Supplementary Information for
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Bacterial Farming by the Fungus Morchella crassipes
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Martin Pion, Jorge E. Spangenberg, Anaele Simon, Saskia Bindschedler, Coralie
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Flury, Auriel Chatelain, Redouan Bshary, Daniel Job, Pilar Junier
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correspondence to: pilar.junier@unine.ch
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Supplementary Figure S1. Transport of bacteria on fungal mycelia.
For the experiment with a compartmentalized Petri dish, a 2 mm slice of the agar
medium was removed from the plate. The fungus and bacteria were inoculated the
same day in one of the compartments (upper compartment). A. Bacteria can cross an
air-filled gap (only) using a fungal dispersal netwotk. If the bacterial inoculum is
located in one compartment (arrow) of a compartmentalized Petri dish, bacteria can
only cross the gap using a fungal network. Inlet shows the bacterial colonization of
the otherwise inaccessible compartment. Scale bar, 500 μm. B. The non flagellated
mutant was not able to disperse along the mycelium and stayed close to the
inoculation point. Scale bar, 500 µm.
Supplementary Figure S2. Bacterial and fungal-specific fatty acids profile.
Fatty acids profile from a bacterial (A) and a fungal (B) pure culture. The fatty acids
are the myristic (14:0), pentadecanoic (15:0), palmitic (16:0), palmitoleic (16:1),
margaric (17:0), 2-hexyl-cyclopentaoctanoic (cy17:0), stearic (18:0), linoleic (18:2),
and 2-octyl-cycloprapenoctanoic (cy19:0) acids. From the bacterial fatty acid
biomarkers (e.g., 15:0, 16:1, 17:0, cy17:0, cy19:0), the relative more abundant (i.e.,
16:1 and cy17:0) were selected to trace the 13C-enrichment by compound specific
isotope analysis using GC-C-IRMS.
Supplementary Figure S3. Inhibition of sclerotial formation in purified agar.
Inhibition of sclerotial formation in purified agar. The absence of sclerotial formation
in purified agar does not depend on bacteria. Actually, without bacteria, sclerotia are
formed in technical agar (B) but not in purified agar (A). However, the phenotype of
sclerotial formation was recovered when Mn(II) was added to the medium (C),
showing the role of Mn on sclerotia formation. Arrows in (B) and (C) indicate
examples of sclerotia.
Supplementary Figure S4. Effect of glucose and dead bacteria in the differential
formation of sclerotia.
The inoculum was placed in the upper part of the plate (shown by an arrow) and
consisted of a suspension of alive bacterial cells (A), saline solution (B), glucose 1 g
L-1 (C); glucose 2 g L-1 (D), glucose 5 g L-1 (E), glucose 10 g L-1 or a slurry of dead
bacteria prepared by sonication (G).
Supplementary Figure S5. Second generation farming. (A) Sclerotia contain an
important bacterial population. Unwashed sclerotia were inoculated in a new Petri
dish. During sclerotia germination and mycelium growth, bacteria dispersed. (B)
Bacteria were harvested in the centre of the Petri dish as shown by the disappearance
of the fluorescence after 9 days. (C) Fluorescence disappearance coincided with the
formation of new sclerotia in the outer rim of the Petri dish. An arrow indicates the
place of inoculation. By contrast, sclerotia of the same age without bacteria formed
new sclerotia homogeneously when repicked in a new plate (data not shown).
Supplementary Figure S6. M. crassipes farming in other bacterial species.
Bacterial dispersion along fungal hyphae is widespread but fungal farming is not
universal to all migrating bacteria. The two other bacterial strains expressing the GFP,
Cupriavidus necator JMP289 and Pseudomonas knackmusii B13, were kindly
provided by Prof. Jan Van der Meer (University of Lausanne, Switzerland).
Pseudomonas knackmusii B13 migrated on M. crassipes (A) and induced farming (B).
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On the contrary Cupriavidus necator JMP289 migrated (C) but did not induce
farming (D). Scale bar: (A) 100 µm; (C) 500 µm. Arrows indicate the bacterial
inoculum.
Supplementary Table S1. Elemental composition of the purified and technical agar.
The concentrations of C, H and N in purified agar were measured on 2.5 mg of dried
agar using a CHN-analyzer (CHNEA1108 elemental analyzer, Carlo Erba
Instruments, Italy). The concentrations of Al, Mn, Fe, Cu, Zn, As, Cd and Pb were
determined using a quadrupole inductively coupled plasma-mass spectrometry (ICPMS HP 4500, Agilent Technologies, USA). For this analysis 1 g of dried agar was
digested in aqua regia (HCl:HNO3, 3:1, v/v).
Supplementary Table S2. Carbon isotopic composition of bacterial and fungal
biomarkers in experiment and control samples. The d13C values (in ‰ VPDB) of
the two bacterial biomarkers show a significant 13C-enrichment during the breeding
(A-D). A. Unlabeled bacterial inoculum. B. Unlabeled bacteria cultivated 5 days
alone in the malt extract medium. C. Same bacteria in the same medium incubated
with an unlabeled M. esculenta. D. Same bacteria in the same medium incubated with
a labelled M. esculenta. The d13C values of the fungal biomarker show a 13Cenrichment after sclerotia harvesting (E-I). E. Unlabeled fungal inoculum. F.
Unlabeled mycelium incubated alone for 5 days. G. Unlabeled M. esculenta
(mycelium and sclerotia combined) incubated alone for 9 days. H. Unlabeled M.
esculenta when incubated with unlabeled bacteria for 9 days. I. Unlabeled M.
esculenta incubated alone for 9 days with labeled bacteria. Control and experiments
were independently repeated at least three times, except for E and F.
Supplementary Table S3. Mean and standard deviation of cell densities calculated
by colony forming unit (CFU) counting to measure the effect of fungus-driven
dispersal on bacterial fitness. The inocula corresponded to: 5.93x106 (single),
3.03x104 (flagellated; competition), and 4.52x104 (non-flagellated; competition).
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Supplementary Figure 1.
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A
18:1
Relative abundance
16:0
16:1
cy17:0
17:0
15.00
20.00
15:0
18:0
14:0
25.00
cy19:0
30.00
Time (min)
B
35.00
40.00
45.00
35.00
40.00
45.00
Relative abundance
18:2
18:1
16:0
18:0
15.00
20.00
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100
25.00
30.00
Time (min)
Supplementary Figure 2.
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Supplementary Figure 3.
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Supplementary Figure 4.
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Supplementary Figure 5.
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Supplementary Figure 6.
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Supplementary Table 1.
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Purified
Al
mg/kg
25.5
Technical 53.4
Mn
mg/kg
0.38
Fe
mg/kg
17.0
Cu
mg/kg
0.31
Zn
mg/kg
0.55
As
mg/kg
0.10
Cd
mg/kg
0.01
Pb
mg/kg
0.06
C
wt.%
42.288
H
wt.%
7.91
N
wt.%
0.109
5.06
27.9
0.51
10.0
0.29
0.02
0.20
41.405
7.765
0.074
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Supplementary Table 2.
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Experiment
Rearing
Description
Unlabeled bacterial inoculumA
Unlabeled bacteria incubation aloneB
Bacteria incubation 0% labeled fungiC
Bacteria incubation 100% labeled fungiD
Time (d)
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5
5
5
Harvesting
Fungal inoculumE
Fungal mycelium incubation aloneF
Fungi aloneG
Fungal sclerotia 0% labeled bacteriaH
Fungal sclerotia 100% labeled bacteriaI
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5
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Bacteria
d C 16:1
d13C cy17:0
–25.4±0.2
–25.2±0.3
–26.2±0.1
–25.4±0.3
–25.0±2.5
–27.8±1
103±18
183±68
Fungi
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d C 18:2
–32.5
–32.0
–30.8±0.3
–29.9±0.5
–20.4±1.0
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Supplementary Table 3.
Agar
Technical
Purified
Technical
Inoculation
Single
Single
Competition
Competition
Bacteria
Flagellated
Flagellated
Flagellated
Non-flagellated
Bacteria alone
1.77x1091.03x109
8.89x1084.30x108
1.01x1078.44x106
2.02x1072.75x106
Bacteria + fungus
1.52x1093.48x108
8.58x1083.53x108
6.22x1073.99x107
2.43x1071.76x107
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Supplemental Material and Methods
Fitness calculation
Bacterial abundance measured by CFU counting was used to calculate the relative
fitness of the bacterium in the presence and absence of the fungus according to [44]:
where the relative fitness of the bacterium in the presence (x) and absence (y) of the
fungus (w(x,y)) is expressed on terms of the ratio between the initial (x0 and y0) and
final (xF and yF) cell densities.
Reagents and material used for biomarkers extraction
Reagent-grade water (RGW) was prepared by three repeated liquid-liquid extractions
of 500 mL Millipore water with 50 mL dichloromethane and vigorously shaking in a
separation funnel. Dichloromethane, methanol, toluene, and hexane of a quality
suitable for chromatography, monobasic and dibasic potassium phosphates (KH2PO4,
K2HPO4) were purchased from VWR International AG (Switzerland). A potassium
phosphate buffer solution (pH 7.4, 0.05 M) was prepared shortly before use from 1 M
monobasic potassium phosphate and 1 M dibasic potassium phosphate stock
solutions. Fatty acids used for preparation of standards were purchased from Sigma–
Aldrich-Supelco Chemie GmbH (Switzerland). All the organic solvents were glassdistilled shortly before use. The glassware used for sample handling was thoroughly
washed and rinsed with deionized distilled water. The washed glassware and the
Pasteur pipettes and GC vials as obtained from the supplier (VWR International AG,
Switzerland and Supelco, Switzerland, respectively) were heated at 480°C for 4 h
before use.
Isotopic analysis of the fungal and bacterial biomarkers
13
C values (the per mil
deviations relative to the VPDB standard) according to the following formula:
where R is the ratio of the heavy to light isotopes (13C/12C) and the standard (std) is
VPDB. The calibration and assessment of the reproducibility and accuracy of the
isotopic analysis were achieved by injection of a mixture of n-alkanoic acids and
deuterated naphthalene of known isotopic compositions and replicate analyses of the
microbial FAME fractions. The standard deviations for repeatability ranged between
0.05 and 0.4‰ for the main FAME with natural 13C abundance. For 13C-labeled
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C values in the 100 to
1000‰ range.
Presence of bacteria inside sclerotia
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Presence of bacteria inside surface-cleaned sclerotia was also assessed on opened
sclerotia using two approaches: suspension/CFU treatment and scanning electron
microscopy observations.
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