Project LIFE13 ENV/IT/000461 “Environmentally friendly biomolecules from agricultural wastes as substitutes of pesticides for plant diseases control” “EVERGREEN”

Project LIFE13 ENV/IT/000461

“Environmentally friendly biomolecules from agricultural wastes as substitutes

of pesticides for plant diseases control”

“EVERGREEN”Start: 01/10/2014End: 31/09/2016

EVERGREEN 12nd month Meeting – 2015, November 5th

Molecular Plant Pathology Lab – DISPAA – University of Florence,Polo Scientifico e Tecnologico, Sesto Fiorentino (Florence)

INSERIRE AGENDA

LIFE13 ENV/IT/000461 – EVERGREEN 12nd month Meeting – 2015, November 5th

DISPAA, University of Florence

B1, B2, B3, B4, B5, B7 B1, B2, B3, B4, B5, B6, B7

(B4) B7 (B4) B7

C1, C5 C1, C2, C3, C4, C6, C7

(C1) C7 C1, C5





















EVERGREEN form for Monthly/Periodical reports



TECHNICAL ACTIVITIES CARRIED OUTB. Implementation actions

B2 Demonstration of the qualitative and quantitative yields of extraction process for the recovery of high quality polyphenolic molecules from not edible vegetable biomass and waste at laboratory scale

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

B5 Demonstration of Kilo-scale extraction of the high quality poly-phenolic bioactive molecules recovered from vegetable not edible biomass and waste

B6 Demonstration of the null toxicity profile of the high quality poly-phenolic bioactive molecules recovered from vegetable not edible biomass and waste, on model organisms and microorganisms.



TECHNICAL ACTIVITIES CARRIED OUT

C. Monitoring of the impact of the project actions

C2 Monitoring of the absence of side effects for the high quality standardised polyphenolic preparations on common targets of any living organism at laboratory level

C3 Monitoring of the absence of a direct selection operated by the polyphenolic preparations towards the emergence of bacteria resistant to the polyphenolic molecules themselves, at laboratory level

C6 Monitoring of the absence of a selection on the polyphenolic preparations on the selection of copper and antibiotic resistant bacteria, on plant and in soil, from laboratory to in field screenings.



Action B31st January 2015 - 31st December 2015
















HR inhibition on Tobacco challenged by Pseudomonas savastanoi












Test No. 202: Daphnia magna Acute Immobilization Test OECD Guidelines for the Testing of Chemicals Retrieved July 4, 2013

Action B6 1st April 2015 - 31st December 2015

Genus Artemia in ecotoxicity testingEnvironmental Pollution 144 (2006) 453-462



Action C21st January 2015 - 31st December 2016



Action C31st April 2015 - 31st March 2016



Inhibition of TTSS

• Inhibition of hrpA promoter activity

(*) Normalized fold versus WT condition (without treatment, only MM medium)

• Inhibition of Quorum Sensing promoter activity

Code Extract/Molecule

hrpa promoter

Effect*

VN Grape seed 0.52 ± 0.011

TV Green tea leaf 0.46 ± 0.013

FO Olive leaf 0.75 ± 0.008

FC Artichoke leaf 0.47 ± 0.017

Kan Kanamycin 0.21 ± 0.018

PCA p-Coumaric acid 1.03 ± 0.022

QS promoterEffect*

1.30 ± 0.007

0.89 ± 0.004

0.68 ± 0.025

0.44 ± 0.031

0.23 ± 0.022

0.98 ± 0.006

Code Peptide vs HrpA

hrpa promoter

Effect*

Li27 Psn23 0,86 ± 0,021

AP17 Psn23 0,64 ± 0,016

Psa21 Psa 0,74 ± 0,018

and QS

• Inhibition of Type Three Secretion System pilus assembly

Inhibition of TTSS

Results were calculated measuring abs ratio (490 nm/600 nm) with this formula:

T3 pilus assembly (%) = [ (Xunk – XΔhrpA) / (XWT – XΔhrpA) ] *100X, abs ratio

Xunk, abs ratio of unknown sample

Congo Red assay

VN TV FO FC0

102030405060708090

100

Extract

T3 p

ilus a

ssem

bly

(%)

Psa21 AP17 Li27 0

102030405060708090

100

Peptide

T3 p

ilus a

ssem

bly

(%)

• Modulation of gene expression by polyphenolic extracts and anti-infective peptides

Inhibition of TTSS

In progress…

In vitro at 24hWT vs WT + molecules

16s rRNA gene housekeepingtwo independent RNA extraction

hrpA hrpL hrpRS hrpV Lon rpoN0.1

1

10

AP17 AP17 60µM

Nor

mal

ized

Fol

d Ex

pres

sion

hrpA hrpL hrpRS hrpV Lon rpoN0.1

1

10

Li27 30µM

Nor

mal

ized

Fol

d Ex

pres

sion

hrpA

hrpL

hrpV

hrpR

Srp

oN Lon

RsmA

RsmB

PssI

PssR

0.1

1

10

VN 100µM

Nor

mal

ized

Fol

d Ex

pres

sion

hrpA hrpL hrpV hrpRS rpoN Lon RsmARsmB PssI PssR0.1

1

10

TV 100µM

Nor

mal

ized

Fol

d Ex

pres

sion

hrpA hrpL hrpV hrpRS rpoN Lon RsmARsmB PssI PssR0.1

1

10FC FO

Nor

mal

ized

Fol

d Ex

pres

sion 100µM

and QS

Polyphenolic-based

plant extracts

Anti-virulence Peptides



And now,

Dissemination and

Financial issues

Dr. Silvia BorselliDr. Costantino Raspi



Documents

Project LIFE13 ENV/IT/000461 “Environmentally friendly biomolecules from agricultural wastes as substitutes of pesticides for plant diseases control” “EVERGREEN”