biopesticides : quand les biotechs se mettent au service d’une agriculture durable par quentin...

Mercredi 10 décembre

Biopesticides : quand les biotechs se mettent au service d’une agriculture durable Quentin ZUNE, ULg Gembloux Agro-Bio Tech

Marc ONGENA, ULg Gembloux Agro-Bio Tech

Avec le soutien de :

Université de Liège – Gembloux Agro-Bio Tech – Unité de Bioindustries Passage des Déportés, 2 - 5030 Gembloux – Belgique. Tél+32(0)81 62 2305 (Fax 6142) - www.fsagx.ac.be

Biopesticides: quand les biotechs se mettent au service d’une agriculture durable

Quentin Zune & Marc Ongena

Walloon Center for Industrial Biology, Gembloux Agro-Bio Tech/University of Liège

LIEGE CREATIVE, 10th December 2014

Need for phytosanitary products

2

35 % of losses due to pests 70 % if no phytosanitary treatments

Plant diseases caused by: insects, fungi, virus, bacteria, nematodes

Problems of chemicals

Ecotoxicity of the product or residues Pathogen adaptation Lack of specificity Health problems for the users Strong interest from consumers for « safe » food and sustainable agricultural practices Registration/homologation constraints

- «Plan Écophyto 2018» in France, to reduce chemicals by 50%

- «Programme de Réduction des Pesticides et Biocides» to reduce by 25%

- European dir. 128/2009/CE, sustainable use of phytosanitary products

Problems of chemicals

Need for eco-friendly and efficient alternatives

Implementation of Integrated Pest Management (IPM): Disease risk monitoring: Comprehensive information on the life cycles of pests and their interaction with the environment

+ Prevention: soil fumigation/strilization, rotating between different crops, use of pest-resistant varieties, and planting pest-free rootstock

+ Pest control: spraying of pesticides, BIOLOGICAL CONTROL

Adapting agricultural practices to favor antagonists of plant pathogens, reduction of disease vector,… Use of disease resistant genetical-modified crops Introducing antagonists of plant pathogens into the environment or onto the plant

5

Biological control/biopesticides as part of IPM

BIOPESTICIDES

Broad def: any living organism or derived products used on plants for its protective effect against diseases

Biopesticides: nature

Microbes

GMOs

Nematodes, predators

Biochemicals

Insects

Bacteria, fungi, yeast, viruses, protozoa

Insect sex pheromones, Plant extracts, Growth regulators Plant resistance activators

7

Microbial biopesticides

Plant-beneficial (rhizo)bacteria Streptomyces, Arthrobacter, Azotobacter, Burkholderia, Pantoea, Serratia,Collimonas

8

Biopesticides: applications

Insect pests

Nematodes

Soil-born microbial pathogens

Post-harvest diseases

Leaf, flower, fruit diseases

Biopesticides: applications

Organic farming

Conventional agriculture

Industrial greenhouses

Forestry

Biopesticides: market

Global market valued at $1.3 billion in 2011 Expected to reach $4,3 billion by 2019 (rate of 16.0% from 2014 to 2019) Europe expected to be the fastest

growing market (stringent regulation, demand from organic products)

North America dominated the global bio pesticides market (around 40%)

How do they work to protect plants?

Microbial biopesticides in action

Microbial biopesticides: multiple benefits

Growth promotion, yield increase

via: - help in nutrient acquisition (N, P, Fe) - phytohormone production (cytokinin, gibberelin, IAA, ethylene) - help under abiotic stress

Protection against phytopathogens, disease resistance

via: - competition for space and ressources - antibiosis (antibiotics, lytic enzymes) - host immunization (systemic)

Beneficial)

rhizobacteria)

Formation of microcolonies/biofilms

Root$coloniza+on$and$compe++on$for$niche$and$nutriments$

On)Agar,)4)dpi)

Pathogen)

Anti-fungal, anti-bacterial, anti-viral activities

Botrytis cinerea (grey mould disease)

Pythium ultimum (seedling disease)

Direct$antagonism$of$phytopathogens$$

Secretion of LMW antimibials, Hydrolytic enzymes

Pathogen)

restric9on))

Host plant immunization

Root treatment with: -over producing mutants -enriched LPs extracts -Purified LPs

A"variety"of"biocontrol0related"ac2vi2es"Plant$systemic$resistance$elicita+on$9$ISR$

in Arabidopsis In Solanaceae

Treated Control Treated Control

Elicitor production

16

Must persist in the niche from inoculation to infection > must be perfectly adapted to the environment where it is introduced

Must resist other organisms, be efficient at low doses

> strong producer of active substances Must not induce resistance in the target pathogen

> act via multiple mechanisms

Biopesticides: requirements

View from the industry: The product must work as good as a comparable chemical pesticide

> use/select/optimize the most active agents The product shouldn‘t be more expensive than a chemical pesticide

> cost-effective production at industrial scale The product should be applicable as easily as a chemical pesticide

> optimized formulation

From the strain to a marketable product

Main steps for microbial biopesticide development

18

Main steps for biopesticide development: step 1, strain selection

Highthrouput screening for main biocontrol activities

Suppressive soils Less infected plant tissues

Where to look for?

How to select?

Colonization Antagonism Host immunization

Taxonomy

GRAS

Cultivable

Spore formation

Structure identification

Biosynthesis, genes

- Low molecular weight metabolites:

Antimicrobials, host immunity elicitors, siderophores

- Hydrolytic enzymes

Mode of action, identification of compounds involved in biocontrol activity

Step 2: characterization of biological activity

Physiological factors Environmental parameters

Regulation of genes/products and expression in vivo

Study of factors modulating expression of biocontrol determinants

Step 3: expression of bioactive compounds in vivo

-  Developement as root-associated microcolonies/biofilms -  Host plant species, cultivar, developmental stage -  Nutritional basis imposed by the plant, growth rate -  Auxiliary microflora -  soil type, pH, T, pO2

Most data on biocontrol metabolites come from in vitro assays

However, in vivo context very different:

2. Fermentation bioprocess

1. Strain selection

3. Downstream process operations and packaging

•  Economic

•  Efficiency

•  Ecofriendly

•  Quality product

•  Practical application

powder

Vegetative cells

Nutrient excess

Endospore formation

Nutrient limitation Nutrient depletion

spores

Fermentation time

Cell division ! Sporulation

Step 4: Production of the biopesticide

1 – 100 mL

1-100 L

Lab-scale Optimization of medium composition, pH and T° for strain growth

! low transfer capacity, no regulation

Pilot-scale Optimization of transfer operations (homogeneity, cells suspension, oxygen transfer rate)

pH, pO2,T°, substrate regulation

Complex, Expensive and Time consuming

Industrial scale Scale-up conserves critical parameters for fermentation (density power, oxygen transfer rate)

! Heterogeneities!!!

Step 4: Development of the fermentation bioprocess 1 – 100 m³

1. Concentration and purification of the microbial cells

Centrifugation Precipitation

2. Drying of the cream to get a powder

3. Packaging

Atomization (high T°)

Lyophilization (low pressure and T°)

Preserves product quality

Step 4: Downstream process operations and packaging

Biopesticides: market

Global market valued at $1.3 billion in 2011 Expected to reach $4,3 billion by 2019 (rate of 16.0% from 2014 to 2019) Europe expected to be the fastest

growing market (stringent regulation, demand from organic products)

North America dominated the global bio pesticides market (around 40%)

But growth rate of the market lower than expected, could be better!!

Limitations of rhizobacteria as phytosanitary products

Efficacy may be limited or inconsistent !

Need for improving our knowledge

Persistence of populations in the niche and colonization of the host tissue (rhizosphere)

Identification of active ingredients (secondary metabolites) Establishing their functions in multitrophic interactions (molecular cross-talk, antibiosis, stimulation of host immunity) Secretion of biocontrol

metabolites in planta

Exploiting Omics and Bioprocesses

Learning more about microbial biopesticides

Whole genome sequencing Annotation of draft sequence (RAST)

Genome mining -Based on known nucleotide sequences in close relatives (genome alignment software Mauve, NRPSpredictor, BLASTx) -Based on predicted amino acid sequences (Antismash, Norine)

Comparative Genomics: PGPR antibiotic potential

8,5% of CDS devoted to synthesis of NRPS/PKS antibiotics

FZB42, YAU Y2

+ orphan NRPS gene clusters

polyketides

lipopeptides

dipeptides

siderophore

lantibiotics

Linear peptides

Comparative Genomics: PGPR antibiotic potential

LC-ESI-MS analysis of Bacillus antibiotics

Metabolomics: PGPR antibiotic potential

Surfactin Fengycin Iturin

Antiviral +++ - -

Antibacterial ++ (+) (+)

Antifungal (+) +++ +++

Anti-yeast + - ++

Anti-mycoplasma ++ - -

Anti-insect +++ - -

Antiprotozoa +++ - -

ISR in plants +++ +/- -

Different activities of lipopeptides on different targets

rich in PG, CL, low content of PC. No sterols

rich in PE, cholesterol and sphingolipids

high levels of PG, PC, and PE. Cholesterol

PC and PE abundant. Ergosterol

PC and PE abundant, PS, shingolipids. Sitosterol, stigmaterol

=> Selectivity of CLPs to certain membrane compositions

Biophysics: molecular mechanisms of antibiotic activity

Model membranes

Biophysics: molecular mechanisms of antibiotic activity

Experimental, in silico

PM#fraction#only12136%

PM#>#DRMs11233%

DRMs#>#PM8225%

DRMs#fraction#only206%

P-type H+-ATPase Aquaporins Calcium dependent protein kinase Small GTP-binding proteins Leucine rich repeat (LRR)-receptor kinase NADPH oxydase NtrbohD Phospholipase D

Analysis of proteome (2D UPLC-MS/MS, NCBI database) in tobacco plasma membrane raft-like microdomains upon elicitation by surfactin

Confirmation of raft-specific proteome signature

Proteomics: molecular mechanisms of plant immunization

High-affinity receptors for non-self detection

Specific aspects of plant immunization by surfactin

Surfactin preferably interacts with the lipid phase of the plasma membrane, inducing transient disturbance, proteome rearrangement and triggering of defense-related signalling cascade

Micelle&of&surfactin

Cytosol

Cholesterol

Membrane&protein

B

Protein with lipidaddressing motif

x

Production of biocontrol metabolites in planta

Are bioactive metabolites produced by bacteria in sufficient amounts, at the right place, at the right time?

Various approaches are available:

Extraction from soil substrate (difficult!)

Comparative analysis of selected mutants (may be tricky to generate!)

Need for accurate spatio-temporal monitoring of antibiotic production in situ

Pump)

Drying)under)

vaccuum)during)

1h30B2h)

Automated)

9AA)matrix)

deposi9on)

MALDIBTOF/TOF)analysis)in)

nega9ve)ion)mode)

Sample):))

colonized)root)on)an)

ITO)coated)glass)slide)

covered)by)agar)

Imaging Mass Spectrometry for antibiotic signature in planta

control$

inoculated$

Imaging Mass Spectrometry for antibiotic signature in planta

Surfactins much more abundant but iturins and fengycins detected in low amounts

MALDI-TOF MS imaging of CLPS Surfactins

Iturins

Fengycins

Imaging Mass Spectrometry for antibiotic signature in planta

Transcriptomics: antibiotic gene expression in planta

qRTPCR RNAseq

Reporter systems (yellow fluorescent protein, YFP)

In vitro experiment

•  Petri dish, microtiter plates and flask cultures

(+) Easiness of implementation, cheap

(+) Fast and high throughput screening

But…

(-) Not representative of real conditions at several levels

Bioprocess technologies to mimic natural systems

•  Omics technology characterize cell physiology at the cell level (in vivo)

! bioprocess study mecanisms at the population level

Planktonic)state)=)free)cells)

suspended)in)the)liquid)medium)Biofilm)=)bacterial)community)growing)

on)solid)surface)

≠Physiology$

Cells

EPS Matrix

• )Gene)expression)

• )Metabolism)(I))

• )Growth)rate)

• )Secre9on)profiles)

Bioprocess technologies to mimic natural systems

Batch)condi9on)=)cell)growth)stops)

when)nutrients)are)depleted)

No)control)of)the)growth)rate)

Chemostat)condi9on)=)con9nuous)

feeding)of)root)exsudate))

Effect)of)growth)rate)on)biofilm)

metabolism))

Kine+c$

≠Substrate)

Batch)(flask))

Root)system)

Need$:)experimental)device)close)to)natural)condi9ons)(biofilm),)

con9nuous)feeding)rate)and)taking)into)account)cell)history)

Biomass)

Bioprocess technologies to mimic natural systems

Flow cell to study biofilm growth

•  Flow rate modulation ! growth rate control of the biofilm

•  Steady-state of biofilm (Chemostat)

•  Coupled with microscopy

•  Pulse adds and on line sampling

•  Design of a bioreactor with a support to immobilize biomass on a biofilm

•  pH, T°, pO2 regulation

Fresh medium

Waste

Fresh medium

Waste

Biofilm colonization of the support

Biofilm reactor to study biofilm growth

Bioprocess technologies to mimic natural systems

! cellular steady state increases relative surfactin gene expression

Steady-state

High expression

Modulation of pump flow rate

! Low growth rate increases surfactin production

Note : time of experiment ! 72 hours

Root system

In vivo

In vitro

Bioprocess technologies to mimic natural systems

Biofilm : great heterogeneity, complex behavior!

Flow cell technology & biofilm reactor ! response at the biofilm level

•  Isogenic population ! phenotypic diversity

secretor non-secretor dormant cell (spore) motile cell EPS producer

Combined with :

•  Confocal microspoy

•  Fluorescent reporter system

•  Flow cytometry

Structure & composition

Response mechanism at the

biofilm scale

Nutritional context of in planta conditions

Confocal microscopy

3D visualization

! biofilm structure (roughness, smoothness, mushroom-like structure)

Specific staining

! characterize and locate phenotypes (secretor, spores, ect.)

Biofilm : great heterogeneity, complex behavior!

Transcriptional reporter system Expression of a fluorescent protein when a specific gene is activated!

! easy detection and quantification

Screening of genes involved in regulation mechanisms

On-line measurement of gene activation under specific conditions

On-line localization of cells expressing the gene

Pstress

GFP coding sequence

Environmental cue

Signal transduction

GFP synthesis

Sectional view

Biofilm : great heterogeneity, complex behavior!

Laser 520nm

Side scatter canal (FSC)

Side scatter (SSC)

Green fluorescence (FL1)

Yellow fluorescence (FL2)

Red fluorescence(FL3) Cells sample

Analysis of 30000 cells / sample (time 30-60s)

Distinction of subpopulations

Biofilm : great heterogeneity, complex behavior!

•  Useful tool to characterize and select phenotypes inside a biofilm

Product( Bioagent/(mode(of(action(

Crop( Company(

Avogreen®) B.#subtilis/#antibiosis) Avocado) Ocean)Agriculture))

Bacillus)SPP®) Bacillus)spp./)antibiosis# Several)crops) Bio)Insumos)Nativa)Ltda,)Chile.))

Ballad®) B.#pumilus#/)antibiosis,)compettition,)growth)promotion)and)resistance)induced#

Cereals,)oil)plant,)corn,)sugar)beet)) AgraQuest)Inc,)USA.))

Bio)safe®) B.#subtilis/#antibiosis) Soybean,)bean,)cotton) Lab.)Biocontrole)Farroupilha,)Brazil)

Biosubtilin))

B.#subtilis/#antibiosis,)competition# Cotton,)cereals,)ornamental)and)vegetable)crops)

Biotech)International)Ltd.))

Botrybell)

)B.velezensis# tomato,)lettuce,)pepper,)grape,)strawberry,)

and)vegetable#)Agricaldes,)Spain))

Cease®) B.subtilis# Several(crops# BioWorks)Inc.,)USA)

Companion®)) B.#subtilis/)antibiosis,)growth)promotion,)resistance)induction,)competition#

Cotton,)bean,)pea,)soybean,)peanut,)corn,)and)others)

Growth)Products)Ltd.,)USA))

EcoGuard)TM)Biofungicide)

B.#licheniformis#/antibiosis)and)enzymes#

golf)courses,)sports)turf,)lawns,)turf)farms)and)arboretums)

Novozymes)A/S,)Denmark.)Novozymes)Biologicals,)USA)

Ecoshot) B.#subtilis# Grape,)citrus,)vegetable,)legumes,)and)others)

Kumiai)Chemical)Industry)Co,)Japan)

FZB24®WG,)li)and)TB)

B.#subtilis# Several)crops) ABiTEP)GmbH,)Germany.))

HiStick'N/T®'/'Subtilex®'/'Pro4Mix®'

B.#subtilis# Soybean,'ornamental'plants'and'other'crops'

4Becker'Underwood,'USA''Premier'Horticulture'Inc.,'Canada''

Kodiak' B.#subtilis/'antibiosis,'growth'promotion,'resistance'induction,'competition#

Cotton' Gustafson'Inc,'USA'

Rhapsody®' B.#subtilis'# Turf,'forest,'ornamen4tals' AgraQuest'Inc,'USA''

Rhizo'Plus®' B.#subtilis#FZB24# Gardening'(Several'crops)' ABiTEP'GmbH,'Germany'

RhizoVital®42'li'' B.#amylolique0faciens# Potato,'corn,'strawberry,'tomatoes,'cucumber,'ornamentals'

ABiTEP'GmbH,'Germany'

Serenade®' B.#subtilis/'antibiosis' Grapes,'Apple,'pears,'bananas,'cherries,'Walnuts,'Peanuts,'Hops,'Leafy'vegetables,''tomatoes,'peppers,'cucurbits,'mango,'bean,'onion,'garlic,'potatoes,'Broccoli,'Carrots.'

AgraQuest'Inc,'USA'

Sonata®' B.#pumilus#' Tomatoes,'peppers,''Potato,'grapes,'strawberry,'cucurbit,'apple,'pear'

AgraQuest'Inc,'USA'

Sublic®' Bacillus#spp.# Several'crops' ELEP'Biotechnologies4,'Itália.''

Yield'Shield®' B.#pumilus'# Soybean' Bayer'CropScience,'USA''

Some success stories for microbial biopesticides

B. subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus...

However still far from optimal exploitation…

Biotechs may be used in situ but also ex vivo to better understand how microbes function to protect plants

Should help to better appreciate:

why some microbial strains are more efficient than others why a given strain is efficient on some crops and not on others why a given strain is efficient in certain soil conditions and not in others

Should benefit industrial producers (production, formulation) and farmers (application)

There is a future, big players are getting involved…

Global theme: “From Omics to the Field”

Sponsoring still welcomed!

Co-organizers: Monica Höfte and Marc Ongena

!!

E-mail for contact: pgpr2015.gembloux@ulg.ac.be let us know your interest in attending the meeting! Website for info: http://www.events.gembloux.ulg.ac.be/pgpr2015/

The rhizosphere zoo:

The rhizosphere microbiome:

Mendes et al, 2013

Metagenomics: for antibiotic effect on auxiliary microflora

heatmap indicates differences in the relative abundances of OTUs

Sampling and DNA extraction

DGGE analysis of 16S rRNA gene fragments amplified from TotalCommunity-DNA

Pyrosequencing and statistical analysis

Schreiter et al, 2014

Metagenomics: for antibiotic effect on auxiliary microflora

Co9culture$characteriza+on$!$microbial$consor+um$management))•  2 strains

•  Complementary behaviors (cross-feeding)

•  Promote biobased compounds synthesis

•  Mutual protection

Combined confocal microscopy and flow cytometry to manage microbial consortia

0$

1$

2$

3$

4$

5$

6$

7$

8$

0$

5$

10$

15$

20$

25$

0$ 5$ 10$ 15$ 20$ 25$ 30$

β9gal$U/10

5$cells)$Ce

lls$x$106$/g$of$roo

t$

Days$post9inocula+on$

coloniza+on$BGS3$rela+ve$srfA$gene$expression$

Expression of srfA genes in the rhizosphere using lacZ reporter system

Higher)surfac9n)gene)expression)when)popula9on)established)

Approx. 1.8 µM in the plant growth medium

Transcriptomics: Bacillus antibiotic production in planta

PGPR: A myriad of chemically divers antibiotics

Lipopeptides formed by Non Ribosomal Synthetases (NRPS)

Surfac9n)assembly)line)

Strieker)et)al.)Cur)Opin)Struct)Biol)2010)

NRPS synthesis serves structural diversity of LPs

Genus)level:)families/groups) Strain)level:)variants/homologues)

Variants:$Pep+de$sequence$

Homologues$:$FA$lenght,$isomery$

Pseudomonas Bacillus

Most)are)cyclic,)some)are)linear)

CLP FaXy$acid Pep+de$sequence Iturin'family'(Bacillus)

Iturin)A C14BC17 Asn Tyr Asn Gln Pro Asn Ser

iturin)C C14BC17 Asp Tyr Asn Gln Pro Asn Ser

mycosub9lin C14BC17 Asn Tyr Asn Gln Pro Ser Asn

Bacillomycin)D C14BC16 Asn Tyr Asn Pro Glu Ser Thr

Bacillomycin)F C14BC17 Asn Tyr Asn Gln Pro Asn Thr

Bacillomycin)L C14BC16 Asp Tyr Asn Ser Glu Ser Thr

Bacillomycin)Lc C14BC16 Asn Tyr Asn Ser Glu Ser Thr

Subtulene)A C15* Asn Tyr Asn Gln Pro Asn Ser

VariantsBhomologues)

Variants:$Pep+de$sequence$

Homologues$:$FA$lenght,$isomery$

O H N H

N H N H

O H

N H

N H 2

O N H

N N H

N H O

N H 2

O O

N H 2

O

O

O

O

O

O O N H

2

O

NRPS synthesis serves structural diversity of CLPs

60

Essais au champs ou en serres industrielles

Activité protectrice des lipopeptides sur diverses cultures d’intérêt

I. Lipopeptides as weapons to survive/compete: primary role (?)

- +

- +

By$inhibi+ng$growth$of$microbial$compe+tors$

An3viral''

An3protozoal'

An3bacterial''

An3oomycete' An3fungal''

Membrane)permeabiliza9on,)cell)lysis)

Biophysic)on)model)membranes)

3DBconforma9onal)studies)

Mechanisms)of)pore)forma9on)and)target)specificity)

An3;nematode'

By$repelling$predators$

Serraween)ac9ng)as)avoidance)cues)

Pradel)et)al.)PNAS)2007)

Physico-chemical interactions between co-produced LPs

In)Bacillus)S499) In)Pseudomonas)CMR12a)

Sessilin)and)orfamide)physically)interact)

and)form)a)white)line))

a

b

c

a

b

c

I F

S

Fengycin)and)surfac9n)coBprecipitate)

Different activities for structural variants on a specific target

Bacterial colony Fungal mycelium

2

1

Fusaricidins)from)P."polymyxa"

MS)Imaging)

II. Role of LPs in motility, dispersal

Surfac9n)for)B."amyloliquefaciens" Orfamide)for)Pseudomonas)CMR12a)

Effect)of)interac9on)on)swarming)poten9al)

D’aes)et)al.)Environ)Microbiol)2014)

III. Role of LPs in biofilm formation

Surfac9n)for)B."amyloliquefaciens" Sessilin)for)Pseudomonas)CMR12a)

Surfac9n)as)signal)driving)cell)

differen9a9on)and)biofilm)forma9on))

Romero,)Res)Microbiol.)2013)

IV. Role of LPs in root colonization

Several)strains)tested)In)different)growing)systems:)

)efficient)surfac9n)producers)are)beher)colonizers)

On)Agar,)4)dpi)

In)perlite,)14)dpi))

In)peat)soil,)5)weeks)pi)

V. Role of LPs in plant immunization

C$$$$$$$$$$$$$$ $$$LP$

● Disease)reduc9on)on)leaves)upon)root)treatment)with)purified)LPs)))

)

● Disease)reduc9on)on)leaves)upon)root)treatment)with)mutants))

) ) ) ) ))

● No)migra9on)of)inducing)agent)from)roots)to)the)infected)leaves)

)

● Efficient)produc9on)of)the)inducing)agent)in)the)rhizosphere)

)

● Protec9on)associated)with)defence)responses)at)the)molecular)level))

Fast production upon contact with washed roots of tomato and AT:

Stimulation of surfactin upon root perception by Bacillus

S C15

S C15

i, control cells ii, cells in contact with root

UPLC-ESI-MS i ii

3h30

4h30

5h30

6h30

In the presence of purified cell wall polymers:

2h30

$Shared$benefits$for$both$the$bacterium$and$the$plant:$$$faster$establishement$in$the$rhizosphere $immuniza+on$without$

$ $ $ $ $ $growth$cost $ $$

VI. Role of LPs in pathogen virulence

From)Pseudomonas"syringae" From)Pseudomonas"cichorii"

Syringomycin/syringopep9n)as)necrosis)inducers)

)Cichopep9n)contributes)to)virulence)

))

Pauwelijn et al. MPMI 2013

Scholz-Schroeder et al. MPMI 2001

Colonization, biofilm

Antiviral

Antiprotozoal

Antibacterial

Antioomycete Antifungal

Chelation, solubilization

Motility Virulence, Immunization

In conclusion

Much more than just molecular pneumatic drills!

CLPs thus also favor the ecological fitness/rhizosphere competence of the producing strains and act as signals or impact various developemental processes in cohabiting organisms without causing any detrimental leakage in membrane, lower [ ] involved.

High cytotoxicity favoring direct antagonism of pathogens > CLPs considered as molecular pneumatic drills, high [ ] required.

In conclusion

Are antibiotics produced in sufficient amounts, at the right place, at the right time? Need for more accurate spatiotemporal monitoring of antibiotic production in situ

Other functions to be discovered at sub-inhibitory concentrations?

The Bacillus CLP pattern may be influenced by:

- nutritional basis imposed by the plant - host plant species - biofilm formation -  growth rate (not shown) -  temperature - pH (not shown) - oxygen availability (not shown)

Summary

May explain why some Bacillus strains are efficient but not others May explain why a given strain is efficient on some crops and not on others May explain why a given strain is efficient in certain soil conditions and not in others

73

Conclusions The demand for organic produce is growing in the main markets (EU, USA, asia) Organic agriculture is in line with mainstream trends (low/no residue, low input, eco-system services) The production is often limited to the most suitable pedoclimatic conditions (least disease and pest pressure) If biocontrol agents can adequately control key pests/diseases, production areas will grow rapidly und thus also extend the demand for existing biocontrol PPPs

Thanks

Supported by:

For your attention…

Phytopathology Lab Ghent University Jolien D’aes Ellen Pauwelijn

)

Gembloux Agro-Bio Tech Laurent Franzil Hélène Cawoy Guillaume Henry Emmanuel Jourdan

Lab Mass Spec at ULg Delphine Debois Edwin De Pauw