Ph.D Course in
Biology and Biotechnology of Fungi

XIX Cycle

University of Torino
Department of Plant Biology

Truffles Volatiles and Exudates

Investigation of their Role in Truffle/Plant
Interactions

Candidate: Richard Splivallo
Supervisor: Prof. Paola Bonfante

Academic period:
2003-2004
2004-2005
2005-2006

Ph.D. cycle coordinator: Prof. Paola Bonfante

The Ph.D. project has been funded by the "Fondation pour des bourses d'études Italo-Suisses" (2 years scholarship to RS), the Italian Ministry of Foreign Affairs (8 months to RS). Research was funded by CEBIOVEM and IPP-CNR

Table of content

I. Introduction ................................................................................................9
1.1 What are truffles? ........................................................................................9
1.2 The reasons to study truffles.........................................................................10
1.3 History: truffles in civilizations.......................................................................10
1.4 History: the nature of truffles in philosophy/science .......................................12
1.5 Description and ecological importance of ectomycorrhizas ...........................12
1.6 Biology and distribution of truffles ................................................................15
1.7 Truffle taxonomy: from the past to the modern times......................................17
1.8 Economical importance of truffles.................................................................19
1.9 Truffle aroma ...............................................................................................20
References.......................................................................................................21

II. Aim of the Work..........................................................................................25
Reference.........................................................................................................26

III. Present state of knowledge. Biological significance of truffle
secondary metabolites ....................................................................................30
1 Introduction ..................................................................................................30
2 Truffles: life cycle and distribution .............................................................31
3 Field observations and open questions ......................................................35
4 An overview of truffle metabolites .............................................................36
4.1 VOCs from fruitbody................................................................................... 36
4.2 Fruitbody non-VOCs................................................................................... 40
4.3 Mycelial VOCs ........................................................................................... 40
5 Metabolites involved in truffle and host plant interaction .........................41
6 Interaction with non-host plants ..................................................................42
6.1 Phytotoxic metabolites in soil from truffle fields? ........................................... 44
6.2 Phytotoxic metabolites from the fruitbody .................................................... 46
6.3 Phytotoxic metabolites from the mycelium..................................................... 48
6.4 Possible contribution to the burnt by other organisms ................................... 48
6.5 Ecological significance of truffle metabolites.................................................. 50
7 Conclusion ....................................................................................................51
References...................................................................................................... 51

IV. Discrimination of truffle fruiting body versus mycelial aromas by
stir bar sorptive extraction ........................................................................... 60
1 The metabolic pathways of major truffle volatiles..................................... 60
1.1 Pathway to 1-octen-3-ol, a major fungal volatile.......................................... 61
1.2 Pathway to benzaldehyde and related aromatic compounds.......................... 62
1.Introduction ................................................................................................. 65
2.Results and discussion ................................................................................ 66
2.1 Search for undescribed VOCs.................................................................. 66
2.2 VOCs profile variation and species markers .......................................... 79
2.3 Mycelium versus fruiting body VOCs...................................................... 85
3.Conclusions.................................................................................................. 86
4.Experimental................................................................................................ 87
4.1 Tuber fruiting bodies identification and sample preparation ................... 87
4.2 Fruiting bodies extraction method ........................................................... 87
4.3 Tuber mycelium and culture conditions .................................................... 88
4.4 Mycelial VOCs extraction methods .......................................................... 88
4.6 Authentic standards and calibration ........................................................ 88
4.7 Statistical analyses ................................................................................... 89
References...................................................................................................... 89

V. Truffle volatiles inhibit growth and induce an oxidative burst in
Arabidopsis thaliana. .................................................................................... 94
Introduction .................................................................................................... 95
Results ............................................................................................................ 96
Effect of the natural aromas of truffles on A. thalian.................................. 96
Effect of synthetic VOCs on A. thalian......................................................... 96
H2O2 and ROS-scavenging enzymes ............................................................. 100
Discussion ...................................................................................................... 104
Phytotoxic effect: natural aromas and synthetic VOCs ................................ 104
Plant responses: H2O2 and ROS-scavenging enzymes................................... 105
Conclusion...................................................................................................... 106
Material and methods ................................................................................... 106
References...................................................................................................... 109

VI. Early Signaling between Truffles and Plants:Responses of the host-
plant Cistus and the non-host Arabidopsis to truffle metabolites...............112
1 Introduction ..................................................................................................113
1.1 Importance of truffles and ectomycorrhizas ................................................. 113
1.2 Recognition between symbiotic partners (not forming ectomycorrhizas)........ 113
1.3 Recognition between symbiotic partners forming ectomycorrhizas................ 114
1.4 The dark side of truffles.............................................................................. 115
2 Experimental design & biological material................................................115
2.1 Plant material ............................................................................................. 115
2.2 Fungal material: fruiting bodies & extractions............................................... 115
2.3 Fungal material: mycelium cultures............................................................... 116
2.4 Bioassay: dual cultures of mycelium/plants .................................................. 116
2.5 Agar diffusion bioassay............................................................................... 117
2.6 Bioassay data analysis ................................................................................ 118
3 Preliminary results and discussion .............................................................119
3.1 Mycelial exudates induce root shortening in host-plant Cistus....................... 119
3.2 Mycelium exudates induce root shortening and root hair elongation in
Arabidopsis...................................................................................................... 120
3.3 Mycelium exudates from the "black truffle" T. melanosporum or the "white
truffle" T. borchii have the same effect on Arabidopsis root length/root hair....... 121
3.4 Fruiting body extract induces in Arabidopsis changes similar to mycelium
exudates. ......................................................................................................... 122
3.5 The bioactive metabolite is not indole-3-acetic acid .................................... 122
3.6 The ethylene pathway could be involved in the perception of truffle
metabolite(s) in Arabidospis. ............................................................................ 123
4 Conclusion ...................................................................................................124
5 Future work .................................................................................................125
References......................................................................................................127

VII. Pitfalls and Open Questions .................................................................130
1 Hormonal pathways involved in the response of Arabidopsis to truffle
volatiles............................................................................................................130
2 Response of Arabidopsis to Botrytis after treatment with 1-octen-3-ol. .132
3. Field Data: Can bioassays reveal volatiles directly in a truffle field? ....133
3. Field Data: Can bioassays reveal volatiles directly in a truffle field? ....134
4 TLC plate bioassays: a swift method to identify bioactive compounds ?
..........................................................................................................................136
References.......................................................................................................138

VIII. Conclusion ........................................................................................... 139
What has been learned?................................................................................ 139
Future work..................................................................................................... 141

Télécharger le document ici