Streptomycetes and their secondary metabolites as drivers of organismic interactions. Mika Tarkka

Transcription

1 Streptomycetes and their secondary metabolites as drivers of organismic interactions Mika Tarkka

2 Bacteria affect plant and fungal growth and health by the production of secondary metabolites the direct responses of plants and fungi to bacteria affect further layers of interactions, e.g. -mushroom parasites -mycorrhiza formation (root-fungus symbiosis) -plant disease development There is a need to work with more complex biological systems consisting of several interacting organisms

3 Streptomycetes Ubiquitous, filamentous gram-positive bacteria Important in soil carbon cycle Producers of diverse secondary metabolites Most aromatic: efficient producers of volatile compounds Protect plants and fungal gardens against parasites, and promote mycorrhiza, plantfungus symbiosis formation

4 Outline Functional characteristics of Streptomyces communities: Mushroom protectors from Harz mountains Inhibition of microorganisms by mycorrhiza-associated streptomycetes Perspectives: On the road to mesocosm The TrophinOak project

5 PART 1 Streptomycetes living on fungi: protecting the mushrooms Fruiting bodies are rich in bacteria Similar bacterial taxa than in the soil PUTATIVE FUNCTIONS Nutrition N, P Promotion of growth and development Mycorrhization helper bacteria Antagonism Protection against pests Xerocomus chrysenteron (Basidiomycota)

6 Mushrooms of Boletales are commonly infected by fungal parasites of the genus Sepedonium The fungal parasite Sepedonium microspermum (teleomorph Hypomyces microspermus) on Xerocomus chrysenteron Our starting hypotheses: 1) The bacteria on the mushrooms protect them from the fungal parasite 2) Streptomycetes play a central role in mushroom protection

7 Isolation of bacteria from Xerocomus badius and Xerocomus chrysenteron PBS-Tween 2 isolation from nonsterile and semisterile (EtOH) surface Four healthy fruiting bodies were collected The fungal parasite was isolated from all fruiting bodies D. Krüger K. Hommel This is not a real mushroom Isolation of 27 bacteria on 6 different culture media

8 Sepedonium growth in co-cultures with bacteria Sepedonium growth (mm radius) 215FB 217FB 221FB 238FB 275FB 278FB 237FB 23FB yellow 95BFB 239FB 11FB 227FB 213FB 219FB 138FB 225FB 1FB 214FB 99FB 154FB 216FB 132FB 155FB 276FB 224FB white 228FB 139FB 131FB 222FB 93FB 224FB yellow 147FB 24FB 91FB 22FB 277BFB 141FB 232FB Control 142FB 156FB 229FB 23FB white 235FB 236FB 144FB 146FB 134FB 143BFB 145FB 223FB 231FB 279FB 133FB 218FB 226FB yellow 226FB white 12FB Co-cultures with Xerocomus associated non-streptomycetes Only subtle influence on the growth of the fungal parasite 35 3 SEPEDONIUM MICROSPERMUM GROWTH IN BACTERIUM FUNGUS CO-CULTURES 25 No difference to free-living control (p >.5, 5 % of bacteria) Inhibition, 45 % of bacteria Stimulation, 5 % of bacteria

9 Xerocomus associated streptomycetes: some strong antagonists of the fungal parasite Bacterial inoculation with none Act153FB Act137FB Act149FB Act14FB a ab b c d Co-culture phenotype None of the nonstreptomycetes belonged to the phenotypes c or d Mycelial growth of Sepedonium microspermum 2B2-DK34 in co-culture with Xerocomus fruitbody associated streptomycetes. Co-culture phenotypes are indicated.

10 Mycelial growth (mm), 8 weeks, YMEA Streptomycetes have only a moderate impact on the growth of the host fungus, Xerocomus 2, a ab 15, b 1, 5,, Bars with different letters are significantly different (p<.5) according to ANOVA and Tukey test. n = 8

11 Intensity The organic extract of Streptomyces 97FB culture inhibits the growth of Sepedonium and suppresses peptaibole production 8 Sepedonium Sepedonium + 97FB extract * * With 97FB extract 4 * * * * No 97FB extract M. Haid Retention time (s) Base peak chromatograms of the metabolite spectra of S. chrysospermum of S. chrysospermum under the influence of the culture extract of Streptomyces 97FB. Asterisks indicate derivatives of antibiotics with peptaibole backbone, identified by mass spectrometry. Peptaiboles are ionophores which participate in mushroom lysis

12 Mushroom protecting streptomycetes Conclusions Strongly antagonistic to the fungal parasite Sepedonium, but not to their host Xerocomus Inhibition of the fungal parasite leads to decreased peptaibole production Perspectives The search for inhibitory compounds (K. Scherlach, C. Hertweck) The analysis of Streptomyces FB15 (T. Wubet, L. Feldhahn, M. Tarkka) and Streptomyces FB97 genomes (K. Scherlach, C. Hertweck)

13 PART 2: Mycorrhiza (plant-fungus symbiosis) associated streptomycetes Questions: Who is there? interactions of a (small) streptomycete community with organisms in their surrounding? In vitro dual cultures Who is active? can secondary metabolite spectrum be connected to observed phenotypes? Substance analysis (HPLC, MS) Tarkka et al. BMC Microbiol 212

14 Streptomycete isolation from forest tree mycorrhizas Spruce Picea abies mycorrhized by Fungus: Piloderma sp. S. Herrmann, UFZ Halle Selective media Morphology 16S rdna sequencing ITS sequencing of mycorrhizal fungus

15 Relative fungal growth (% control) The Streptomyces AcM collection: Variation in responses of mycorrhizal fungi Amanita muscaria Hebeloma cylindrosporum Laccaria bicolor Piloderma spec. no major effect towards host mycorrhizal fungus Piloderma

16 Relative fungal growth (% control) The Streptomyces AcM collection from mycorrhizas: Inhibition of plant pathogenic fungi Fusarium oxysporum Heterobasidion abietinum Heterobasidion annosum Mostly inhibition of pathogenic fungi

17 Relative % ohne Bakterium Relative fungal growth ( % control ) Does the in vitro bioassay inhibition range give us information of the potential novelty of compounds? 125 Fusarium oxysporum Heterobasidion abietinum Heterobasidion annosum 1 75 NO IMPACT 5 25 Streptomyces sp. AcM11 Fungus killer None AcM1 AcM5 AcM8 AcM9 AcM11 AcM12 AcM2 AcM25 AcM29 AcM3 AcM31 AcM33 AcM34 AcM35 AcM37 Bacterium in co-culture AcM streptomycete isolate in co-culture

18 Fungus killer Streptomyces sp. AcM11 produces many reported secondary metabolites mau 2 DAD1 A, Sig=21,1 Ref=off (X:\ACM2\ D) DAD1 B, Sig=23,1 Ref=off (X:\ACM2\ D) DAD1 C, Sig=26,2 Ref=off (X:\ACM2\ D) DAD1 D, Sig=28,2 Ref=off (X:\ACM2\ D) DAD1 E, Sig=31,2 Ref=off (X:\ACM2\ D) DAD1 F, Sig=36,4 Ref=off (X:\ACM2\ D) DAD1 G, Sig=435,4 Ref=off (X:\ACM2\ D) DAD1 H, Sig=5,4 Ref=off (X:\ACM2\ D) 1 Norm *DAD1, (44 mau,apx) of D *Cycloheximide Molecular weigth: CYCLOHEXIMIDE nm FERULIC ACID 1 5 Norm *DAD1, (24.9 mau,apx) of D *Ferulic acid Molecular weigth: Norm 4 3 min *DAD1, (47.8 mau,apx) of D *Acta 293-B1 Molecular weigth: Norm *DAD1, 9.9 (77.2 mau,apx) of D *Actiphenol Molecular weigth: ACTA 293-B1 4 nm ACTIPHENOL nm nm Organic extracts were prepared from bacterial suspension cultures, and the metabolites were identified based on their retention times, UV-Vis-spectra, LC-MS analyses and comaprisons to reference compounds

19 Pilzwachstum in Co-Kultur % ohne Bakterium Relative fungal growth ( % control ) Streptomyces sp. AcM2 has only a subtle inhibitory activity against fungi 125 Fusarium oxysporum Heterobasidion abietinum Heterobasidion annosum 1 75 NO IMPACT 5 25 Impact of Streptomyces sp. AcM2 on pathogenic fungi None AcM1 AcM5 AcM8 AcM9 AcM11 AcM12 AcM2 AcM25 AcM29 AcM3 AcM31 AcM33 AcM34 AcM35 AcM37 Bacterium in co-culture AcM streptomycete isolate in co-culture

20 Only one of the by Streptomyces sp. AcM2 produced compounds is in public databases mau DAD1 A, Sig=21,1 Ref=off (X:\ACM2\ D) DAD1 B, Sig=23,1 Ref=off (X:\ACM2\ D) DAD1 C, Sig=26,2 Ref=off (X:\ACM2\ D) DAD1 D, Sig=28,2 Ref=off (X:\ACM2\ D) DAD1 E, Sig=31,2 Ref=off (X:\ACM2\ D) DAD1 F, Sig=36,4 Ref=off (X:\ACM2\ D) DAD1 G, Sig=435,4 Ref=off (X:\ACM2\ D) DAD1 H, Sig=5,4 Ref=off (X:\ACM2\ D) mau DAD1, (67.4 mau,apx) of D Unknown nm 2 1 OM-Medium min Norm *DAD1, (17.1 mau,apx) of D *Ferulic acid FERULIC ACID Siderophore nm

21 Other by the Streptomyces sp. AcM2 produced metabolites are not in public databases mau DAD1 A, Sig=21,1 Ref=off (X:\ACM4\ D) DAD1 B, Sig=23,1 Ref=off (X:\ACM4\ D) DAD1 C, Sig=26,2 Ref=off (X:\ACM4\ D) DAD1 D, Sig=28,2 Ref=off (X:\ACM4\ D) mau DAD1 E, Sig=31,2 Ref=off (X:\ACM4\ D) DAD1 F, Sig=36,4 Ref=off (X:\ACM4\ D) DAD1 G, Sig=435,4 Ref=off (X:\ACM4\ D) 15 DAD1 H, Sig=5,4 Ref=off (X:\ACM4\ D) DAD1, (1867 mau,apx) of D mau DAD1, (48 mau,apx) of D nm nm DAD1, (166 mau,apx) of D mau mau DAD1, (1658 mau,apx) of D nm min SGG Medium nm

22 Summary: Mycorrhiza associated Streptomyces community: The AcM strains Streptomyces fungus interactions Mycorrhizal fungus Piloderma selects for streptomycete community, which inhibits many other spruce associated fungi The community has only low impact on plant disease resistance Strong antagonists of fungi produce well known antibiotics and siderophores

23 PART 3. MYCORRHIZA HELPER BACTERIUM Mycorrhiza formation with AcH 55 Streptomyces sp. AcH 55 More fine roots More mycorrhizas Faster colonisation of roots Metabolites of AcH 55: -Auxofuran promotes fungal growth -WS-5995 B inhibits fungal and bacterial growth Mycorrhiza, symbiosis between fungi and plants Maier et al.mycol Prog 24 Riedlinger et al. AEM 26 Schrey et al., New Phytol 25 Lehr et al., New Phytol 27 Kurth et al. in preparation

24 TrophinOak: Multitrophic Interactions with Oaks Page 24

25 TrophinOak: Soil substrate based culture system Erysiphe alphitoides Oak powdery mildew (parasite) Pedunculate oak Quercus robur Streptomyces AcH 55 Piloderma croceum (mycorrhizal fungus) Oak P. croceum mycorrhiza Mycorrhizal fungus Piloderma croceum Mycorrhiza Helper Bacterium Streptomyces Microbial soil filtrate * * bacteria and fungi < 1.2 µm

26 TrophinOak Highlights RNASeq analysis of mycorrhiza formation Streptomyces sp. AcH 55 Mycorrhiza helper bacterium RNA-Seq analysis of : -Mycorrhization helper effect -Plant protection against powdery mildew Piloderma croceum Mycorrhizal fungus 2 µm Quantification and visualization of Mycorrhization helper bacterium c 2 µm F. Kurth, Vaclav Kristufek

27 TrophinOak Facilitated by genome projects ORGANISMS GENOMES TRANSCRIPTOMES Host tree Pedunculate oak Quercus robur Stage: Sequencing OakContigDF159 contig assembly Mycorrhiza and root transcriptomes* Mycorrhizal fungus Piloderma croceum Stage: Genome assembled Contig Assembly Mycorrhiza and pure culture transcriptomes Mycorrhization helper bacterium Streptomyces sp. AcH 55 Stage: Genome assembled UFZ *Tarkka et al. New Phytol, accepted

28 Conclusion? Ingredients for successful analysis of secondary metabolism in biotic interactions Culturable organisms in a stable seminatural culture system Methods to evaluate metabolite production in the substrate Secondary metabolite clusters sequenced and organisms preferably amenable to genetic manipulation

29 Acknowledgements Florence Kurth Kerstin Hommel Dirk Krüger Norbert Arnold Silvia Schrey Hans-Peter Fiedler Nadine Horlacher Lasse Feldhahn Tesfaye Wubet Jessica Gutknecht Mark Haid Rüdiger Hampp Tilmann Weber Thomas Neu François Buscot Sylvie Herrmann Sabine Recht Ines Krieg Bärbel Krause TrophinOak Thank you for your attention Page 29