ARIMNet2 Mid-Term Project Evaluation Meeting 12 October ... · PROJECT OBJECTIVES ARIMNet2 Mid-Term...

1

STOmP

Sustainable Tomato Production: plant defense

enhancement, development of new biopesticides

and optimization of environmental, water and

chemical inputs

ARIMNet2 Mid-Term Project Evaluation Meeting

12 October 2017, Montpellier, France

Coordinator Lucia ZAPPALA’

University of Catania, Italy

[email protected]

CONSORTIUM & OTHER

STAKEHOLDERS

Partner 1 (Coordinator): University of Catania, Dept. of

Agriculture, Food and Environment , ITALY (UNICT) /

Entomology, sustainable crop protection, ecotoxicology

Partner 2: University of Sfax, Faculty of Sciences, TUNISIA

(FSS) / Phytochemistry, Botany

Partner 3: University of Reggio Calabria, Dept. of

Agriculture, ITALY (UNIRC) / Entomology, sustainable

crop protection, plant physiology, horticulture

Partner 4: Italian Agricultural Research Council –

Horticulture Research Center, ITALY (CREA-ORT) /

Agronomy, protected crops technology

Partner 5: University of Nice Sophia-Antipolis, Institute of

Chemistry, FRANCE (ICN) / Phytochemistry, Analytical

Chemistry, Botany

Project start date: 01/052016 - Project end date: 30/04/2019

Overall budget: 842 k€ - ARIMNet2 grant: 592.5 k€

ARIMNet2 Mid-Term Evaluation Meeting, 12 October 2017, Montpellier

CONSORTIUM & OTHER

STAKEHOLDERS

• Partner 6: French National Institute for Agricultural

Research, Sophia-Antipolis, FRANCE (INRA) / Plant

physiology, entomology, ecotoxicology

• Partner 7: University of Napoli “Federico II”, Dept. of

Agriculture, ITALY (UNINA) / Entomology, biological

control, plant resistance

• Partner 8: Institut Agronomique et Vétérinaire Hassan II -

Complexe Horticole d’Agadir, MOROCCO (IAVHII)

/ Entomology, IPM, horticulture

• Partner 9: Hellenic Agricultural Organisation, GREECE

(DEMETER) / Insect physiology, insecticide resistance

Project start date: 01/052016 - Project end date: 30/04/2019

Overall budget: 842 k€ - ARIMNet2 grant: 592.5 k€


BACKGROUND

Tomato has a very high social and economic relevance

in Europe and the whole Mediterranean area

New tomato diseases and invasive pest is promoted by

climate change and globalized trade

Pest and disease control often relies on agrochemicals

which can

– disrupt the existing integrated pest management programs

(IPM);

– induce resistance in target pests

– Increase production costs

– Can have side effects on farmers, consumers and non-target

organisms.


PROJECT OBJECTIVES


Goal:

Developing and implementing environmental friendly methods

for the management of tomato key pests and pathogens,

limiting the use of chemicals and improving integrated control

techniques

Objective 1

Characterize insecticide resistance of two invasive tomato

pests (T. absoluta and B. tabaci) in populations coming from

various Mediterranean countries

Objective 2

Include insecticides derived from plants in tomato IPM

schemes, and develop new carriers and formulations for

increased pest control efficacy and minimized toxicity to plants

and non-target insects

PROJECT OBJECTIVES


Objective 3

Apply beneficial fungi and microbial consortia to enhance

the defense barriers of tomato plants against biotic (pests and

pathogens) stress agents

Objective 4

Optimize irrigation and fertilization in terms of nutritional

value or chemical defenses, and their consequences on

herbivorous and insect natural enemies

Objective 5

Validate a newly designed insect-proof ventilated

greenhouse in terms of tomato yields, pest and natural

enemies population dynamics

SCIENTIFIC APPROACH


- WP1 Insecticide resistance characterization

- WP2 Insecticide potential of vegetal extracts

- WP3 Plant defense enhancement against biotic (pests

and pathogens) stress agents

- WP4 Reduced irrigation and fertilization bottom-up

effects at the multi-trophic levels

- WP5 Evaluation of a newly designed greenhouse

model

- WP6 Project management, Communication and

Dissemination Plan

ACTIVITIES – M&M


WP1 Insecticide resistance characterization Task 1.1. Characterization of resistance using

classical assays

– Tuta absoluta

– Chlorantraniliprole

– Flubendiamide

– Indoxacarb

– Emamectin benzoate

– Spinosad

– Bemisia tabaci – Imidacloprid (adults)

– Spiromesifen (L2)

– Spirotetramat (L2)

– LC50 and Resistance Ratio (RR)

Tuta absoluta Bemisia tabaci

ACTIVITIES – RESULTS


WP1 Insecticide resistance characterization Task 1.1. Characterization of resistance using

classical assays

– Tuta absoluta

– Resistance to chlorantraniliprole widespread in Italy and Greece

with high RR (> 64-fold) . Also detected in Israel (RR: 22,573-fold).

– Low/moderate resistance to emamectin benzoate and to

indoxacarb was detected for the first time. Reports of poor field

performance are currently absent.

– Bemisia tabaci – Very high resistance to spiromesifen (RR > 5000) and spirotetramat

(RR > 1000) reported for the first time in strains from the MED

region.

– Key finding for future resistance management in ketoenol group.



WP1 Insecticide resistance characterization Task 1.2. Molecular analysis of resistance

– Tuta absoluta

– Mechanisms of diamide resistance were elucidated

– Four mutations in the RyR gene, are involved in the resistant

phenotypes

– The role of biochemical resistance needs to be investigated

– Bemisia tabaci – Alternative resistance mechanism could be associated with

the ketoenol resistant phenotype.

– The role of detoxification enzymes is currently investigated



WP1 Insecticide resistance characterization Dissemination

– Data will be included and available for end users on

‘Galanthus’ an online open resistance database including

data from several Mediterranean countries on several pests

Access at:

http://en.galanthos.gr

ACTIVITIES – M&M


WP2 Insecticide potential of vegetal extracts Task 2.1. Plant extract preparation and

characterization

Task 2.2. Increasing pest control efficacy

Task 2.3. Evaluating and reducing

phytotoxicity

Task 2.4. Evaluating non-target toxicity on

natural enemies survival and biological

control services

ACTIVITIES – M&M


WP2 Insecticide potential of vegetal extracts Task 2.1. Plant extract preparation and characterization

Dilution of EOs

ACTIVITIES – M&M


WP2 Insecticide potential of vegetal extracts Task 2.1. Investigated plants

Thymus zygis Juniperus

phoenicea

Artemisia

herba-alba Eucalyptus alba

Rosmarinus

officinalis Pelargonium

capitatum Mentha peprita Pistacia lentiscus

Lavandula angustifolia Pinus nigra Olea europaea

ACTIVITIES – M&M


WP2 Insecticide potential of vegetal extracts Task 2.1. Plant extract preparation and characterization

WP2 Insecticide potential of vegetal extracts Task 2.1.



ACTIVITIES – M&M


WP2 Insecticide potential of vegetal extracts Task 2.1. Formulation

ACTIVITIES – M&M



Nanoemulsion

ACTIVITIES – M&M



Nanoemulsion

Sonicator

ACTIVITIES – M&M


WP2 Insecticide potential of vegetal extracts Task 2.1. Emulsion characterization

– Appearance (aspect, color, transparency)

– Stability in time (phase separation, sedimentation, creaming)

– Stability at centrifugation (3000 rpm during 5 minutes)

– Size of the emulsion – by Dynamic Light Scattering

– by Zetasizer

Zetasizer Nano

series Malvern®



WP2 Insecticide potential of vegetal extracts Task 2.1.

ACTIVITIES – M&M


WP2 Control potential of vegetal extracts Task 2.2. Increasing pest control efficacy

– On bacteria and fungi

– On insect pests

– sap-feeders

– chewers

Task 2.3. Evaluating and reducing

phytotoxicity

– Phytotoxicity index (P𝑖)

Macrosiphum euphorbiae

Bemisia tabaci

Solanum lycopersicum

Tuta absoluta

Ephestia kuehniella

ACTIVITIES – M&M


WP2 Insecticide potential of vegetal extracts Task 2.2.; Task 2.3. Experimental setup

Thyme Lavender Rosemary Fennel Anise Clove Mugwort.

Insects direct exposure

to treated tomato leaflets

(Leaf-dip method)

Insects indirect exposure

to essential oil vapour-phase (fumigation with treated filter paper)

Citrus.

Translaminar, ingestion and

contact toxicity, oviposition

repellency



WP2 Insecticide potential of vegetal extracts direct exposure to treated tomato leaflets

− On aphids

Unbalanced entomotoxicity and phytotoxicity tradeoff

No effective EOs in a non-phytotoxic dose range

VS

0.0 0.2 0.4 0.6 0.8 1.0

0

20

40

60

80

100

0.0

0.2

0.4

0.6

0.8

1.0

% Of formulated EO

M. eu

ph

orb

iae

mo

rtality

rate

(%

) 2

4h

Ph

yto

toxic

ity in

dex



WP2 Insecticide potential of vegetal extracts indirect exposure to essential oil vapour-phase

Fumigation method sucessfully balanced the entomotoxicity and phytotoxicity trade-off

VS Effective EOs in a non-phytotoxic dose range

0 1 2 3 4

0

20

40

60

80

100

0.0

0.2

0.4

0.6

0.8

1.0

Essential oil concentration (µL.L-1air)

M. e

up

ho

rbia

em

orta

lity ra

te (

%) 2

4h

Phyto

toxic

ity ind

ex

LD50 rosemary mugwort µL(EO).L-1(air) 2,92 0,91

LD50 fennel Anise µL(EO).L-1(air) 2,43 1,79



0 20 40 60 80 100

Ctrl waterSpinosad 2x fr

NE-Anise EO 0.5%NE-Anise EO 1%NE-Anise EO 2%NE-Anise EO 5%

NE-Anise EO 10%NE-Anise EO 20%

NE-Artemisia EO 10 %NE-Artemisia EO 20 %

NE-Fennel EO 0.5%NE-Fennel EO 1%

NE-Fennel EO 2 %NE-Fennel EO 5 %

NE-Fennel EO 10 %NE-Fennel EO 20 %

NE-Rosemary EO 20%NE-Lavender EO 20%

NE-Peppermint EO 20%NE-Sage EO 5 %

NE-Sage EO 10%NE-Sage EO 20%

% mean mortality

Contact toxicity on Tuta absoluta eggs

WP2 Insecticide potential of vegetal extracts Translaminar, ingestion and contact toxicity, oviposition repellency



WP2 Insecticide potential of vegetal extracts On T. absoluta

− Citrus peel EOs as emulsions and included in

polyethylene glycol (PEG) nanoparticles (EO-

NPs) ‒ overall good insecticidal activity

‒ higher mortality through contact on eggs and larvae by EO

emulsions and through ingestion on larvae by EO-NPs

‒ the nanoformulation also significantly reduced the visible

toxic effects on the plants

ACTIVITIES – M&M


WP2 Insecticide potential of vegetal extracts Task 2.4. Evaluating non-target toxicity on natural

enemies survival and biological control services ‒ Side effects on the predator Nesidiocoris tenuis and on

bumblebees were evaluated under laboratory conditions at

IAVHII

‒ On the predator ‒ Residual contact toxicity was evaluated releasing adults in cages with

treated plants

‒ On pollinators ‒ Residual contact toxicity

‒ Ingestion via treated pollen and nectar

‒ Dermal contact: spraying on the abdominal parts of the insect

‒ Products tested ‒ Garlic extract

‒ Thymus vulgaris essential oil

‒ Abamectin 5%,

‒ Azadirachtin 0.1%,



WP2 Insecticide potential of vegetal

extracts Task 2.4.

‒ On N. tenuis ‒ the highest mortality was recorded for garlic

(94.0%) and Abamectin (92.8%); Azadirachtin had moderate toxicity (64.8%), followed by thymus (38.8%)

‒ On bumblebees ‒ direct dermal contact caused high mortality

rate for all the treatments ‒ in the ingestion toxicity trials through pollen

and nectar the highest mortality was given by Abamectin (40.1%) followed by Azadirachtin and thymus

‒ Garlic extract and Azadirachtin have a slightly lower acute toxicity than Thymus extract and Abamectin on bumblebees

ACTIVITIES


WP3 Plant defense enhancement against biotic (pests

and pathogens) stress agents Task 3.1. Use of beneficial fungi (PGPF, Plant Growth

Promoting Fungi) ‒ The root symbiont Trichoderma harzianum T22 enhances the

production of VOCs that are responsible of superior attraction

of parasitoids towards aphid infested tomato plant

ACTIVITIES


WP3 Plant defense enhancement against biotic (pests

and pathogens) stress agents Task 3.1. Use of beneficial fungi

‒ DEGs involved in VOCs related pathways (cis-3-exenol, β-

caryophyllene, methyl salicylate) and identified in tomato T22,

tomato aphids, tomato T22 + aphids

GENE fold change

SMT22+Aph

Annotation

Solyc07g052150.2.1 7,383503225 Sesquiterpene synthase

Solyc07g052140.2.1 6,18848554 (-)-germacrene D synthase

Solyc01g005230.2.1 2,009382507 S-adenosyl-L-methionine salicylic acid carboxyl methyltransferase

Solyc01g014330.2.1 1,668374865 S-adenosyl-L-methionine carboxyl methyltransferase family protein

Solyc08g080670.1.1 2,603222167 Osmotin-like protein

Solyc01g087840.2.1 2,576193326 Subtilisin-like protease

Solyc01g106620.2.1 1,565662871 Pathogenesis-related protein 1a

Their up-regulation explains A. ervi flight behaviour

ACTIVITIES


WP3 Plant defense enhancement against

biotic (pests and pathogens) stress agents Task 3.2. Use of beneficial bacteria (PGPR,

Plant Growth Promoting Rhizobacteria)

Verticilium dahliae: an important vascular

fungal pathogen (a biotic stress) ‒ In vitro study

‒ The antifungal activity of Bacillus amyloliquefaciens strain

C2 has been characterized.

‒ In vivo study ‒ The efficacy of the strain C2 of B. amylolisquefaciens in

protecting tomato against V. dahliae, and promoting its

growth has been evidenced

ACTIVITIES


WP4 Irrigation and fertilization bottom-up effects at the

multi-trophic levels Task 4.1. Effects of reduced irrigation and fertilization

on plant physiology - primary and secondary

metabolism

Task 4.2. Effects of reduced irrigation and fertilization

inputs on pests (T. absoluta and whiteflies) and their

natural enemies (predators and parasitoids)

Objective: Can we detect bottom-up effects of moderate limitation of nitrogen and/or water

supply on the first, second, and third trophic level?

ACTIVITIES


WP4 Irrigation and fertilization bottom-up effects at

the multi-trophic levels

Optimization of seed

germination

Optimization of the

hydroponic system and

nutrient solution

ACTIVITIES




Optimization of drought and N stress

ù

ACTIVITIES




Optimization of electrophysiological measurementnt

Experimental design

Nutrient solution:

10 vs 2.5 mM nitrogen

-H2O = 50% of +H2O

Location: climate controlled

greenhouse

Macrosiphum

euphorbiae

Tuta absoluta Spodoptera

littoralis

Bemisia

tabaci

Aphelinus

abdominalis

Necremnus tutae

• Nutritional value:

Carbon & Nitrogen

• Direct defense:

Phenolics & Alkaloids (LC-MS)

• Indirect defense:

Herbivore-induced volatiles

(SPME, GC-MS)

Infestation:

• B. tabaci: 120 adults, 10 days

• M. euphorbiae: 30, mixed stages, 7 days

• T. absoluta: 6 larvae, L2-L3, 2 days

• S. littoralis: 1 larvae, L1-L2, 2 days

Parasitism:

• A. abdominalis: 2 couples, 24h

• N. tutae: 2 couples, 48h

Sap-feeders: populationsize

Leaf-chewers: larval weight increase, consumed leaf area

Successful parasitism,

emergence rate




the multi-trophic levels Diluted bottom-up effects

Nutritional value Defense

compounds

Nutritional value

(Growth rate)

(Successful parasitization)

(Parasitoid emergence rate)

Population size

Nutritional value

Consumed leaf area

(Nutritional value)(Growth rate)

Clear effect of irrigation and

fertilization on 1st trophic

level.

Some clear effects 2nd level.

Some weak and somepotential (indirect) effects on

3rd level.

Herbivore-induced

plant volatiles?

(Biocontrol success rate)

(Parasitoid emergence rate)

Herbivore-induced

plant volatiles?

Two papers are currently in progress

ACTIVITIES – M&M


WP5 Evaluation of a newly designed greenhouse

model Task 5.1 Yields evaluation

Task 5.2 Insect pest population dynamics

Experiment carried out in 2016 and 2017

in a natural ventilation greenhouse divided into two

bodies (14.4 x 20.0 m each) One closed frontally, sideways and at the roof with an

aphid net having a porosity of 46% (Net Greenhouse - NG),

with a double door.

The second one closed at the front, side and top with a

95% bumblebee net (No Net Greenhouse - NNG).



WP5 Evaluation of a newly designed greenhouse

model Production, LAI and T. absoluta attack were

significantly greater in NNG than in NG;

The differences between treatments in NG were not

significant and therefore only the treated net

determined that result. The net as a bug insect limitation tool was only partially

successful.

Net performance greatly improves when locally treated with

a suitable insecticide.

PUBLICATIONS


DISSEMINATION ACTIVITIES


Workshops “Chemical Ecology Inputs in Biological control” (ChEcKInBio) on 13

July 2017 (organized by ISA and ICN)

Website https://www.researchgate.net/project/Sustainable-Tomato-

Production-plant-defense-enhancement-development-of-new-

biopesticides-and-optimization-of-environmental-water-and-

chemical-inputs-STomP

Congress oral or poster presentations on the project activities Italian Congress of Entomology, Padova (I), June 2016;

Intl Congress of Entomology, Orlando (FL), Sept 2016;

Intl congress of the French Ecological Society, Marseille, (F), 24-29

Oct 2016;

Workshop on “Essential oils: phytochemistry and new

applications”, Catania (I) 30 June 2017;

International symposium on insect-plant interactions (SIP), Tours

(F) 2-6 July 2017.

TRAINING & MOBILITY


Natural products and Biocontrol congress,

Perpignan, France 20-24 Sept 2016;

SFE international congress, Marseille, France, 24-

29 Oct 2016;

Meeting “GDR Mediatech”, Marseille, France, 24-

29 Oct 2016;

Meeting “GDR Mediatech”, Montpellier, France, 2-

4 Nov 2017;

ERINI Grasse, Some days in May, August, Sept

and Oct 2016 of a post-doc at INRA to train on

Chemical analyses and Metabolomics

NEXT STEPS / PERSPECTIVES


Next steps

Ganttchart SCHEDULE

Year 1 Year 2 Year 3

WP / Task 1 2 3 4 5 6 7 8 9 10 11 12

WP1

Leader: DEMETER

Contributing partners: UNICT

x x x x x x x x x x x x

WP1 - T1.1

Leader: DEMETER x x x x x x x

WP1 - T1.2

Leader: DEMETER x x x x x x x x x x

WP2

Leader: ICN

Contributing partners: FSS,

UNICT, UNIRC, IAVHII, ISA


WP2 - T2.1

Leader: FSS x x x x x x x x

WP2 - T2.2

Leader: ISA x x x x x x x x x

WP2 - T2.3

Leader: ICN x x x x x x x x

WP2 - T2.4

Leader: UNICT x x x x x x x x x

SCHEDULE

Year 1 Year 2 Year 3

WP / Task 1 2 3 4 5 6 7 8 9 10 11 12

WP3 Leader: UNINA Contributing partners: ISA, FSS, IAVHII


WP2 – T3.1 Leader: UNINA


WP2 – T3.2 Leader: FSS


WP4 Leader: UNIRC, ISA Contributing partners: ICN, UNICT, IAVHII


WP4 – T4.1 Leader: UNIRC

x x x x x x x x x

WP4 – T4.2 Leader: ISA

x x x x x x x x x x x

WP5 Leader: CREA-ORT Contributing partners: UNINA


WP6 Leader: UNICT Contributing partners: ALL




Next steps

WP1: further data on resistance phenotype;

progress in mechanisms

WP2: Biopesticide formulation and activity testing

still in progress; large scale production of

extracted EOs; non-target toxicity trials

WP3: characterization of Trichoderma strain that

enhances direct pest control; selection of

beneficial Bacillus strains; protocols for field

applications of PGPF



Next steps

WP4: Physiological and biochemical traits of water

and nutrient stressed tomato plants; effects of

reduced irrigation and fertilization of Tuta absoluta

and its natural enemies

WP5: production level evaluation in the newly

designed greenhouse

WP6: a progress meeting will be organized in

Naples in July 2018 (European Congress of

Entomology, 2-6 July 2018); a final workshop will

be organized, most likely in Crete




Next steps

WP4: Physiological and biochemical traits of water and

nutrient stressed tomato plants; effects of reduced

irrigation and fertilization of Tuta absoluta and its

natural enemies

WP5: production level evaluation in the newly designed

greenhouse

WP6: a progress meeting will be organized in Naples in

July 2018 (European Congress of Entomology, 2-6 July

2018); a final workshop will be organized, most likely in

Crete

Perspectives

Networking among partners for further project proposals

PROJECT FUNDERS


Thank you for your attention!

Photo by G. Siscaro Photo by G. Siscaro Photo by G. Siscaro Photo by G. Siscaro

Photo by L. Santonicola Photo by G. Siscaro

Photo by G. Siscaro Photo by G. Siscaro Photo by A. Biondi Photo by A. Biondi

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