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
EVERGREEN (LIFE)
CEBAS-CSIC (EVERGREEN)
Dr. Carlos Garcia
Dra. Teresa Hernández
Dr. J. Moreno
Lda. Mª Dolores Coll
Lda. Blanca
D. Manuel Campoy
Dña. C. Montesinos
Project LIFE13 ENV/IT/000461
CEBAS-CSIC GROUP: EVERGREEN
Project LIFE13 ENV/IT/000461
WHAT CEBAS-CSIC GROUP WILL DO IN THE
EVERGREEN PROJECT??
EVERGREEN: This project will demostrate the successful use of biomolecules
recovered from agricultural vegetable wastes as substitutes of pesticides for the
environmentaly friendly and sustainable control of plant diseases
ACTIONS B1, B3, B4, (B6)
B1) Demostration of the performances of traditional pesticides for the control of bacterial and nematodes
diseases of plats important for the EU.
B4) Demostration of the biological activity of the high quality polyphenols extracts recovered from not edible
biomass and wastes, against plant pathogenic bacteria and nematode, in plant.
B3) Polyphenols activity??
ACTIONS C1, C2 and C4:
C1: Monitoring on the environmental impact of cooper and nematicides for the crop defence against
phytopathogenic bacteria and nematodes
C2 (B6?). Monitoring of the absences of side effects for the high quality polyfhenolic preparations on common
targets of the living organisms at laboratory level.
C4. Monitoring of the short-term environmental beneficts from the use of the high quality polyphenols
preparations in plant disease control at pilot scale level in field screening
ACTION D…: Communication and dissemination actions
EVERGREEN (LIFE)
Project LIFE13 ENV/IT/000461
B1) and C1 Demostration and monitoring of the performances of traditional
pesticides for the control of bacterial and nematodes diseases of plats important
for the EU. (From October 2014 to March 2015: DONE)
AGRICULTURE: WE NEED TO PRODUCE FOOD
(9000 MILL IN 2050)
1) Use of agrochemical to combat plant diseases; is it useful? YES
2) Are good the agrochemicals for the environment?? What about soil
quality?? (Cu and nematicides) (OUR ACTYVITY B1 and C1)
SUSTAINABLE PRODUCTION: WE MUST PRODUCE
WITH NO RISK FOR THE ENVIRONMENT
EVERGREEN
Project LIFE13 ENV/IT/000461
C2-(B6) ACTIVITY (FROM APRIL 2015)
Monitoring of the absences of side effects for the high quality polyphenolic preparations on common targets of the living organisms at
laboratory level (EFFECT ON SOIL MICROORGANISMS).
CEBAS EXPERIMENT (FINISHED))
CONTROL SOIL
SOIL + POLYPHENOLS
SOIL + Pathogen (bacteria and nematicide)
SOIL + POLYPHENOLS + Pathogen
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,
Nº
Description
1
Control (soil)
TC (chesnut A)
TCO (chesnut+olive)
TCC (chesnut+artichoke)
TAN (chesnut B)
PFV (red vine leaves)
EPV (grapefruit seeds)
PV (orujo grape poder)
2
3
4
5
6
7
8
Paramete
rs
MICROCOSM
TIMES: 0,1 AND 2 MONTHS
pH
CE
Macro and microelements
Ct, Nt, COT
Water Soluble carbon and nitrogen
Β- Glucosidase
Fosfatase
Des-hidrogenase
Urease
Microbial respiration
PLFAs
EVERGREEN (LIFE)
Project LIFE13 ENV/IT/000461
N water soluble
TOC water soluble
100
70
60
80
ppm Cs
ppm Nt
50
40
30
20
60
40
20
10
0
0
Control
TC
TCO
TCC
T0
T30
TAN
PFV
EPV
PV
Control
TC
T60
T0
Urease
TCC
T30
TAN
PFV
EPV
PV
PFV
EPV
PV
T60
β-Glucosidase
5
2,0
µ mol PNF/ g dry soil h
µmol N-NH4/ g dry soil h
TCO
4
3
2
1
1,5
1,0
0,5
0,0
0
Control
TC
TCO
TCC
T0
T30
TAN
T60
PV
EPV
PFV
Control
TC
TCO
T0
TCC
T30
TAN
T60
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)
Table 18. Bacterial, fungal, Gram+, Gram-, satured and monosatured PLFAs concentration in soils T0.
PLFA (nmol g-1)
Control
TC
TCO
TCC
TAN
PFV
Bacteria
0,42 a
4,61 c
3,71 bc
2,27 ab
3,63 bc
3,93 bc
EPV
1,81 ab
2,45 abc
Gram+ Bacteria
0,38 a
3,08 c
1,72 abc
1,12 ab
2,13 abc
2,31 bc
1,44 abc
1,67 abc
Gram- Bacteria
0,04 a
1,53 bc
1,99 c
1,15 abc
1,50 bc
1,61 bc
0,36 ab
0,78 abc
Fungi
Total saturated
PLFAs
Total monoin,
PLFAs
Actinobacteria
0,00 a
0,04 a
0,03 a
0,01 a
0,01 a
0,07 a
0,00 a
0,06 a
0,79 a
5,84 bc
7,42 c
4,62 abc
6,30 bc
0,03 a
1,01 b
1,15 b
0,64 ab
0,68 ab
0,013 a
0,050 a
0,008 a
0,004 a
0,007 a
4,46 abc
1,22 b
0,018 a
2,65 ab
0,09 a
0,001 a
PV
3,35 ab
0,24 a
0,002 a
Table 20. Bacterial, fungal, Gram+, Gram-, satured and monosatured PLFAs concentration in soils T30.
PLFA (nmol g-1)
Bacteria
Control
4,79 c
TC
1,70 a
TCO
4,57 c
TCC
1,79 a
TAN
3,32 bc
Gram+ Bacteria
2,38 bc
1,27 ab
3,09 c
1,09ab
2,22 bc
Gram- Bacteria
Fungi
Total saturated
PLFAs
Total monoin,
PLFAs
Actinobacteria
2,11 cd
0,17 e
0,43 a
0,07 d
1,47 bc
0,18 e
0,69 ab
0,00 a
1,10 ab
0,07 d
7,30 c
2,90 ab
5,41 bc
3,87 ab
4,21 ab
0,95 ab
0,37 a
0,96 ab
0,68 ab
0,54 ab
0,40 d
0,08 abc
0,14 c
0,11 bc
0,10 abc
PFV
4,80 c
2,47 bc
2,33 d
0,02 b
5,31 bc
1,16 b
0,14 c
EPV
2,80 ab
1,60 ab
1,18 ab
0,05 c
4,99 abc
0,59 ab
0,03 a
PV
1,44 a
0,72 a
0,71 ab
0,07 d
2,66 a
0,30 a
0,06 ab
Table 22. Bacterial, fungal, Gram+, Gram-, satured and monosatured PLFAs concentration in soils T60.
PLFA (nmol g-1)
Bacteria
Control
7,73 ab
TC
7,86ab
TCO
9,07ab
TCC
10,13 b
TAN
9,67 ab
Gram+ Bacteria
4,93 ab
5,30 ab
6,12 ab
7,14 b
6,24 ab
Gram- Bacteria
2,80 a
2,55 a
2,95 a
2,98 a
3,43 a
Fungi
0709 b
0,22 ab
0,17 ab
0,36 b
0,20 ab
7,03 a
7,62 a
8,36 a
10,92 a
10,59 a
2,86 a
2,54 a
2,99 a
3,69 a
3,45 a
Total saturated
PLFAs
Total monoin,
PLFAs
PFV
EPV
9,52ab 6,39 ab
6,98 b 4,12 a
2,54 a
0,34 ab
10,33 a
2,55 a
2,26 a
0,21 ab
6,37 a
2,03 a
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 biodiversity that control soil. Only fungi
could be affected
PV
5,59 a
4,00 a
1,58 a
0,23 ab
6,64 a
1,66 a
Polyphenols added to the soil
have not influence on soil biobiodiversity and soil population
microorganisms
EVERGREEN (LIFE)
Project LIFE13 ENV/IT/000461
Polyphenol Total PLFAs T60
Polyphenol Fungi/Bac ratio T60
0.15
16
0.1
nmol/g
nmol/g
12
8
0.05
4
0
0
Control
TC
TCO
TCC
TAN
PFV
EPV
Control
PV
TC
TCO
TCC
TAN
PFV
EPV
PV
Polyphenol Sat PLFA/ Mono Sat PLFA
ratio T60
Polyphenol G+/G- ratio T60
4
5
4
2
nmol/g
nmol/g
3
3
2
1
1
0
Control
TC
TCO
TCC
TAN
PFV
EPV
PV
0
Control
TC
TCO
TCC
TAN
PFV
EPV
PV
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.
EVERGREEN
Project LIFE13 ENV/IT/000461
POLYPHENOLS “ACTIVITY”
B3 ACTIVITY??? (from April 2015)
CEBAS EXPERIMENT
a) POLYPHENOLS ANALYSIS
b) PHYTOTOXICITY EFFECT
c) ECOTOXICOLOGICAL ASPECTS
d) CHEMICAL STABILITY
a) EVERGREEN-ANALYSIS
30
35
GALLOTANINOS
25
Project LIFE13 ENV/IT/000461
mg/g dry 30
matter
ELLAGITANINOS
25
20
mg/g dry
matter
20
15
15
10
10
5
5
0
0
TAN
TC
TCC
GL1
GL2
TAN
TCO
TC
TCC
EG1
GL3
EG2
TCO
EG3
Total EG
mg/g dry
matter
TOTAL GALLOTANINOS
60
50
80
70
mg/g
dry
60
mg/g dry
40
matter
matter
50
40
30
30
20
20
10
10
0
TAN
0
TAN
TC
TCC
TC
TCC
TCO
POLYPHENOLS from chestnuts showed a good quantity of gallotannins and ellagitannins.
TCC showed the highest value.
TCO
a) EVERGREEN-ANALYSIS
Project LIFE13 ENV/IT/000461
Ellagic acid
8
7
6
mg/g dry matter
5
4
3
2
1
0
TAN
TC
TCC
TCO
The ellagic acid was found in polyphenols from chestnuts used in EVERGREEN.
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.
b) EVERGREEN-PHYTOTOXICITY
Project LIFE13 ENV/IT/000461
TC (chesnut)
TCO (chesnut+oil)
TCC (chestnut)
TAN (chestnut+ artichoke)
PFV (Polvere Foglie vite rossa).
EPV (Estrato in polvere vinacce)
PV (Polvere vinaccioli).
Germination tests on filter paper: In Petri dishes with filter paper and 15 seeds of rye-grass (Lollium perenne) 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.
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
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
b) EVERGREEN (LIFE)
Project LIFE13 ENV/IT/000461
Germination Index (%)
250,0
200,0
%
150,0
100,0
50,0
0,0
Control Control
agua
TC
TCO
TCC
TAN
PFV
EPV
PV
Best results both parameters (IG (%) and Lmr (cm), Table 1-2 and figures 14)) 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.
Reference
Ecotoxicity (mg/l)
TC 1g/l
351110
292600
TC 0,1 g/l
6990000
5120000
TCO 1g/l
343350
269280
TCO 0,1g/l
4720000
5140000
TCC 1g/l
431120
624830
TCC 0,1g/l
3790000
5820000
TAN 1g/l
366810
349200
TAN 0,1g/l
239570
249100
PV 0,1g/l
447948110
316232270
EPV 1g/l
83990
80680
EPV 0,1g/l
295670
406140
PFV 1g/l
5190000
3800000
PFV 0,1g/l
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 Photobacterium phosphoreum was measured using
a luminometer (Kapanen and Itävaara, 2001) after adding extracts of the samples. This
assay uses a suspension of luminescent bacteria (Photobacterium phosphoreum) 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
5290000
6010000
PV 1g/l
c) EVERGREEN-ECOTX
7480000
8380000
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.
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)
Polyphenol
TC
TCO
TCC
TAN
Tª C
ppm Cs
ppm Ns
pH
ppm Cs
ppm Ns
25
30
37
45
50
25
30
37
45
50
25
30
37
45
50
25
30
37
45
50
287,74
287,16
304,20
300,78
299,66
276,25
277,14
278,95
287,53
294,85
315,49
336,38
338,65
347,65
326,63
307,04
310,77
311,25
319,18
323,70
0
0
0
0
0
0
0
0
0
0
1,48
1,40
1,36
1,33
1,39
0
0
0
0
0
4
5,5
7
8,5
10
4
5,5
7
8,5
10
4
5,5
7
8,5
10
4
5,5
7
8,5
10
254,50
233,30
232,20
232,55
232,32
288,20
292,40
290,30
291,98
289,29
239,80
240,50
238,90
239,52
221,66
286,20
288,50
286,30
289,80
284,17
0
0
0
0
0
0
0
0
0
0
0,53
0,40
0,22
0,31
0,34
0
0
0
0
0
Polyphenol
PV
EPV
PFV
Tª C
25
30
37
45
50
25
30
37
45
50
25
30
37
45
50
ppm Cs
289,90
295,19
296,33
308,27
309,06
372,73
375,74
357,01
384,92
385,36
241,24
239,13
238,11
249,01
247,41
ppm Ns
0
0
0
0
0
2,41
2,35
2,64
2,33
2,33
0
0
0
0
0
pH
4
5,5
7
8,5
10
4
5,5
7
8,5
10
4
5,5
7
8,5
10
ppm Cs
318,40
327,60
338,00
337,53
339,69
344,50
348,30
353,80
354,03
352,74
210,90
209,60
209,30
212,81
213,45
ppm Ns
0
0
0
0
0
2,24
2,13
1,79
2,15
2,26
0
0
0
0
0
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.
EVERGREEN (LIFE) B4
Project LIFE13 ENV/IT/000461
Effects ON PLANTS: SOME INITIAL
EXPERIMENTS
B4 ACTIVITY
KIWI, OLIVE AND
TOBACCO
CEBAS-CSIC: PLANTS EXPERIMENT WITH OLIVE AND KIWI
(BACTERIA INOCULATION)
Project LIFE13 ENV/IT/000461
EVERGREEN (LIFE) B4
Project LIFE13 ENV/IT/000461
PLANT KIWI AND OLIVE
EVERGREEN (LIFE) B4
Project LIFE13 ENV/IT/000461
PLANT KIWI AND OLIVE (PREPARATION)
EVERGREEN (LIFE) B4
First results with kiwi and polyphenols
(This experiment will be done another time; summer is not good for this culture)
Polyphenols/ nº days
7d
30d
60d
Control
3
4
5
5
5
5
5
5
5
3
3
5
4
3
5
4
5
5
1
1
3
3
3
5
2
3
1
Bacteria
TC
TCO
TCC
TAN
PV
EPV
PFV
Control SOIL
Bacteria
Polyphenols TC
EVERGREEN (LIFE) B4
Olive plants inoculate with bacteria and treated with polyphenols
No symptoms at this moment were showed.
EVERGREEN (LIFE) B4
B4 ACTIVITY FOR EVERGREEN
a) TOBACCO PLANTS: plants preparation
b) NEMATODE: plants inoculation
c) POLYPHENOLS USED: TC and TCO
EVERGREEN (LIFE) B4
a) TOBACCO PLANTS (PREPARATION)Project LIFE13 ENV/IT/000461
1.Tobacco seeds
7. Petri dishes 48
h later.
2. Seeds aireation
in Enlenmeyer flask
8. hydrated
seedling
3. Tobacco seeds
pipetting
9. Remove tobacco
seedling
4. Petri dishes
5.Incubation
10. Seedling
transplant to
vermiculite
6. Seeds in Petri
dishes
11. Vermiculite
tray
EVERGREEN (LIFE) B4
Project LIFE13 ENV/IT/000461
b). Nematode experimentt at greenhouse level on tobacco plants
Nº
Treatments
Commercial
product
0
Control – (without nematode)
1
Control + (with nematode)
2
Mocap
granulado
Etoprofos 10%
3
Neenazal
Azadirachtine
(p/v)
4
TC (chestnut polyphenol)
0,1 g/ L
5
TCO (Chestnut polyphenol+olive)
0,1 g/ L
----
Made
Doses
---
----
CERTIS
1%
30 k/ Ha
0,3g/100 ml
EVERGREEN (LIFE) B4
c). Nematode (M. incognita) inoculation on tobacco plants.
d)
Project LIFE13 ENV/IT/000461
EVERGREEN (LIFE) B4
Project LIFE13 ENV/IT/000461
PLANTS ROOT WITH NEMATODE (TOBACCO ASSAY)
EVERGREEN (LIFE) B4
Project LIFE13 ENV/IT/000461
PLANTS ROOT WITH NEMATICIDES (TOBACCO ASSAY)
EVERGREEN (LIFE) B4
Project LIFE13 ENV/IT/000461
PLANTS ROOT WITH POLYPHENOLS (TOBACCO ASSAY)
EVERGREEN (LIFE)
Project LIFE13 ENV/IT/000461
ACTIVITY D (DISSEMINATION)
MATERIALS FOR DISSEMINATION
• Notebooks for EVERGREEN (Done)
• POSTER (Done)
• NOTICE BOARD (Done)
• Cap of coffee for EVERGREEN (Done)
EVENTS (Future):
• Meeting: agricultural stakeholder (January, 2016)
• Some technical meeting from CSIC
EVERGREEN (LIFE). D-ACT.
Project LIFE13 ENV/IT/000461
NOTICE BOARD: EVERGREEN FROM CEBAS
CEBAS WORKING GROUP
THANK YOU FOR YOUR ATTENTION