHw 3.0 – 3.5 ORGANIC CARBON (C) MIN. 50 % TANNIN CONTENT MIN. 75 % * Authorization: D.M. 30-06-2011, publ. on Italian Official Gazette n° 215 Sept. 15, 2011 (modifications of D.Lgs. 75/2010)

INSTM ACTIVITY FOR THE PROJECT Month 1-6 D2 – Notice boards & information displayed on INSTM website (Link: http://www.instm.it/0000000988.aspx), at the Chestnut Tannin production site, and at the Tobacco experiment site D7 - Articles and press releases Submission of the following manuscript: BARGIACCHI E., CAMPO M., ROMANI A., MILLI G., MIELE S., 2015. Hydrolysable tannins from sweet Chestnut (Castanea sativa Mill.) to improve tobacco and food/feed quality. Note 1: Fraction characterization, and Tobacco biostimulant effect for gall-nematode resistance. Journal of Agricultural and Food Chemistry, submitted D8 – Networking: with Regione Umbria’s PSR funded Project QualiTaba (Traced quality of tobacco in a context of sustainable production in Central Italy) D10 - International conferences and fairs Participation to CORESTA Congress (www.CORESTA.org), Oct. 12-16, 2014, Quebec City (Canada) with the oral presentation: MILLI G., BARGIACCHI E., MIELE S. , 2014. Comparative tests among nematode agrochemicals and alternative products on Virginia Bright tobacco. Presentation AP07, CORESTA web. Submission of an abstract for a poster presentation at the International Fertilizer Society, London (UK), 2015 Technical Conference, June 23-24, 2015 Submission of an abstract for an oral presentation at CORESTA Agro-Phyto Meeting, Izmir (Turkey), Oct. 25-29, 2015 Submission of an abstract for an oral presentation at the “2nd World Congress on the Use of Biostimulants in Agriculture”, Firenze (Italy), Nov. 16-19, 2015.

INSTM ACTIVITY FOR THE PROJECT Month 6-12 (Sept. 30, 2015) B1 – Demonstration of the performances of traditional pesticides for the control of bacterial and nematode diseases of plants important for the EU (2nd CYCLE EXPERIMENT-beginning date: May 2015). A.I.: Ch Tannins, Etoprofos, Azadirachtine, Bacillus firmus, Kendal Nem (pool of unidentified biomolecules) B3 – Demonstration of the biological and of the chemical stability of the crude polyphenolic extracts and of their fractions, recovered from not edible vegetable biomass and waste, at laboratory scale (other polyphenols) B4 - Demonstration of the biological activity of the high quality polyphenolic extracts recovered from not edible biomass and waste, against plant pathogenic bacteria and nematode, in planta (Ch tannins + other polyphenols) B7 - Demonstration of the in vivo performances of the high quality poly-phenolic bioactive preparations, recovered from vegetable not edible biomass and waste,at pilot scale level in fieldscreenings  preliminary activity  FORMULATIONS for enhancing protective results (Ch tannins + other polyphenols)

INSTM ACTIVITY FOR THE PROJECT Month 6-15 D2 – Notice boards & information displayed: on INSTM website, and at the Chestnut Tannin microgranulate fertilizers production site, and the crop experiment sites D3 Demonstration workshop in Italy  Visit to Ch tannin production site, Tobacco experiments, and Ch Tannin microgranulate fertilizer production site (proposed date: July, with 12M-Meeting) D7 - Articles and press releases poster presentation at the International Fertilizer Society, London (UK), 2015 Technical Conference, June 23-24, 2015 web release from the oral presentation at CORESTA Agro-Phyto Meeting, Izmir (Turkey), Oct. 25-29, 2015 article on Acta Horticulturae from the oral presentation at the “2nd World Congress on the Use of Biostimulants in Agriculture”, Firenze (Italy), Nov. 16-19, 2015. D8 – Networking: proposed networking with 2020 Horizon “From Biodiversity to Chemodiversity: Novel Plant Produced Compounds with Agrochemical and Cosmetic interest(AGROCOS)” KBBE-2009-3-1-04 Prospecting for novel plant-produced compounds – ended 2014-03-31

INSTM ACTIVITY FOR THE PROJECT Month 6-15 D10 - International conferences and fairs AOCS Annual Meeting, Orlando (USA), May 2-6, 2015, with an oral presentation: BARGIACCHI E., CAMPO M., ROMANI A., MIELE S., 2015. Calibration of a fluorescence-based sensor and  non-invasive rapid method for detecting anti-oxidants and maturation in tobacco leaf . Session ANA4-B International Fertilizer Society, London (UK), June 23-24, 2015 Technical Meeting, with a poster presentation: MIELE S., BARGIACCHI E., 2015. Microgranulated fertilizers based on Chestnut tannins: technical and legal status problems for their production and use CORESTA Agro-Phyto Meeting, Izmir (Turkey), Oct. 25-29, 2015, with an oral presentation: BARGIACCHI E., CAMPO M., ROMANI A., MIELE S., 2015. Life+2013 EVERGREEN focusses on polyphenol botanicals for a sustainable tobacco production. 2nd World Congress on the Use of Biostimulants in Agriculture, Firenze (Italy), Nov. 16-19, 2015, with the oral presentation: BARGIACCHI E., CAMPO M., ROMANI A., MIELE S., 2015 Fractions and Formulations to Enhance Biostimulant Activity of Sweet Chestnut (Castanea sativa Mill.) Tannin Extracts. D11 – Dissemination to institutions and policy makers Event at Accademia dei Georgofili (proposed date: Nov. 13, 2015), present: Political representatives, major Farmers’ Associations and Unions, Federchimica, FIRAB, AIAB, LegaAmbiente

DAL 2003 AL 2014 SONO STATE DEPOSITATE 5 DOMANDE DI BREVETTO CON PRIORITA’ ITALIANA ED ESTENSIONE EUROPEA O PCT (Brevetti acquisiti o in progress) MIELE S., BARGIACCHI E., COSTA G.L., PAMPANA S., FOSCHI L., 2003. Procedimento per l’utilizzo di estratti organici ottenuti per lisciviazione acquosa di legno, finalizzato alla preparazione di soluzioni organiche acidificanti per l’agricoltura biologica e di concimi ad alta efficienza ed a basso impatto ambientale. Domanda di brevetto industriale n° MI2003A 000640, depositata il 31-03-03, brevetto italiano 0001347939 del 07 ottobre 2008 a nome Gruppo Mauro Saviola. BARGIACCHI E., BERTOLA R., COSTA G., DELLA CROCE R., MIELE S., POMPEIANO A., ZAMBELLI P., 2005. Metodo per la preparazione di fertilizzanti organo-minerali granulari con azoto a lenta cessione, ad alta efficienza e a minimo impatto ambientale. Domanda di brevetto per invenzione industriale MI2005A 001892 del 7-10-2005. Brevetto italiano n° 0001366444 del 30-09-2009. BARGIACCHI E., COSTA G., MIELE S., MAGNI S., 2010. Uso di estratti naturali di tannini e non tannini atti a migliorare la fertilità del suolo e determinare un effetto starter sulle colture, e fitocomplesso di tannini e di non tannini per tale uso. Brevetto per invenzione industriale MI2010A000040 del 15-01-2010 MIELE S., ROMANI A., BARGIACCHI E., CAMPO M., 2012. Metodo di produzione del tabacco per la riduzione di nitrosammine. Brevetto italiano MI2012A001419 del 08-08-2012. BARGIACCHI E., BELLOTTI P., PINELLI P., COSTA G., MIELE S., ROMANI A., ZAMBELLI P., SCARDIGLI A., 2014. Uso di estratto di tannini di castagno come additivo antiossidante, antimicrobico e per ridurre nitrosammine e micotossine. Domanda di brevetto per invenzione industriale No.  MI2014A000177, Data di deposito: 7 febbraio 2014

CARATTERISTICHE ANTIOSSIDANTI E ANTIRADICALICHE DEI 9 FLUSSI DI PROCESSO PROCESS STREAMS GAE1 (g/100g EXTR) GAE2 (g/mM Tot.Tann.) EC50 (µM) 1 2.382 0.636 0.695 2 0.640 0.413 2.007 3 9.148 1.182 0.444 4 7.905 0.938 1.000 5 2.370 0.565 1.429 6 30.991 1.319 1.516 7 0.067 0.250 2.399 8 7.367 0.616 1.510 9 0.493 0.052 0.545 DPPH (1,1-diphenyl-2-picrylhydrazyl) in methanol GAE1: Gallic Acid Equivalents (g gallic acid/100g sample); GAE2: Gallic Acid Equivalents (g gallic acid/mM total tannins by HPLC/DAD); EC50: polyphenolic concentration inhibiting DPPH· activity to 50% (μM) A. Romani & M.Campo, 2012 (mod.)

CONSORTIUM INSTM Italian Interuniversity Consortium for Science and Technology of Materials (www.instm.it) Sergio Miele - full professor of agronomy & agrobiotechnology expert in natural & fossil-based fertilizers & agrochemicals, innovative formulations and application methods (>20 EU, US & PCT patents, 5 on tannins) responsible of research contracts, scientific responsible of the associated beneficiary of 6 research projects Enrica Bargiacchi agronomist & crop processing chain expert innovative fertilizer & agrochemical formulation (> 20 EU, US & PCT patents, 5 on tannins)