Microbial biopesticides
Plant Pathology
Instituto de Tecnología Agroalimentaria
Universidad de Girona (Spain)
http://www.u...
What is a biopesticide?
BIOPESTICIDE:A pesticide of biological origin
and not synthetic (may be semi-synthetic)
PESTICIDE:Active ingredient that k...
Microbial (e.g. Bacillus amyloliquefaciens QST713, CpGV virus)
Non-microbial (natural extracts or compounds)
 Botanical (...
Why we need
biopesticides?
Actual losses of agricultural productivity
(x 106 Tn per year)
Production
5311
Diseases
1294
Insect pests
“Weeds”
and othe...
VIRUS
BACTERIA
FUNGI
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...
How we develop a microbial biopesticide?
The plant as a microbial ecosystem
Martínez-Alonso et al. 2011.
Yashiro et al. 2011
Shade et al. 2013
(0,001-10% culturabl...
PythiumOomycota
PhlebiopsisBasidiomycota
Ulocladium
Trichoderma
Penicillium
Myrothecium
Muscodor
Gliocladium
Epicoccum
Cha...
MECHANISMS OF ACTION
(pathogens/disease control)
HOST MEDIATED
RESISTANCE
-Induced systemic resistance (ISR)
-Systemic acq...
From the plant to the product...
Development of microbial pesticides for disease control
Specific analytical
methods and
traceability
Selected
microbial
strain
Pilot tests
Identification and
characterization
Bio...
Molecular marker assisted screening:antimicrobials, insecticidal, nematicidal...
Samples
PCR assisted
screening
IsolationS...
srfAA bmyB fenD XX XX 3.2%
Bacillus with simultaneous presence of genes for
antimicrobial peptides
5
10
15
20
25
Frequency...
Strain singularity. Exemples among Bacillus sp.
PCA
MALDI-TOF of cultures
rpoB sequence (or other genes) and gene
profilin...
Stages in development of products
(disease control)
Phase 1: in vitro
Phase 2: ex vivo
Phase 3: In planta and field
Selecting the best strain for disease control performance (Ex vivo ass
Lactobacillus plantarum strain collection
IMMATURE ...
Lactobacillus plantarum – Control of fireblight of apple and pear
Severityofinfections 1
2
3
Populationlevel
(logCFUperflo...
from the lab to the field ...
High water availability and very reach
culture media
Osmotic stress and poor
growth conditio...
Biopest
Biopest
Inoculum
preparation
Liquid phase
fermentation
Solid phase
fermentation
Homogeneization
Centrifugation
Con...
PRODUCTION METHODS BASED ON MICROBIAL FERMENTATION
AND DOWNSTREAM TECHNOLOGIES
Fermentation
Products and cell
separation
D...
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
...
Physiological improvement by
nutritional enhancement and osmoadaptation
Bonaterra et al. FEMS Microbiol. Letters. 250 (200...
Application of microbial pesticides
real time PCR
plate counting
0 10 20 30
2
4
6
8
Time (days)
Survival-VBNC
0 10 20 30
Death
Populationlevel
(LogcellsorCFUo...
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 ...
Microorganism Origin Disease/effect Plant crop
P. agglomerans EPS125 fruit, 1993 postharvest rot fruits
P. fluorescens EPS...
REGISTRATION FOR COMMERCIALIZATION
- Species (strain) identification
- Biological properties
- Analytical methods
- Residues
- Traceability. Fate and behavio...
Biocontrol agents and biosafety in Europe
Toxicology and pathogenicity
in mammals
Pathogenicity to plants
(Directive 2001/...
The EFSA QPS approach
(Qualified Presumption of Safety)
Definition of the taxonomic unit
Sufficiency of the body of knowle...
An example:Bacillus subtilis QST713
Taxonomy, species, strain Bacillus subtilis QST713
Identification Classical microbiolo...
Commercial microbial biopesticide products
Microorganism Activity Company
Aureobasidum pullulans B Bioferm (2012)
Bacillus amyloliquefaciens QST713 F/B AgraQuest (20...
Advantages
- ecological approach
- self-reproduction/dead
- low aggressive toxicological profile (?)
- multiple mechanisms...
http://www.udg.edu/cidsav
Centre d’Innovació i Desenvolupament en Sanitat Vegetal (CIDSAV)
Gràcies!
Emilio Montesinos - Ingeniería de nuevos sistemas microbianos para una agricultura sostenible
Emilio Montesinos - Ingeniería de nuevos sistemas microbianos para una agricultura sostenible
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Emilio Montesinos - Ingeniería de nuevos sistemas microbianos para una agricultura sostenible

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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.

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Emilio Montesinos - Ingeniería de nuevos sistemas microbianos para una agricultura sostenible

  1. 1. Microbial biopesticides Plant Pathology Instituto de Tecnología Agroalimentaria Universidad de Girona (Spain) http://www.udg.edu/cidsav Emilio Montesinos Seguí
  2. 2. What is a biopesticide?
  3. 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. 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. 5. Why we need biopesticides?
  6. 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. 7. VIRUS
  8. 8. BACTERIA
  9. 9. FUNGI
  10. 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. 11. How we develop a microbial biopesticide?
  12. 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. 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. 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. 15. From the plant to the product... Development of microbial pesticides for disease control
  16. 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. 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. 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. 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. 20. Stages in development of products (disease control) Phase 1: in vitro Phase 2: ex vivo Phase 3: In planta and field
  21. 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. 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. 23. from the lab to the field ... High water availability and very reach culture media Osmotic stress and poor growth conditions Storage conditions
  24. 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. 25. PRODUCTION METHODS BASED ON MICROBIAL FERMENTATION AND DOWNSTREAM TECHNOLOGIES Fermentation Products and cell separation Drying 1010 -1011 cfu/g
  26. 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. 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. 28. Application of microbial pesticides
  29. 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. 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. 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. 32. REGISTRATION FOR COMMERCIALIZATION
  33. 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. 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. 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. 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
  37. 37. Commercial microbial biopesticide products
  38. 38. 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
  39. 39. 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
  40. 40. http://www.udg.edu/cidsav Centre d’Innovació i Desenvolupament en Sanitat Vegetal (CIDSAV)
  41. 41. Gràcies!

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