Black truffle-associated bacterial communities during the development and maturation of Tuber melanosporum ascocarps and putative functional roles
Sanjay Antony-Babu
INRA, Interactions Arbres – Microorganismes, UMR1136, F-54280 Champenoux, France
Interactions Arbres – Microorganismes, Université de Lorraine, UMR1136, F-54500 Vandoeuvre-lès-Nancy, France
Sanjay Antony-Babu and Aurélie Deveau contributed equally to this work.Search for more papers by this authorCorresponding Author
Aurélie Deveau
INRA, Interactions Arbres – Microorganismes, UMR1136, F-54280 Champenoux, France
Interactions Arbres – Microorganismes, Université de Lorraine, UMR1136, F-54500 Vandoeuvre-lès-Nancy, France
Sanjay Antony-Babu and Aurélie Deveau contributed equally to this work.For correspondence. E-mail [email protected]; Tel. (+33) 038339 4088; Fax (+33) 038339 4069.Search for more papers by this authorJoy D. Van Nostrand
Institute for Environmental Genomics, Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73072 USA
Search for more papers by this authorJizhong Zhou
Institute for Environmental Genomics, Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73072 USA
Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720 USA
State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing, 100084 China
Search for more papers by this authorFrançois Le Tacon
INRA, Interactions Arbres – Microorganismes, UMR1136, F-54280 Champenoux, France
Interactions Arbres – Microorganismes, Université de Lorraine, UMR1136, F-54500 Vandoeuvre-lès-Nancy, France
Search for more papers by this authorChristophe Robin
Agronomie & Environnement, Université de Lorraine, Nancy-Colmar, UMR 1121, F-54500 Vandoeuvre-lès-Nancy, France
INRA, Agronomie & Environnement, Centre INRA de Nancy-Lorraine, Nancy-Colmar, UMR 1121, F-54500 Vandoeuvre-lès-Nancy, France
Search for more papers by this authorPascale Frey-Klett
INRA, Interactions Arbres – Microorganismes, UMR1136, F-54280 Champenoux, France
Interactions Arbres – Microorganismes, Université de Lorraine, UMR1136, F-54500 Vandoeuvre-lès-Nancy, France
Search for more papers by this authorStéphane Uroz
INRA, Interactions Arbres – Microorganismes, UMR1136, F-54280 Champenoux, France
Interactions Arbres – Microorganismes, Université de Lorraine, UMR1136, F-54500 Vandoeuvre-lès-Nancy, France
Search for more papers by this authorSanjay Antony-Babu
INRA, Interactions Arbres – Microorganismes, UMR1136, F-54280 Champenoux, France
Interactions Arbres – Microorganismes, Université de Lorraine, UMR1136, F-54500 Vandoeuvre-lès-Nancy, France
Sanjay Antony-Babu and Aurélie Deveau contributed equally to this work.Search for more papers by this authorCorresponding Author
Aurélie Deveau
INRA, Interactions Arbres – Microorganismes, UMR1136, F-54280 Champenoux, France
Interactions Arbres – Microorganismes, Université de Lorraine, UMR1136, F-54500 Vandoeuvre-lès-Nancy, France
Sanjay Antony-Babu and Aurélie Deveau contributed equally to this work.For correspondence. E-mail [email protected]; Tel. (+33) 038339 4088; Fax (+33) 038339 4069.Search for more papers by this authorJoy D. Van Nostrand
Institute for Environmental Genomics, Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73072 USA
Search for more papers by this authorJizhong Zhou
Institute for Environmental Genomics, Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73072 USA
Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720 USA
State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing, 100084 China
Search for more papers by this authorFrançois Le Tacon
INRA, Interactions Arbres – Microorganismes, UMR1136, F-54280 Champenoux, France
Interactions Arbres – Microorganismes, Université de Lorraine, UMR1136, F-54500 Vandoeuvre-lès-Nancy, France
Search for more papers by this authorChristophe Robin
Agronomie & Environnement, Université de Lorraine, Nancy-Colmar, UMR 1121, F-54500 Vandoeuvre-lès-Nancy, France
INRA, Agronomie & Environnement, Centre INRA de Nancy-Lorraine, Nancy-Colmar, UMR 1121, F-54500 Vandoeuvre-lès-Nancy, France
Search for more papers by this authorPascale Frey-Klett
INRA, Interactions Arbres – Microorganismes, UMR1136, F-54280 Champenoux, France
Interactions Arbres – Microorganismes, Université de Lorraine, UMR1136, F-54500 Vandoeuvre-lès-Nancy, France
Search for more papers by this authorStéphane Uroz
INRA, Interactions Arbres – Microorganismes, UMR1136, F-54280 Champenoux, France
Interactions Arbres – Microorganismes, Université de Lorraine, UMR1136, F-54500 Vandoeuvre-lès-Nancy, France
Search for more papers by this authorSummary
Although truffles are cultivated since decades, their life cycle and the conditions stimulating ascocarp formation still remain mysterious. A role for bacteria in the development of several truffle species has been suggested but few is known regarding the natural bacterial communities of Périgord Black truffle. Thus, the aim of this study was to decipher the structure and the functional potential of the bacterial communities associated to the Black truffle in the course of its life cycle and along truffle maturation. A polyphasic approach combining 454-pyrosequencing of 16S rRNA gene, TTGE, in situ hybridization and functional GeoChip 3.0 revealed that Black truffle ascocarps provide a habitat to complex bacterial communities that are clearly differentiated from those of the surrounding soil and the ectomycorrhizosphere. The composition of these communities is dynamic and evolves during the maturation of the ascocarps with an enrichment of specific taxa and a differentiation of the gleba and peridium-associated bacterial communities. Genes related to nitrogen and sulphur cycling were enriched in the ascocarps. Together, these data paint a new picture of the interactions existing between truffle and bacteria and of the potential role of these bacteria in truffle maturation.
Supporting Information
| Filename | Description |
|---|---|
| emi12294-sup-0001-fs1.tif7.3 MB | Fig. S1. Multivariate analysis of bacterial communities associated to Tuber melanosporum studied with TGGE. A. Bacterial communities associated to T. melanosporum samples collected in November 2010. The relative distribution of the major phyla was determined on ascocarpic, ectomycorrhizae and soil samples. Principal component axes 1 and 2 explain most of the variance in the data cumulatively (F1 = 21.1% and F2 = 19.2%). B. Bacterial communities associated to the ascocarp along maturation. The relative distribution of the major phyla was determined on ascocarps (gleba and peridium), ascocarpic adhering soil and bulk soil samples collected from October 2010 to January 2011. Principal component axes 1 and 2 explain most of the variance in the data cumulatively (F1 = 22.6% and F2 = 12.9%). |
| emi12294-sup-0002-fs2.tif507 KB | Fig. S2. In situ hybridization of sections from mature truffle collected in January 2011 with group-specific 16S rRNA oligonucleotide probes. A. Partial colonization of the peridium by Bacteroidetes. Sample was hybridized with the universal eubacteria Eub338mix-FITC probe (green) and the Bacteroidetes-specific probe CF319-cy3 (red). White arrow points at bacteria from Bacteroidetes. B. Colonization of the peridium by Beta-proteobacteria. Sample was hybridized with the universal eubacteria Eub338mix-FITC probe (green) and the Beta-proteobacteria-specific probe BET42a-cy3 (red). White arrow points at bacteria from Beta-proteobacteria. C. Bacteria are present around ascii but not on spores. Sample was hybridized with the universal eubacteria Eub338mix-FITC probe (green) and the alpha-proteobacteria-specific probe ALF1a-cy3 (red) and the fungal cell walls were stained with calcofluor white. The white arrow points at bacterial cells. As: ascospore. D. Massive colonization of gleba by alpha-proteobacteria. Sample was hybridized the alpha-proteobacteria-specific probe ALF1a-cy3 (red) and the fungal cell walls were stained with calcofluor white. White arrow point at interspaces between hyphae heavily colonized with bacteria. E. Dispersed colonization of gleba by beta-proteobacteria. Sample collected in January 2011 was hybridized with the Beta-proteobacteria-specific probe BET42a-cy3 (red) and the fungal cell walls were stained with calcofluor white. White arrow points at single bacterial cells in between fungal cells. |
| emi12294-sup-0003-fs3.tiff1.5 MB | Fig. S3. Functional assignment of probes hybridized by DNA from Bradyrhizobium, Bacteroidetes and Actinobacteria colonizing mature truffle peridium according to GeoChip analysis. Data are expressed as the percentage of probes providing a significant signal for the two biological replicates in each functional category. |
| emi12294-sup-0004-fs4.tif7.2 MB | Fig. S4. Truffle life cycle and sample description. A. Life cycle of Tuber melanosporum and the stages considered in sampling. Main stages are denoted by smaller case alphabets. a: Spore germinates, b: mycelium spreads, c: truffle forms, d: truffle grows, e: truffle maturation starts, f: ripening process starts, g: end of truffle maturation and h: truffle disintegrates and spore disperses. Sampling period for the ectomycorrhizal-associated and ascocarp-associated samples are presented by red and green asterisk, respectively. B. Representation of aerial view of the tree. The numbers denote the sampling sites for ectomycorrhizae at November time point. C. Ectomycorrhizal root tip with soil adhering to it (ectomycorrhizosphere soil), D. Surface of truffle with white external mycelium. E, F. Cross-section of a truffle (E: early stage, F: full mature). |
| emi12294-sup-0005-ts1.docx18.2 KB | Table S1. Comparison of the bacterial communities colonizing the truffle habitats at the phylum, class, order and genus levels in November 2010. The relative distribution of the sequences in the different taxonomic levels considered was analyzed with a one-factor ANOVA (and a Fisher Test, p < 0.05). Sample types with similar letters are not significantly different. The symbols ‘>’ , ‘=’ and ‘≥’ mean ‘significantly more abundant’, ‘not significantly different’ and ‘not significantly different from the preceding sample type but significantly more abundant than the first preceding sample type with a single letter’. EcA : ectomycorrhizae, AEcS : adherent soil to ectomycorrhizae, BS : bulk soil surrounding the ectomycorrhizae, G : gleba, P : peridium, AS : ascocarpic soil, ASBS : bulk soil surrounding the ascocarps. Grey shading highlights significantly enriched communities in the ascocarp compared to the soil and/or the ectomycorrhizae. |
| emi12294-sup-0006-ts2.doc42 KB | Table S2. Sample description (A) and description of the ascocarp maturation stages (B). |
| emi12294-sup-0007-ts3.xlsx128 KB | Table S3. Lists of phyla, classes, orders, families and genera, which are significantly differently represented in the bulk soil and ascocarp according to a one-way ANOVA (sample, p < 0.05) in samples collected from October 2010 to January 2011. D3: October, D4: November, D5: December, D6: January. G: gleba, P: peridium, AS: ascocarpic soil, ASBS : bulk soil surrounding the ascocarps. |
| emi12294-sup-0008-ts4.doc32.5 KB | Table S4. Colonization of mature truffle collected in january as observed by FISH. Scoring was performed on 25μm sections of three independant ascocarps. Each section was hybridized with universal eubacterial probe eub338-FITC and a phylum specific probe coupled to cy3 (alpha-proteobacteria: ALF968 and ALF1, beta-proteobacteria: BET42a, Bacteroidetes: CF319, Firmicutes: LGC354). For each specific probe, at least three different sections were observed on their entirety. A score was given for each probe as following: 4: massive colonization, more than 70% of cells hybridizing with eub338 universal eubacterial probe also hybridize with the specific probe; 3: dense colonization (20–50%); 2: low density (5–20%); 1: few independent cells (<5%); 0: no cells hybridized with the specific probe. |
| emi12294-sup-0009-ts5.xlsx482.3 KB | Table S5. List of probes with at least a 5-fold increase in probe intensity in the peridium compartment compared to the ascocarpic soil. Each value is the mean value of two replicates. |
| emi12294-sup-0010-ts6.doc48.5 KB | Table S6. Oligonucleotide FISH probes used in this study. |
| emi12294-sup-0011-ts7.xls41.5 KB | Table S7. Band pattern of TTGE gels. Each gel was imaged with GelDoc transilluminator and bands were detected using the QuantityOne software from BioRad. A total of 35 bands were identified in the different samples. ‘1'and ‘0’ values correspond to presence or absence of the corresponding band, respectively. EcA: ectomycorrhizae, AEcS: mycorrhizospheric-soil, BS: bulk soil associated to the ectomycorrhizae, G: gleba, P: peridium, AS: ascocarpic adhering soil, ASBS: bulk soil associated to the ascocarp. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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