Effect of climate change and management on plant-associated microbial communities Gabriele Berg Environmental Biotechnology INTRODUCTION: Impact of climate change Bogs Impact on global cycles carbon cycle nitrogen cycle 1
Introduction: Plant-microbe interaction Soil-borne pathogens Disease Induced resistance Root exudation Plant Plants form a unique Biocontrol habitat in terrestrial Plant growth ecosystem. promotion Competition Manyfold interactions/interplays take Hormomal place. stimulation Change of composition The balance of structure and functions is important for plant growth and health. Plant-associated microorganisms 2
INTRODUCTION: Impact of climate change Multiple Interactions Host Pathogen Temperature (warming) Moisture (Heavy rains Storms) Drought Elevated CO 2 Changing conditions Climate change Vectors INTRODUCTION: Impact Up to 422,000 species Pittmann & Jørgensen Science 2002 Pathogens Geography Plant species Plant-associated communities Animals Grazers Human activities Treatments Climate Soil quality [Smalla et al. AEM 2001] [Berg et al. AEM 2002] [Berg et al. AEM 2005] [Opelt et al. The ISME J. 2007] Annual cycle 3
INTRODUCTION: Impact of climate change Impact on plant pathogens Temperature: bigger areal, higher aggressiveness/virulence More HGT, new pathogens Changing conditions: More niches: Multi-species infections More host plants Storms: Better distribution INTRODUCTION: Impact of climate change Impact on soil Salinisation Drought Impact on plant beneficial bacteria Better performance -> rev. by Compant et al. 2010 FEMS 4
I. Impact on plant pathogens and biocontrol? 1. Example: Soil-borne pathogens: Verticillium dahliae Verticillium dahliae Serratia plymuthica HRO C48 Strawberry, rape olives 5
INTRODUCTION: Target pathogens Yield losses caused by soil-borne pathogens worldwide: 4 billion $ 1st target pathogen: Verticillium dahliae Kleb. cause Verticillium wilt on a broad host range Methyl bromide was used banned HIGH RISK PATHOGEN No possibility to suppress the pathogen Model-organism: organism: Serratia plymuthica HRO-C48 (RhizoStar) RHIZOSTAR Serratia plymuthica HRO-C48 Isolated from the rhizosphere of rape European Patent 98124694.5 No risk for human health and environment (risk group 1) Solution: Biocontrol by naturally occurring bacteria Adaptation to new host plants Serratia Rape and Olives 6
Model-organism: organism: Serratia plymuthica HRO-C48 (RhizoStar) Influence of Quorum Sensing (QS) on the interaction of S. plymuthica on the interaction with eucaryotes Plants Serratia plymuthica HRO-C48 Fungi Rhizosphere competence Biofilms QS QS QS QS Proteases Chitinases IAA production [Müller et al. FEMS Microb. Ecol. 2009] QS Pyrrolnitrin 7
Model-organism: organism: Serratia plymuthica HRO-C48 (RhizoStar) 200 l of Serratia plymuthica HRO- C48 Suspension VOC s Control Serratia Examples for VOCs produced by Serratia plymuthica: 2-Heptanon, Di-Methylpyrazin, 2-Nonanon, 2- phenylethanol, Benzylnitril, Undecanon Impact on rhizosphere communities: microbial fingerprints 5 weeks 10 weeks 15 weeks Bacterial communities: Strawberry + Potato rhizosphere only short-term changes in the composition of bacterial and fungal communities 8
Impact on rhizosphere communities: microbial fingerprints 6.0 log10 CFU g -1 root fresh weight 5.0 4.0 3.0 2.0 1.0 0.0 Non-infested soil V. longisporum-infested soil Soil treatment Non-inoculated Serratia plymuthica-inoculated V.l. Ctrl. V.l. Ctrl. V.l. Ctrl. V.l. Ctrl. Bacterial communities: Oilseed rape Presence of the pathogen enhance the abundance of the antagonist S. plymuthica HRO- C48 Impact on rhizosphere communities: microbial fingerprints Similarity of microbial communities Climate change? community field site growth stage treatment bacteria 60 66-75 78-100 pseudomonads 62 70-78 82-100 fungi 59 70-72 69-79 Compared to the growth stage and the field site, the treatments showed only negligible, short-term effects the composition of microbial communities [Scherwinski et al. Biocontrol 2005; FEMS Microb. Ecol. 2009] 9
2. Example: Soil-borne pathogens: Rhizoctonia solani Rhizoctonia solani Serratia plymuthica Pseudomonas trivialis Trichoderma gamsii Sugar beet BIOTECHNOLOGY: Biocontrol of Rhizoctonia solani * * Formulation into the pill of the sugar beet seed Colonization of the rhizosphere Protection against Rhizoctonia solani 10
BIOTECHNOLOGY: Biocontrol of Rhizoctonia solani [Zachow et al. FEMS Microb. Ecol. 2010] BIOTECHNOLOGY: Biocontrol of Rhizoctonia solani 11
Serratia plymuthica and Trichoderma gamsii in the sugar beet rhizosphere BIOTECHNOLOGY: Biocontrol of Rhizoctonia solani 120 Parcel field trial Tabertshausen-Kasten (TAK) 2009 RI in relation to Std BERETTA n beets in relation to Std BERETTA 100 80 60 40 20 0 A cocktail of antagonistic microorganisms is much more effective than a treatment using single BCA. 12
Impact on rhizosphere communities: microbial fingerprints Endophytic communities: Lettuce rhizosphere Compared to the sampling time and site effects, the treatments showed only negligible, short-term effects the composition of microbial communities [Grosch et al. Mycol Res 2006] 3. Example: Multi-species infections?? Oil seed pumpkin 13
Biocontrol of pumpkin diseases High yield losses of the Styrian oilseed pumpkin caused by : Climate (Temperature, Moisture ) and thunder storms hail a complex of pathogens: Didymella bryoniae Fuckel, (1870) (anamorph : Phoma cucurbitacearum) Biocontrol of pumpkin diseases High yield losses of the Styrian oilseed pumpkin Erwinia carotovora Pseudomonas spp. Xanthomonas cucurbitae 14
Biocontrol of pumpkin diseases Isolation and identification of D. bryoniae strains from different fields used for infection studies I. Characterization of isolates Isolation from infected pumpkins Morphological characterization Sequenz analysis/identification II. Establishment of a pathosystem 14 days old plant D. bryoniae on QPDA plate Conidia suspension were injected in the leaf stalk Biocontrol of pumpkin diseases Interaction between D. bryoniae and bacterial pathogens Bacteria use the fungal hyphae as highway 15
Biocontrol of pumpkin diseases Development of a biocontrol strategy Isolation of pumpkin-associated microorganisms (bacteria and fungi) In vitro screening for their antagonistic potential against D. bryoniae and bacteria Ad planta experiments with BCA strains Effective antagonists - biological product and evaluation under field conditions Biocontrol of pumpkin diseases Development of a biocontrol strategy Gleisdorfer Ölkürbis Gleisdorfer Diamant Gleisdorfer Maximal roots 9x10 4 2.2x10 4 6.5x10 4 blossoms 1.1x10 7 1.2x10 7 1.6x10 7 pulps of fruits 2.0x10 4 9.6x10 3 3.1x10 4 Altogether, 2.321 isolates 43 with a broad-spectrum antagonistic potential 6 were chosen for further experiments Greenhouse and field trials 16
Biocontrol of pumpkin diseases Development of a biocontrol strategy Greenhouse experiments + and infection Field experiments Gleisdorf: - infection Stadl-Paura: + and - infection II. New pathogens from the rhizosphere? 17
Biosafety: Pathogens from the rhizosphere? Facultatively human pathogenic bacteria USA: 85.000 patients die per year EU: infection rate on intensive care units: 46% Germany: 1 Million nosocomial infections per year Bacillus spp. Burkholderia cepacia Pseudomonas aeruginosa Serratia marcescens Staphylococcus spp. Stenotrophomonas maltophilia Stenotrophomonas a pathogen from the root? Ecosystem functioning: Stenotrophomonas plays an important ecological role in the nitrogen and sulphur cycle Pathogen defence: biological control of several soil-borne plant pathogens Plant growth promotion: strawberries, Brassicaceae Plant stress protection: salinated conditions Bio- & phytoremediation: xenobiotics, RDX, cocaine... 18
Stenotrophomonas a pathogen from the root? control S. rhizophila DSM14405 T Stenotrophomonas a pathogen from the root? S. maltophilia is also an emerging human pathogen S. maltophilia has been associated with bacteremia and pneumonia infections of immunocompromised patients, these infections have a very high rate of mortality Is it possible to differentiate between clinical and environmental strains? 19
Stenotrophomonas a pathogen from the root? I. Differentiation between S. maltophilia S. rhizophila ARDRA: Restriction of the 16S rrna gene using Pst I S. rhizophila S. maltophilia DSM 14405 DSM 50170 II. Differentiation between S. maltophilia S. rhizophila Key enzyme of osmolyt synthesis (Glucosylglycerol = GG) Only S. rhizophila isolates produce GG II. What turn Stenotrophomonas strains into Multidrug opportunistic efflux pumppathogens? Only S. maltophilia isolates have the efflux pump [Ribbeck-Busch et al, Environ Microb 2005] Biosafety: Pathogens from the rhizosphere? Plant rhizobacteria Pathogenicity Fitness Recognition Adherence Antibiosis/Toxicity Resistance against antibiotics Biofilm formation Competition for nutrients and minerals (siderophores) Production of hydrolytic enzymes Osmotolerance Versatility Facultative pathogens Humans Induced resistance in plants Production of phytohormons Growth at 37 C [Berg et al. Environ Microb 2005] 20
Stenotrophomonas a pathogen from the root? Endophytic colonisation of S. maltophilia Three-dimensional reconstruction of a CLSM-stack based on isosurfaces and dots showing Stenotrophomonas cells colonizing a new coming root hair of tomato (red: bacteria; beige: tomato root). Panel a shows the full dataset, panel b and c show a 3D-crop along the X-axes (orthogonal cut) and the Z-axes (oblique cut), respectively Stenotrophomonas a pathogen from the root? Mutation frequencies Low mutation frequencies were particularly frequent among environmental S. maltophilia strains (58.3%), whereas hypermutators were only found among clinical isolates. [Turrientes et al. AEM 2010] CONCLUSIONS S. rhizophila is a promising Biological control and Stress protecting agent. S. maltophilia should be excluded from direct biotechnological applications. These results indicate that clinical environments might select bacterial populations with high mutation frequencies. 21
CONCLUSIONS, COOPERATIONS AND THANKS Prof. Dr. Kornelia Smalla (BBA Braunschweig) Prof. Dr. Leo Eberl (Uni Zürich) Prof. Rafael Imenez Diaz (Uni Cordoba) Dr. Rita Grosch (IGZ Großbeeren) Prof. Leda Mendoça-Hagler (Uni Rio de Janeiro) Prof. Ben Lugtenberg (Uni Leiden) Dr. Dilfuza Egamberdiyeva (Uni Taschkent) Prof. Martin Grube (Uni Graz) Prof. Erich Leitner (TU Graz) Dr. Arne Peters E-nema GmbH Robert Dahl Erdbeerhof Rövershagen Dr. Ralf Tilcher Plants form a unique habitat in terrestrial ecosystem. KWS Saat AG Manyfold interactions/interplays take place. Dr. Wolfgang Vogt Sourcon-Padena AG The balance of structure and functions is important for plant growth and health. This balance will be influenced by climate change. Plant-associated microorganisms are an important factor influencing the response of plants to climate change. 22