Los días 20 y 21 de mayo de 2014, la Fundación Ramón Areces organizó el Simposio Internacional 'Microorganismos beneficiosos para la agricultura y la protección de la biosfera' dentro de su programa de Ciencias de la Vida y de la Materia.

1. Microbial biopesticides
Plant Pathology
Instituto de Tecnología Agroalimentaria
Universidad de Girona (Spain)
http://www.udg.edu/cidsav
Emilio Montesinos Seguí

2. What is a biopesticide?

3. BIOPESTICIDE:A pesticide of biological origin
and not synthetic (may be semi-synthetic)
PESTICIDE:Active ingredient that kILLS pests
(direct action)
PEST: An animal, plant or microbial organism
having a deletereous effect on plants
SOME DEFINITIONS

4. Microbial (e.g. Bacillus amyloliquefaciens QST713, CpGV virus)
Non-microbial (natural extracts or compounds)
 Botanical (e.g. laminarin, clove oil)
 Animal&Food Industry byproducts (e.g. blood meal, chitosan)
 Microbial fermentation (e.g. spinosad, strobilurins)
 Biotechnological (e.g. harpins)
TYPES OF BIOPESTICIDES

5. Why we need
biopesticides?

6. Actual losses of agricultural productivity
(x 106 Tn per year)
Production
5311
Diseases
1294
Insect pests
“Weeds”
and other
863
1122
c.a. 35 % losses
Oerke E.C. 2006. J.Agr.Sci.

7. VIRUS

8. BACTERIA

9. FUNGI

10. Population increase until 2050
of 2.200 x 106 hab.
2.700 x 106 Tn
1.22 Tn/hab.
Increase of the crop
surface 600 x 106 ha
4 Tn/ha
Improvement of the crop productivity
2 Kg pesticides/ha
BIOTECHNOLOGY
Increase of 1.200 x 106 Kg
of pesticides
INTEGRATED CROP PRODUCTION
BIOPESTICIDES AND BIOFERTILIZERS
POPULATION GROWTH AND FOOD SECURITY

11. How we develop a microbial biopesticide?

12. The plant as a microbial ecosystem
Martínez-Alonso et al. 2011.
Yashiro et al. 2011
Shade et al. 2013
(0,001-10% culturable (thousands of species/g)
Metagenomics. 100-1000 OTUs (20-50 OTUs dominant)
Phylosphere
Rhizosphere

13. PythiumOomycota
PhlebiopsisBasidiomycota
Ulocladium
Trichoderma
Penicillium
Myrothecium
Muscodor
Gliocladium
Epicoccum
Chaetomium
AmpelomycesAscomycotaFungi (molds)
Sporobolomyces
Rhodotorula
Pseudozyma
CryptococcusBasidiomycota
Pichia
Metschnikowia
Kloeckera
Debaryomyces
Conyothyrium
Candida
AureobasidiumAscomycotaYeast
StreptomycesStreptomycetaceae
AgrobacteriumRhizobiaceae
Pseudomonas
BurkholderiaPseudomonadaceae
LeuconostocLeuconostocaceae
LactobacillusLactobacillaceae
Serratia
Rahnella
Pantoea (Erwinia)
EnterobacterEnterobacteriaceae
CellulomonasCellulomonadaceae
Paenibacillus
Brevibacillus
BacillusBacillaceaeBacteria
GenusFamily/ClassGroup
PythiumOomycota
PhlebiopsisBasidiomycota
Ulocladium
Trichoderma
Penicillium
Myrothecium
Muscodor
Gliocladium
Epicoccum
Chaetomium
AmpelomycesAscomycotaFungi (molds)
Sporobolomyces
Rhodotorula
Pseudozyma
CryptococcusBasidiomycota
Pichia
Metschnikowia
Kloeckera
Debaryomyces
Conyothyrium
Candida
AureobasidiumAscomycotaYeast
StreptomycesStreptomycetaceae
AgrobacteriumRhizobiaceae
Pseudomonas
BurkholderiaPseudomonadaceae
LeuconostocLeuconostocaceae
LactobacillusLactobacillaceae
Serratia
Rahnella
Pantoea (Erwinia)
EnterobacterEnterobacteriaceae
CellulomonasCellulomonadaceae
Paenibacillus
Brevibacillus
BacillusBacillaceaeBacteria
GenusFamily/ClassGroup
Montesinos and Bonaterra (2009). Encyclopedia of Microbiology
Microbial groups in which strains have been implicated
in biological control of plant diseases

14. MECHANISMS OF ACTION
(pathogens/disease control)
HOST MEDIATED
RESISTANCE
-Induced systemic resistance (ISR)
-Systemic acquired resistance (SAR)
EXCLUSION
-Colonization of entry sites on host
-Competition for nutrients (C,N,P, iron)
-Physical barriers (biofilm)
DIRECT CELL-TO-CELL
INTERACTION
- Hyperparasitism
- Interference with cell/hyphal
growth SIGNAL
INTERFERENCE
- Quorum sensing
ANTIBIOSIS
- Lytic enzymes
-Antimicrobial peptides
and proteins
- Phenolic compounds
- Polyketides
- others
Montesinos and Bonaterra (2009). Encyclopedia of Microbiology

15. From the plant to the product...
Development of microbial pesticides for disease control

16. Specific analytical
methods and
traceability
Selected
microbial
strain
Pilot tests
Identification and
characterization
Biosafety and
environmental
impact
Registration for commercial use
Mass
production
Specific analytical
methods and
traceability
Discovery,
screening and
isolation in
pure culture
Selected
microbial
strain
Patenting
Patenting
Pilot testsPilot tests
Bioassays and
mechanism of action
Bioassays and
mechanism of action
Identification and
characterization
Identification and
characterization
Preservation
and formulation
Preservation
and formulation
Preservation
and formulation
Biosafety and
environmental
impact
Biosafety and
environmental
impact
Modified from Montesinos and Bonaterra (2009). Encyclopedia of Microbiology

17. Molecular marker assisted screening:antimicrobials, insecticidal, nematicidal...
Samples
PCR assisted
screening
IsolationStrain collection
+
Genomics
(GenBank)
Metagenomics
Ciclolipopeptides
Bacteriocins
Poliketides
Macrolides
Bt toxins
Chitinases
...
Pseudomonas fluorescens
Pantoea agglomerans
Bacillus subtilis/amyloliquefaciens
Leuconostoc mesenteroides
Lactobacillus plantarum
Bacillus thuringiensis
Streptomyces spp.
How we fish selectively biological control agents...

18. srfAA bmyB fenD XX XX 3.2%
Bacillus with simultaneous presence of genes for
antimicrobial peptides
5
10
15
20
25
Frequency
(numberofstrains)
5
10
15
20
25
Pattern/Number of genes
- + - - - - - + + + + + - - - - - - + + + + + + - - - - + + + - - + + +
- - + - - - - + - - - - + + + + - - + + + - - - + + + + + + + + + + + -
- - - + - - - - + - - - + - - - + + + - - + - - + + + - + + - + - + + +
- - - - + - - - - + - - - + - - + - - + - + + - + - - + + - + + + + + +
- - - - - + - - - - + - - - + - - - - - - - + + - + - - - + + + + + - +
- - - - - - + - - - - + - - - + - + - - + - - + - - + + - - - - + - + +
0 1 2 3 4 5
- + - - - - - + + + + + - - - - - - + + + + + + - - - - + - -
- - + - - - - + - - - - + + + + - - + + + - - - + + + + + + + + + -
- - - + - - - - + - - - + - - - + + + - - + - - + + + - + + - + - + + +
- - - - + - - - - + - - - + - - + - - + - + + - + - - + + - + + + + + +
- - - - - + - - - - + - - - + - - - - - - - + + - + - - - + + + + + - +
- - - - - - + - - - - + - - - + - + - - + - - + - - + + - - - - + - + +
srfAA
bacA
bmyB
fenD
spaS
ituC
0
Markergene
N=25 N=49 N=55 N=36 N=6
127
113
102
74
28
18
Numberofisolates
Mora et al. 2011. Int. Microbiol.

19. Strain singularity. Exemples among Bacillus sp.
PCA
MALDI-TOF of cultures
rpoB sequence (or other genes) and gene
profiling (e.g. AMPs)
In vitro antagonism

20. Stages in development of products
(disease control)
Phase 1: in vitro
Phase 2: ex vivo
Phase 3: In planta and field

21. Selecting the best strain for disease control performance (Ex vivo ass
Lactobacillus plantarum strain collection
IMMATURE FRUITSLEAVES
FLOWERS
TC54
TC92
BC24
PC29
CC31
PM35
TC41
TC60
AC73 AC81
TC97CM165
PC263
BC50
CM205
AM266
AC316
CM209FC212
EM214
RC526
TC40
PM357
PM366
CM359
PC579
FC560
CM466
PM411
CC575
EC586
TM358

22. Lactobacillus plantarum – Control of fireblight of apple and pear
Severityofinfections 1
2
3
Populationlevel
(logCFUperflower)
Estreptomicina
L.plantarumTC92
L.plantarumTC54
L.plantarumPM411
P.vagansC9-1
P.fluorecensEPS62e
B.subtilisQST713
Notractat
2
4
6
8
a
b
cd
bc
cd
d
d
d
Roselló et al. 2013. European J. Plant Pathol.
Semi-field assay
Estació Experimental Fundació Mas Badia

23. from the lab to the field ...
High water availability and very reach
culture media
Osmotic stress and poor
growth conditions
Storage conditions

24. Biopest
Biopest
Inoculum
preparation
Liquid phase
fermentation
Solid phase
fermentation
Homogeneization
Centrifugation
Concentrated
suspension of
cells/spores
Drying
Liquid
formulation
Dry
formulation
Stock culture
PRODUCTION OF A MICROBIAL PESTICIDE
(cells or metabolites)
Culture
supernatant
Purification of
metabolites

25. PRODUCTION METHODS BASED ON MICROBIAL FERMENTATION
AND DOWNSTREAM TECHNOLOGIES
Fermentation
Products and cell
separation
Drying
1010 -1011 cfu/g

26. HO
O
OH
OH
OH
O
HO
O
OH
OH
OHO
-+
H3C
CH3
CH3
N
GB
T
GG
OH
OH
HO
OH
O
HO
O
OH
NAGGN
H3C
NH
O
O
H2N
H
N
OO
O
NH2
H2N
GB
HO
O
OH
OH
OH
O
HO
O
OH
OH
OH
T
GB-Glycine betaine
T-Trehalose
GG- Glucosyl-glycerol
NAGGN-N-acetilglutaminilglutamin amide
P. agglomerans EPS125
P. fluorescens EPS62e
0.5 M NaCl
Control
Biofilm
0.5 M NaCl
Control
Biofilm
Osmotic stress induces osmolyte accumulation
in cells
O
O
-+
H3C
CH3
CH3
N
O

27. Physiological improvement by
nutritional enhancement and osmoadaptation
Bonaterra et al. FEMS Microbiol. Letters. 250 (2005)
Cabrefiga et al. Appl. Environ. Microbiol. 78 (2011)
Pseudomonas fluorescens EPS62e
Semi-field assay. Fireblight control
Estació Experimental Fundació Mas Badia

28. Application of microbial pesticides

29. real time PCR
plate counting
0 10 20 30
2
4
6
8
Time (days)
Survival-VBNC
0 10 20 30
Death
Populationlevel
(LogcellsorCFUorgan-1
Leaves
Populationlevel
(LogcellsorCFUorgan-1
Time (days)
0 10 20 30 40 50 60
5
6
7
8
9
Bloom
Post-
bloom Fruit set Fruit growth
Active growth
Flowers-fruits
Epiphytic fitness of Pseudomonas fluorescens EPS62e
Pujol et al. FEMS Microbiol. Letters. 249 (2005)
Pujol et al. Appl. Environ. Microbiol. 72 (2006)

30. Field dispersion trials of EPS62e
Pujol et al. FEMS Microbiol. Ecol. 59 (2007)
0 10 20 30 40 50
-3
-2
-1
0
1
2
Days after inoculation
-3
-2
-1
0
1
2
-3
-2
-1
0
1
2
PEAR FLOWERS
PEAR LEAVES
APPLE FLOWERS
0 10 20 30 40 50
-3
-2
-1
0
1
2
Log%(EPS62e/totalculturable)
-3
-2
-1
0
1
2
-3
-2
-1
0
1
2
Dominant
wind
Mas Badia Agricultural Experiment Station

31. Microorganism Origin Disease/effect Plant crop
P. agglomerans EPS125 fruit, 1993 postharvest rot fruits
P. fluorescens EPS62e fruit, 1997 fire blight apple, pear, others
P. fluorescens EPS817 root, 1998 growth promotion banana
P. fluorescens EPS894 root, 1999 root rot strawberry, tomato
P. fluorescens EPS818 root, 1998 nematodes Prunus , tomato
L. mesenteroides EPS160 fruit, 2004 food-borne pathogens Vegetables, fruit
W. cibaria EPS128 fruit 2004 postharvest rot fruits
B. subtilis EPS20XX leaves, 2008 fungal diseases Several
postharvest rot
Bacterial strains for
microbial biopesticide development
L. plantarum TC92 pear, 2004 fire-blight Pome-fruits

32. REGISTRATION FOR COMMERCIALIZATION

33. - Species (strain) identification
- Biological properties
- Analytical methods
- Residues
- Traceability. Fate and behaviour in the environment
- Efficacy
- Adverse potential effects on human health and
non-target organisms. Ecotoxicology and mammalian
toxicology
Directive and regulation for authorization
of Plant Protection Products (PPPs)
( Regulation (EC) No 1107/2009, Directive 2009/128/EC )
Specific regulations at country level of Plant Strengtheners
(considered as “other means of control”, not biocides, not fertilizers, generally low risk profile)
(Spain: LEY 43/2002 DE SANIDAD VEGETAL )
Regulatory issues for commercialization
of microorganisms as biological control agents
in the European Union

34. Biocontrol agents and biosafety in Europe
Toxicology and pathogenicity
in mammals
Pathogenicity to plants
(Directive 2001/36/EC and 2000/54/EC)
Environmental impact

35. The EFSA QPS approach
(Qualified Presumption of Safety)
Definition of the taxonomic unit
Sufficiency of the body of knowledge
Safety concerns
Suitable for QPS
Not suitable for QPS
No
Yes
Yes
Body of Knowledge
History of
use
Industrial
applications
Scientific
literature
and databases
Clinical
aspects
Ecology
Taxonomy
Body of knowledge
Pathogenicity
End use
Case-by-case assessment
Simplyfied assessment
No
Yes

36. An example:Bacillus subtilis QST713
Taxonomy, species, strain Bacillus subtilis QST713
Identification Classical microbiological, DNA based method
(ribotyping)
Methods of analysis Quantitative method is not strain specific
Is a GMO? No
Origin of isolation Not reported. Soil?
Relevant metabolites of concern Not reported. Cyclic lipopeptides
Toxinogenic Not produce toxins. Very low due to cyclic
lipopeptides
Mechanism of action Competition, predation, antibiosis. ISR.
Host/target range Saprophytic growth
Resting stages and survival Endospores. Dispersal by dust and air streams
Production quality/safety control Microbiological control
Pathogenicity/infectivity potential Oportunistic with no pathogenic potential.
Growth at 37 ºC.
Acute Toxicity Acute oral-dermal LD50 10 log cfu/kg bw.
Inhalation 8 log cfu/kg bw. Low to birds, aquatic
organisms. Acceptabl to worms and arthropods.
Irritation Very silght irritating. Relevant metabolites
allergic reactions
Genotoxicity Not reported
Formulation-technical Wettable powder (Serenade)
Application method Spraying
Exposure of the environment Common soil inhabitant
Exposure of operator Acceptable
Residues Not expected relevant
End use Control of plant-pathogenic fungi and fire-blight

38. Commercial microbial biopesticide products

39. Microorganism Activity Company
Aureobasidum pullulans B Bioferm (2012)
Bacillus amyloliquefaciens QST713 F/B AgraQuest (2006), Bayer Cropscience
Bacillus amyloliquefaciens D747 F/B Certis, Intrachem (2011, provisional)
Bacillus amyloliquefaciens FZB24 F/B Abitep Pending
Bacillus amyloliquefaciens MBI600 F/B Pending
Bacillus pumilus QST2010 FU Pending
Pseudomonas chlororaphis MA342 F BioAgri (2004)
Streptomyces griseoviridis K61 F Verdera Oy (2009)
Streptomyces lydicus WYEC108 FU Pending
Saccharomyces cerevisiae LAS02 FU Pending
Ampelomyces quisqualis AQ10 F Ecogen Europe (2005)
Candida oleophila O F Bionex sprl
Coniothyrium minitans (FU) F Prophyta GmbH (2003)
Gliocladium catenulatum J1446 F Kemira Agro Oy (2005)
Phlebiopsis gigantea (several str.) F Forectry Comm. (2)(2009)
Pseudomonas DSMZ 13134 F/B Sourcon-Padena GmbH, 2014
Pseudozyma flocculosa F Maasmond-Westland
Pythium oligandrum F Biopreparaty Spol s.r.o.
Trichoderma harzianum F Binab Bioinnovation AB (8) (2009)
Trichoderma polysporum F Binab-Bio-Innovation AB (2009)
Trichoderma atroviride F Agribiotec S.r.l. (2)(2009)
Trichoderma asperellum F (2009)
Trichoderma gamsii F (2009)
Verticillium albo-atrum WCS850 F Arcadis Pl. B.V.
Bacillus firmus I-1582 NE BayerCropscience, 2013
Verticillium lecanii (Lecanicillium muscarum) I Koppert Beheer BV, 2009
Adoxophyes orana GV I GAB Consulting
Cidia pomonella GV I (2009)
Helicoverpa NPV I Andermatt Biocontrol GmbH
Spodoptera litoralis NPV I Andermatt Biocontrol GmbH
NPV of Spodoptera exigua I Biosys (2007)
Bacillus thuringiensis subsp. aizawai I Isagro (4)(2009)
Bacillus thuringiensis subsp. Israeliensis I Sipcam SpA (2)(2009)
Bacillus thuringiensis subsp. kurstaki I Alfajarin Químics SA (9)(2009)
Bacillus thuringiensis subsp. tenebrionis I Valent Biosci.(2009)
Beauveria bassiana I Agribiotech srl (4)(2009).Others pending
Metarhizium anisopliae BIPESCO I Agrifutur srl (3)(2009)
Paecilomyces fumosoroseus 97 I Thermo Trilogy Co. (2001)
Paecilomyces fumosoroseus Fe9901 I FuturEco S.L.
Paecilomyces fumosoroseus 251 I Prophyta GmbH (2008)
Zucchini yellow mosaic virus V Bio-Oz Biotec. Ltd.
Active ingredients of microbial type approved or under evaluation in the
European Union. Annex 1. Directive 2009/128

40. Advantages
- ecological approach
- self-reproduction/dead
- low aggressive toxicological profile (?)
- multiple mechanisms of action (in general)
- no residue (?)
Inconvenients
- moderate efficacy compared to conventional pesticides
- low consistency of results from trial to trial (agroecological conditions,
complex interactions)
- restricted conditions of use
- relatively short shelf-life of the formulation
- biosafety (?)
ADVANTAGES, INCONVENIENTS
AND FUTURE PROSPECTS

42. http://www.udg.edu/cidsav
Centre d’Innovació i Desenvolupament en Sanitat Vegetal (CIDSAV)

43. Gràcies!