ectomycorrhizal mushrooms xerocomus, paxillus, tuber–

Ectomycorrhizal mushrooms Xerocomus, Paxillus, Tuber –

aspects of multipartite interactions

between soil yeasts, bacteria and filamentous fungi

MSA, Fairbanks, AK, August 2011

Dirk Krüger 1*, Aleksandar Pavić 2, Andrey Yurkov 3, Kerstin Hommel 1,Dominik Begerow 3, Norbert Arnold 4, Žaklina Marjanović 2, and Mika Tarkka 1

1 UFZ-Helmholtz-Centre for Environmental Research, Department of Soil Ecology, Theodor-Lieser-Str. 4, D-06120 Halle (Saale), Germany2 Institute for Multidisciplinary Research, Department for Life Sciences, Kneza Viseslava 1a, 11 000 Belgrade, Serbia3 Geobotany Section, Ruhr-Universität Bochum, Universitätsst. 150, D-44801 Bochum, Germany4 IPB-Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3, D-06120 Halle (Saale), Germany

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Case 1 – Actinobacteria in Truffles

Truffle Market Alba, Italy (from internet)

- unsuccessful in crop domestication- Italy ( e.g. Alba, Piedmont),

Croatia, Slovenia, Serbia, Hungary- EM partners:

Quercus, Corylus, Populus- in Serbia in deep mineral soils with CaCO3

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ISOLATION OF BACTERIA

100 μl aliqots of PBS-Tween 20 applied to gleba, pipetted off,

plated on YMEA with 50mg/l cycloheximide and 40 mg/l

Nystatin, incubated 48h at 25˚C.


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4

RESULTS

46 isolates grew on mannitol - CaCO3 agar

2 isolates with actinomycete morphology selected for experiments


Rhodococcus strain TMG025 II

Curtobacterium strain TMG034

Partial 16S rDNA phylogeny

RESULTSRhodococcus TMG025 II, Curtobacterium TMG 034 metabolism

- produce siderophores

- solubilize tricalcium-orthophosphate in NBRIP medium

- truffle storage sugar mannitol stimulates tricalcium-orthophosphate solubilization


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Rhodococcus TMG025 II Curtobacterium TMG 034

RESULTSRhodococcus TMG025 II, Curtobacterium TMG 034 metabolism

- both strains degrade trehalose and beta-glucan

- Curtobacterium shows protease, chitinase and pectinase activites as well

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Isolate Urease

Gelatinase

Caseinase

Tween80

Lipa

sea

Lechitina

se

Treh

alase

β‐glucan

ase

Chitina

se

Pectinase

CMC‐cellulase

Side

roph

ore

TMG025 II + ‐ ‐ ‐ ‐ ‐ + + ‐ ‐ ‐ +

TMG034 ‐ ‐ + + ‐ ‐ + + + + ‐ +

Rhodococcus TMG025 II

Curtobacterium TMG 034


CONCLUDING

Bacteria, e.g. Actinobacteria (other data not shown here),

may foster nutrient uptake for mushrooms.

Bacteria may play a role in defending truffles against some, but may foster

the growth of other mushroom parasites (not shown here).

Bacteria may decay the fungus and be involved in hypogeous spore release.

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- EM fungus Paxillus- from UFZ grounds (Halle, Germany) under Tilia- by sequence Paxillus obscurisporus C. Hahn

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Case 2 – Yeasts and Sepedonium on Paxillus

PBS-Tween 20 isolation from inside

mushroom

PBS-Tween 20 isolation from non-

sterile and semisterile (EtOH) surface

This is not a real mushroom.

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ISOLATION OF BACTERIA AND YEASTS

RESULTSisolated yeasts:

basidiomycetes

ascomycete

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Strain Identification results Collection number

YFB213* Kluyveromyces dobzhanskii CBS 11758YFB238* Trichosporon porosum n.a.YFB251 Mastigobasidium intermedium n.a.YFB252 Rhodotorula graminis n.a.YFB253 Holtermanniella festucosa n.a.YFB254 Rhodosporidium babjevae n.a.YFB255 Holtermanniella festucosa n.a.YFB256 Leucosporidiella muscorum n.a.YFB257 Mastigobasidium intermedium CBS 11759YFB258 Cystofilobasidium macerans n.a.YFB259 Mastigobasidium intermedium n.a.YFB260 Rhodotorula graminis n.a.YFB261 Rhodotorula graminis n.a.YFB262 Holtermanniella festucosa CBS 11757YFB263 Mastigobasidium intermedium n.a.YFB269 Cryptococcus victoriae n.a.YFB270 Rhodotorula graminis CBS 11678

note: yeasts in (decomposing) mushrooms known since 1940s (Anderson & Skinner 1947 in Mycologia)

*: isolated from Xerocomus (Boletales)

RESULTSbipartite cultures, tripartite cultures

controls

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LOW NUTRIENT LEVEL MEDIUM (MNM)

Yeast Fungal growth (mm)YFB255 28,0 +/‐5,04 a (+)YFB259 25,6 +/‐3,50 a (+)YFB253 22,8 +/‐5,78 abNone 22 +/‐ 2,02 bYFB258 17,8 +/‐ 2,25 c (‐)YFB213* 14,5 +/‐ 2,73 cd (‐)YFB261 12,8 +/‐ 5,04 cd (‐)YFB263 12,4 +/‐ 3,16 d (‐)YFB269 12,1 +/‐ 2,17 d (‐)YFB256 11,6 +/‐ 0,92 d (‐)YFB254 11,5 +/‐0,93 d (‐)YFB252 11 +/‐ 2,27 d (‐)YFB270 11 +/‐1,07 d (‐)YFB251 10,6 +/‐ 0,74 d (‐)YFB257 10,6 +/‐0,74 d (‐)YFB260 10,4 +/‐1,51 d (‐)YFB262 10,1 +/‐0,64 d (‐)YFB238* 8,75 +/‐ 0,71 d (‐)


Only 2 yeast strains promote the growth:(In MEA 6 yeast strains).

As many as 14 yeast strains inhibit the growth:(In MEA 1 yeast strain).

Inhibition of Sepedonium is related to nutrient competition.

CONCLUDING

Yeasts are present in still fresh, not decomposing fruiting bodies.

Rhodotorula graminis : first report from Europe.

Note: Sepedonium infections cause early lysis of basidia, affecting fitness of host.

Yeasts thus can modify this effect.

Yeasts differ in their abilities to stimulate or inhibit Sepedonium.

Paxillus may be negatively impacted by common presence of Sepedonium and yeasts.

Yeasts rather support the growth of molds and increase the negative effects on

Paxillus. Many yeasts are actually from groups known to also possess adaptations

to mycoparasitism (e.g. haustoria in Holtermaniella).

Nutrient poor media rather imply that yeasts mainly have nutrient competition effect

on Sepedonium and Paxillus. In tripartite assays, antibiotics appeared

stronger component in interaction. 13


Case 3 – Bacteria and Sepedonium on Xerocomus

- EM fungus Xerocomus chrysenteron andothers.- from Harz and Kyffhäuser Mts. in Germany

Xerocomus chrysenteronwith Hypomyces

microspermus (Anamorph Sepedonium

microspermum)

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PBS-Tween 20 isolation from inside

mushroom

PBS-Tween 20 isolation from non-

sterile and semisterile (EtOH) surface

This is not a real mushroom.

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MATERIALS AND METHODSCase 3 – Bacteria and Sepedonium on Xerocomus

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RESULTSCase 3 – Bacteria and Sepedonium on Xerocomus

Genbank Fungus accessiona

Xerocomus chrysenteron and X. badius fruiting bodies used for isolations

X. chrysenteron 2B1 Harzgerode HQ207691



X. chrysenteron 6A Kyffhäuser HQ207694X. chrysenteron 6B Kyffhäuser HQ207695X. badius 6C Kyffhäuser HQ207696

X. badius 8 Wernigerode HQ207697

X. badius 9A Wernigerode HQ207698

Genbank Fungus accessiona

Sepedonium microspermum isolates and their source fruiting bodies

Strain DK32 from X. chrysenteron 2B1 Harzgerode HQ207690



Strain DK38 from X. chrysenteron 6A Kyffhäuser HQ207687A Accession numbers of the ribosomal DNA sequences.

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RESULTS

Genbank Fruiting interaction

Strain Identitya accession bodyb Tissuec phenotyped

92FB Pseudomonas AB566042 Xerocomus chrysenteron 2B2 o p

93FB Pseudomonas AB566043 Xerocomus chrysenteron 2B2 o p

94FB Mycetocola AB566044 Xerocomus chrysenteron 2B2 i p

95FB Pseudomonas AB566045 Xerocomus chrysenteron 2B2 o v

96FB Mycobacterium AB566046 Xerocomus chrysenteron 2B2 o p

97FB Streptomyces AB566047 Xerocomus chrysenteron 2B1 os y


99FB Enterobacteriaceae AB566049 Xerocomus chrysenteron 2B1 os v

100FB Enterobacteriaceae AB566050 Xerocomus chrysenteron 2B1 os v

101FB Streptomyces AB566051 Xerocomus chrysenteron 2B1 os v

102FB Streptomyces AB566052 Xerocomus chrysenteron 2B1 os p

104FB Streptomyces AB566053 Xerocomus chrysenteron 2B1 os z

106FB Streptomyces AB566054 Xerocomus chrysenteron 2B1 os n.a.

134FB Burkholderia AB566055 Xerocomus chrysenteron 2B4 os n.a.




138FB Pseudomonas AB566059 Xerocomus chrysenteron 2B4 os n.a.

139FB Bacillus AB566060 Xerocomus chrysenteron 2B4 os n.a.

140FB Enterobacteriaceae AB566061 Xerocomus chrysenteron 2B4 os n.a.

141FB Micrococcus AB566062 Xerocomus chrysenteron 2B4 os n.a.

142FB Burkholderia AB566063 Xerocomus chrysenteron 2B4 os n.a.

143FB Enterobacteriaceae AB566064 Xerocomus chrysenteron 2B4 i n.a.

144FB Enterobacteriaceae AB566065 Xerocomus chrysenteron 2B4 i n.a.

145FB Micrococcus AB566066 Xerocomus chrysenteron 2B4 os n.a.

147FB Caulobacter AB566067 Xerocomus chrysenteron 2B4 os n.a.


149FB Streptomyces AB566069 Xerocomus chrysenteron 2B4 os x

150FB Streptomyces AB566070 Xerocomus chrysenteron 2B4 os x




154FB Phyllobacterium AB566074 Xerocomus chrysenteron 2B4 os v

155FB Burkholderia AB566075 Xerocomus chrysenteron 2B4 os v

156FB Pseudomonas AB566076 Xerocomus chrysenteron 2B4 os n.a.

214FB Enterobacteriaceae AB566077 Xerocomus chrysenteron 6A o n.a.215FB Pseudomonas AB566078 Xerocomus chrysenteron 6A o v217FB Pseudomonas AB566079 Xerocomus chrysenteron 6A o w218FB Plantibacter AB566080 Xerocomus chrysenteron 6A o n.a.219FB Burkholderia AB566081 Xerocomus chrysenteron 6B os v224FB Sphingomonas AB566082 Xerocomus chrysenteron 6B o n.a.226FB Micrococcineae AB566083 Xerocomus chrysenteron 6B o n.a.227FB Curtobacterium AB566084 Xerocomus chrysenteron 6B o v

228FB Burkholderia AB566085 Xerocomus badius 6C o n.a.

229FB Pedobacter AB566086 Xerocomus badius 6C o n.a.230FB Pseudomonas AB566087 Xerocomus badius 6C o v231FB Variovorax AB566088 Xerocomus badius 6C o n.a.232FB Erwinia AB566089 Xerocomus badius 6C o n.a.233FB Streptomyces AB566090 Xerocomus badius 6C os n.a.234FB Streptomyces AB566091 Xerocomus badius 6C os x236FB Pedobacter AB566092 Xerocomus badius 6C o n.a.237FB Sphingomonas AB566093 Xerocomus badius 6C o w240FB Stenotrophomonas AB566094 Xerocomus badius 6C o n.a.241FB Streptomyces AB566095 Xerocomus badius 6C os n.a.275FB Burkholderia AB566096 Xerocomus badius 8H o w276FB Burkholderia AB566097 Xerocomus badius 8H o n.a.277FB Burkholderia AB566098 Xerocomus badius 8H o n.a.278FB Ewingella AB566099 Xerocomus badius 9A o vaBased on partial 16S rNA sequencebXerocomus chrysenteron 2B1, 2B2, 2B4, 6A, 6B, and X. badius 6C, 8H, 9Aco, outer surface; os, outer surface pre‐sterilized; i, from inside the tissue

dp, positive, n.a., not affected; v‐z, increasingly inhibitive

43 strains from Xerocomus chrysenteron,15 strains from Xerocomus badius

Gram positive taxa the collection contained dominantly Actinobacteria(Streptomycetaceae, Mycobacteriaceae, Micrococcaceae, Microbacteriaceae), and Bacillaceae.

Streptomyces highlighted : 16 from X. chrysenteron, 3 from X. badius.


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Most bacteria have negative impact on Sepedonium (triangle = control).

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0,0

5,0

10,0

15,0

20,0

25,0

132F

B15

3FB

151F

BNon

e13

7FB

152F

B24

1FB

149F

B10

6FB

98FB

150F

B10

5FB

148F

B13

6FB

97FB

104F

B23

4FB

135F

B

Xero

com

us b

adiu

s gr

owth

(mm

)

** ** ** ** ** ** **

Influence of streptomycetes on the growth of Xerocomus badius:Growth of X. badius in co‐culture with 17 mushroom associated streptomycete isolates was measured 8 weeks after fungal inoculation. The mean of each experiment with 8 replicates is indicated. To the control treatment without bacteria (“none”) significantly different values according to student’s T‐test (p < 0.01) are indicated by two asterisks.

0

2

4

6

8

10

Chi

tinas

e ac

tivity

102FB135FB

150FB149FB

234FB98FB

104FB97FB

a aa

ab b

bcbc

c

Chitin degradation by the Sepedonium microspermuminhibiting streptomycetes.

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Base peak chromatograms of the metabolite spectra of S. microspermum (dotted line) and of S. microspermum under the influence of the culture extract of Streptomyces. Asterisks indicate derivatives of antibiotics with peptaibole backbone, identified by mass spectrometry.

CONCLUDING

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Xerocomus surfaces bind a diverse community of bacteria, and the

taxonomic groups of bacteria are distinct in their ability to inhibit the growth of the

mycoparasite Sepedonium microspermum. From our limited collection,

the streptomycetes were among the most antagonistic against S. microspermum.

Streptomyces present on fruiting bodies are not only active in inhibiting

the growth of Sepedonium, but are less inhibitory to their host mushroom

than to the parasite.

Currently the volatile and soluble secondary metobolites produced by the players

are beiing investigated. A strain of Streptomyces has also been sequenced

(which one?).


ALL CONCLUDING

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ACKNOWLEDGEMENTS

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Mycelial growth of Sepedonium microspermum 2B2-DK34 in co-culture with Xerocomus fruitbody associated actinomycetes (all were Streptomyces). Significantly different co-culture phenotypes are indicated.

a a b dc

Co-culture phenotype

none Act153FB Act137FB Act104FBAct149FB

Bacterial inoculation with

from a previous poster

UPGMA on pairwisedistances of inhibition

correlates well with 16S phylogeny (ME)

Next?Any specificity of bacterial assemblages in Boletales?

RESULTSInfluence of fruiting body associated yeasts on the growth of the mycoparasite Sepedonium chrysospermum: impact of low nutrient levels

(MMN 1/10) and cross-wall separation between the microorganisms. Significant differences according to ANOVA and Tukey test ( p <0.05).

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LOW NUTRIENT LEVEL MEDIUM

Yeast strain Fungal growth (mm)YFB255 28,0 +/‐5,04 a (+)YFB259 25,6 +/‐3,50 a (+)YFB253 22,8 +/‐5,78 abNone 22 +/‐ 2,02 bYFB258 17,8 +/‐ 2,25 c (‐)YFB213 14,5 +/‐ 2,73 cd (‐)YFB261 12,8 +/‐ 5,04 cd (‐)YFB263 12,4 +/‐ 3,16 d (‐)YFB269 12,1 +/‐ 2,17 d (‐)YFB256 11,6 +/‐ 0,92 d (‐)YFB254 11,5 +/‐0,93 d (‐)YFB252 11 +/‐ 2,27 d (‐)YFB270 11 +/‐1,07 d (‐)YFB251 10,6 +/‐ 0,74 d (‐)YFB257 10,6 +/‐0,74 d (‐)YFB260 10,4 +/‐1,51 d (‐)YFB262 10,1 +/‐0,64 d (‐)YFB238 8,75 +/‐ 0,71 d (‐)

YEAST VOLATILES

Yeast strain Fungal growth (mm)YFB255 30,9 +/‐ 4,05 aNone 29,7 +/‐ 4,52 aYFB253 29,4 +/‐ 0,92 aYFB213 29,0 +/‐ 3,34 aYFB259 28,3 +/‐ 2,25 aYFB251 27,1 +/‐ 8,2 abYFB238 26,8 +/‐ 1,28 aYFB260 22,6 +/‐ 6,16 abYFB258 19,9 +/‐ 2,30 b (‐)YFB256 19,2 +/‐ 5,61 b (‐)YFB270 19,1 +/‐ 8,01 b (‐)YFB252 16,5 +/‐ 2,83 b (‐)YFB254 15,5 +/‐ 9,76 bc (‐)YFB263 15,0 +/‐ 6,36 b (‐)YFB262 14,4 +/‐ 8,12 bc (‐)YFB261 12,4 +/‐ 9,15 bc (‐)YFB269 11,9 +/‐ 4,36 bc (‐)YFB257 8,00 +/‐ 0,76 c (‐)

RESULTSbipartite cultures, tripartite cultures

controls

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RESULTSRhodococcus TMG025 II, Curtobacterium TMG 034 interaction with molds

Case 1 – Streptomycetes in Truffles

Verticillium sp.(color producer)

TMG016 II(Pseudomonas

fluorescens)

red lesion of Tuber magnatum

fruiting body

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Dirk, this hasNothing to do with the actinos

RESULTSRhodococcus TMG025 II, Curtobacterium TMG 034 interaction with molds

Case 1 – Streptomycetes in Truffles

red lesion of Tuber magnatum

fruiting body

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ectomycorrhizal mushrooms xerocomus, paxillus, tuber–

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