Biocontrol and P. infestans diversity: the potential of antagonistic bacteria

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1 Federal Department of Economic Affairs, Education and Research EAER Agroscope Biocontrol and P. infestans diversity: the potential of antagonistic bacteria Agroscope, Institute for Plant Production Sciences, Wädenswil & Nyon I good food, healthy environment

2 Why bacteria and where to find them? the plant microbiome Diversity of microorganisms selected by the plant potentially modified upon infection with a pathogen potentially beneficial and/or protective 2

3 Isolation of microbiome inhabitants Overall most active strains belong to the Control genera Pseudomonas, (also Bacillus, Arthrobacter and Microbacterium) Selected 9 Pseudomonas strains with Phyllosphere strains varying anti-phytophthora activity Rhizosphere strains Lukas Hunziker Denise Bönisch Hunziker et al., 2015 Guyer et al.,

4 Exploring the bacteria s potential Mode of action What stage of the Phytophthora lifecycle do the bacteria affect? How are these life stages affected? In vitro assays Colonisation ability Is the anti-phytophthora activity maintained on leaf tissue? Are the strains capable of establishing themselves on potato plants? In planta assays Lab strain Rec01 (Zürich, 2001) but are these effects maintained when using a different P. infestans strain? 4

5 Collection of P. infestans 2015 In 2015 : - 60 infestations reported - 27 samples (infected leaves) collected from 19 fields - 22 isolates were successfully retrieved Ramona Gloor Characterization of : Mating type Morphology Pathogenecity Virulence SSR Fungicide resistance 5

6 How aggressive are the isolates? Black s differential set 11 R-genes : R1 R11 R0 & Bintje Susceptible cultivars (no R-genes) Gasore & Sarpo Mira Resistant cultivars Pathogenecity test Leaf disc assay - Infection/no infection - 15 discs per lineage and isolate 7 days 6

7 How aggressive are the isolates? The most aggressive isolates are among the Blue_13 MLG 7

8 How aggressive are the isolates? Resistance to Mefenoxam Presence of A1 and A2 8

9 Is pathogenicity correlated to virulence? What about virulence? Leaf disc assay 7 days 49.4 % 2.8 % The most virulent isolates are not necessarily the most aggressive 9

10 Is virulence correlated to morphological traits? Correlation with morphological traits? Fast growing strains produce less sporangia Higher sporangia production results in smaller sporangia Smaller sporangia appear to be more virulent Isolates producing many small sporangia are more virulent Correlation with mating type? A1 isolates are more virulent Correlation with origin? Virulence 10

11 Back to the bacteria Mycelium Sporangia Protection on plant Survival on plant Mycelial growth on RA+ Sporangia production Sporangia germination Mycelial growth on RA- Coinoculation Spraying % Germ tube R32 P. vranovensis R47 P. chlororaphis R76 P. fluorescens R84 P. marginalis S04 P. frederikbergensis S19 P. frederikbergensis S34 P. jessenii S35 P. marginalis S49 P. fluorescens Stems Scoring according to performance Selected 3 bacteria based on varying effects on mycelial growth and sporangia germination 11

12 Inhibiton of mycelial growth by the bacteria Dual assay Control 12

13 Zooming in on sporangia germination Both sporangia germination and sporangia length are affected by the bacteria 13

14 Is the antagonistic effect maintained on plant tissue? The bacteria successfully reduce symptom development SG-9475-An, mating type A1 is less susceptible 14

15 Outlook CLSA and GC-MS to identify volatile organic compounds (VOCs) Diffusible compounds TLC Genome mining (9 strains) Looking for genes Comparative genomics comparison of genomes of strains showing varying activity Field trial : foliar treatments using bacterial strains - Effect of combinations of strains - Survival of strains under field conditions HCN 1-undecene Phenazines MMTS HPR DMDS DMTS Pyrrolnitrin R47 Aurélien Adithi Ravikumar Bailly 15

16 Acknowledgements Institute for Plant Production Sciences, Nyon & Wädenswil Dr. Katia Gindro and Mycology Group Nicolas Vuille Ramona Gloor Josep Massana Codina Dr. Jürg Frey Adithi Ravikumar Varadarajan Dr. Christian Ahrens University of Fribourg Prof. Laure Weisskopf University of Zürich Dr. Aurélien Bailly Institute for Sustainibility Sciences, Reckenholz Anouk Guyer Alessia Gandolfi 16

17 Thank you for your attention 17

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19 Mode of action? Dual cultures in Petri dishes 9 Pseudomonas strains Different P.infestans strains Control : Red : the pathogen is strongly inhibited Green : the pathogen is growing well 19

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21 Mode of action? Sporangia germination % germinated sporangia Sporangia viability Propidium iodide staining Alive R47 52 % Dead 48 % Length of germtube (µm) S49 S04 98 % 8 % 2 % 92 % Zoospore motility and chemotaxis Aspartic acid Control R47 supernatant 21

22 Is the anti-phytophthora activity maintained on leaf tissue? In vivo test on leaf discs : coinoculation on leaf tissue 4,0 Victoria Bintje Lady Claire Surface convered with mycelium (%) 3,0 2,0 1,0 0, ,0 4,0 3,0 2,0 1,0 0,0 Pinf S34 R02 S35 R74 R76 R84 S50 S04 S49 R32 R01 S19 R95 S22 R47 neg Pinf S34 R02 S35 R74 R76 R84 S50 S04 S49 R32 R01 S19 R95 S22 R47 neg Pinf S34 R02 S35 R74 R76 R84 S50 S04 S49 R32 R01 S19 R95 S22 R47 neg 22

23 Can the bacteria colonise potato plants? Tuber inoculation Bacterial suspensions (OD=1) Stem cuts Leaf application Josep Masana Codina Bacterial suspensions (OD=1) Leaf discs Not sterilised Surface sterilised R32 R47 R76 R84 - S04 S19 S34 S35 S49 retrieved after 3 weeks retrieved after 4 weeks retrieved after 5 weeks CFU/cm 2 of leaf surface CFU count 1,0E+08 1,0E+07 1,0E+06 1,0E+05 1,0E+04 1,0E+03 1,0E+02 1,0E+01 1,0E+00 Day 0 Day 1 Day 4 R32 R47 R76 S35 23