1. ACTIVITY OF CONSORTIUM INSTM Oct. 1, 2014 – March 31, 2015
EU project LIFE13 ENV IT / “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
- Sergio Miele, Enrica Bargiacchi –

2. 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

3. THE PROCESS (B2-B3-B5) STANDARDIZED EXTRACTS1
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

4. 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

5. THE SITES OF INSTM 2014-2015 ACTIVITY

6. 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

7. 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

8. 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).

9. 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

10. 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

11. 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%) % mm
0.8
Authorization: Italian D.Lgs. 75/2010 as Iron-Tannin complex fertilizer and Nitrogen-Phosphate Fertilizer w/ Iron-Tannin complex

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

13. INSTM ACTIVITY FOR THE PROJECT Month 1-6
D2 – Notice boards & information displayed on INSTM website (Link: 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., 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 ( Oct , 2014, Quebec City (Canada) with the oral presentation:
MILLI G., BARGIACCHI E., MIELE S. , 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 , 2015
Submission of an abstract for an oral presentation at the “2nd World Congress on the Use of Biostimulants in Agriculture”, Firenze (Italy), Nov , 2015.

14. 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)

15. 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 , 2015
article on Acta Horticulturae from the oral presentation at the “2nd World Congress on the Use of Biostimulants in Agriculture”, Firenze (Italy), Nov , 2015.
D8 – Networking: proposed networking with 2020 Horizon “From Biodiversity to Chemodiversity: Novel Plant Produced Compounds with Agrochemical and Cosmetic interest(AGROCOS)” KBBE Prospecting for novel plant-produced compounds – ended

16. 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., 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., Microgranulated fertilizers based on Chestnut tannins: technical and legal status problems for their production and use
CORESTA Agro-Phyto Meeting, Izmir (Turkey), Oct , 2015, with an oral presentation:
BARGIACCHI E., CAMPO M., ROMANI A., MIELE S., 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 , 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

19. 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., 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 , depositata il , brevetto italiano del 07 ottobre 2008 a nome Gruppo Mauro Saviola.
BARGIACCHI E., BERTOLA R., COSTA G., DELLA CROCE R., MIELE S., POMPEIANO A., ZAMBELLI P., 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 del Brevetto italiano n° del
BARGIACCHI E., COSTA G., MIELE S., MAGNI S., 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 MI2010A del
MIELE S., ROMANI A., BARGIACCHI E., CAMPO M., Metodo di produzione del tabacco per la riduzione di nitrosammine. Brevetto italiano MI2012A del
BARGIACCHI E., BELLOTTI P., PINELLI P., COSTA G., MIELE S., ROMANI A., ZAMBELLI P., SCARDIGLI A., 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

20. 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.)

21. CONSORTIUM INSTM Italian Interuniversity Consortium for Science and Technology of Materials (
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)