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 fractionsCAST/VESC=Castalagin/Vescalagin; PEDUNC-ISO= Peduncolagin isomer
TETRA-GALLOYL-GLUCOSE
GALLIC ACID
ELLAGIC ACID
THE PROCESS (B2-B3-B5)
1
TRACED
SOURCE
8
2
REVERSE
OSMOSIS &
NANOFILTRATION
STANDARDIZED
EXTRACTS
MULCHING
9
FEED FIBER (1%
TANNINS)
PRETREATMENTS
5
7
4
6
WATER
EXTRACTION
3
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
Table 1. Nematode field experiments. 2014 treatments on Virginia Bright tobacco
Treatm.
No.
AI & Formulation
a
Commercial
Product (CP)
0
Control
1
Etoprofos 10% MG
Mocap
2
Oxamyl 5% MG
Vydate
3
CHT extract 18% MG + CHT 75% WP
Saviostart 18 + Saviotan WP
4
Bacillus firmus I-1582 5% WG
FlocteR
5
Azadirachtine 2.4% WG
Oikos
a
MG = microgranules ; WG = wettable granules ; WP = wettable powder
Manufacturer
Certis Europe
DuPont CropProt
Gruppo Saviola
Bayer CropScience
Sipcam Italy
CP Rate
-1
(kg ha )
60
60
30 + 45
80
10
• 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
– Etoprofos
– Azadirachtine
Non-infested control
Infested control
• 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
18
12
Nitrogen (N)
0
7
Phosphate (P2O5)
0
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
0.8
Tannin extract
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. 1619, 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 plantproduced 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 fluorescencebased 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)
1) 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.
2) 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.
3) 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 15-012010
4) 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.
5) 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
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
PROCESS STREAMS
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.)