Federal Department of Economic Affairs, Education and Research EAER Agroscope The potato microbiome and its potential impact on late blight resistance Agroscope, Institute for Plant Production Sciences, Wädenswil & Nyon 08.08.2016 22.01.2015 www.agroscope.ch I good food, healthy environment
Exploring the microbiome The microbiome : not only in the soil and rhizosphere The phyllosphere is a harsh environment but still 10 7 microbes per cm 2 of leaf Impact on their host Diversity of microorganisms Selected by the plant Potentially modified upon infection with a pathogen Potentially beneficial and/or protective Healthy resistant cultivar Symptomatic moderately susceptible cultivar against late blight? 2
Can we find potential biocontrol strains in the potato s microbiome? 3
Isolation of microbiome inhabitants Control Phyllosphere strains Rhizosphere strains Overall most active strains belong to the genera Pseudomonas, Bacillus, Athrobacter and Microbacterium Pseudomonas accounts for the most active strains Selected 15 Pseudomonas strains with varying anti-phytophthora activity Are there potential biocontrol agents among them? Lukas Hunziker Denise Bönisch Hunziker et al., 2015 Guyer et al., 2015 4
Colonisation ability Mode of action Exploring the bacteria s potential What stage of the Phytophthora lifecycle do the bacteria affect? How are these life stages affected? Is the anti-phytophthora activity maintained on leaf tissue? Are the strains capable of establishing themselves on potato plants? Bio assays 5
Mode of action Effects of the bacteria on different stages of the P. infestans life cycle : Mycelial growth Sporangia production Zoospore production Control : 6
Length of germtube (µm) % germinated sporangia Mode of action Sporangia germination 60 40 20 0 Sporangia viability Propidium iodide staining Alive R47 52 % Dead 48 % 300 200 S49 98 % 2 % 100 0 S04 8 % 92 % Zoospore motility and chemotaxis Aspartic acid Control R47 supernatant 7
Victoria Bintje Lady Claire Surface convered with mycelium (%) Is the anti-phytophthora activity maintained on leaf tissue? In vivo test on leaf discs : coinoculation on leaf tissue 4.0 3.0 2.0 1.0 0.0 25 Pinf S34 R02 S35 R74 R76 R84 S50 S04 S49 R32 R01 S19 R95 S22 R47 neg 20 15 10 5 0 5.0 4.0 Pinf S34 R02 S35 R74 R76 R84 S50 S04 S49 R32 R01 S19 R95 S22 R47 neg 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 8
CFU/cm 2 of leaf surface 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 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 9
Do different P. infestans strains differing in virulence show varying susceptibility to biocontrol bacteria? 10
Monitoring of P. infestans in Switzerland Characterisation of virulence Leaf disc assay Ramona Gloor Black s differential set R0, Bintje R1 to R11 Gasore, Sarpo Mira Infection/no infection for 15 leaf discs 0 leaf discs infected All 15 leaf discs infected 11
Susceptibility of P. infestans strains varying in virulence towards our antagonists Leaf disc assay with subset of P.infestans strains ** ** All P.infestans strains are inhibited The response varies from bacterial strain to bacterial strain regardless of the apparent virulence of the P. infestans strains 12
Outlook - Mechanisms? Aurélien Adithi Ravikumar Bailly 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 anti-phytophthora activity HCN 1-undecene Phenazines MMTS HPR DMDS DMTS Pyrrolnitrin R47 13
Acknowledgements Institute for Plant Production Sciences, Nyon & Wädenswil Dr. Laure Weisskopf Ramona Gloor Josep Massana Codina Dr. Katia Gindro and Mycology Group Dr. Jürg Frey Adithi Ravikumar Varadarajan Dr. Christian Ahrens Dr. Aurélie Gfeller University of Zürich Dr. Aurélien Bailly Prof. Leo Eberl Institute for Sustainibility Sciences, Reckenholz Anouk Guyer Tomke Musa Alessia Gandolfi 14
Thank you for your attention Agroscope good food, healthy environment 15
Monitoring of Phytophthora infestans population diversity in Switzerland In 2015 : Ramona Gloor 22 isolates were successfully retrieved 3 A1, 18 A2 Majority of EU_13_A2 Increased resistance against Mefenoxam in the EU_13_A2 isolates EU_13_A2 miscellaneous 16
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Antagonistic leaf disc assay: method Leaf discs Sporangia-bacterial suspension Anouk Guyer Aurélien Bailly Mout De Vrieze Evaluation of % the surface covered with mycelium... 7 days 49.4 % Greenhouse plants Inoculation of the leaf disc (Ø 2 cm) 2.8 % with automated picture analysis 18