全 文 ：The Microbial Communities and Fruiting of Edible
Ectomycorrhizal Mushrooms
Zambonelli Alessandra1
?
, Iotti Mirco1 , Barbieri Elena2 , Amicucci Antonella2 ,
Stocchi Vilberto
2
, Peintner Ursula
3
, Hall Ian R .
4
(1 Dipartimento di Protezione e Valorizzazione Agroalimentare, via Fanin 46 , I -40127 Bologna, Italy;
2 Dipartimento di Scienze Biomolecolari , Via Saffi 2 , 61029 Urbino ( PU) , I taly; 3 Instituteof
Microbiology, Leopold-Franzens-University Innsbruck, Technikerstr . 25 , 6020 Innsbruck, Austria;
4 Truffles & Mushrooms Consulting Ltd, P . O . Box 268 , Dunedin 9054 , New Zealand)
Abstract : The competition between edible ectomycorrhizal fungi ( EMF) and other ectomycorrhizal fungi have been studied
in greenhouse and in the field taking into account different environmental conditions and using molecular tools as well as
morphological methods . Approximately 100 ectomycorrhizal specieswere identified in soil fungal communities in productive
areas of Boletusedulis sensu lato and in truffières of Tuber magnatumand Tuber borchii . The bacterial communities occur-
ring in T. magnatum and T. borchii fruiting bodies during different stages of their maturation were also characterized .α-
Proteobacteria, were found as major constituents of the bacterial community associated with truffle ascoma, independentely
of the degree of maturation . These bacteria include members of the Sinorhizobium?Ensifer and Rhizobium?Agrobacterium
groups, and Bradyrhizobiumspp ., which are well known for their ability to fix atmospheric nitrogen .
Our studies raise two important questions about truffle biology: 1 ) can the composition of microbial soil communities
infuence EMF fruiting body production ?2) can nitrogen fixing bacteria play a role in truffle nutrition ?
Key words: Edible ectomycorrizal fungi communities; Competition; Bacteria; Truffle; Porcino
CLC number : S 646 Document Code : A Article ID: 0253 - 2700 ( 2009) Suppl.ⅩⅥ- 081 - 05
Introduction
The edible ectomycorrhizal mushrooms ( EEMM )
have a market measured in billions of euros . Many,
such as the European truffles Tuber magnatum, Tuber
borchii , Tuber melanosporum and Tuber aestivum, and
porcini ( Boletus edulis s. l .) , all of which can be
found in Italy, are appreciated worldwide for their aro-
ma and flavours ( Hall et al. , 2007) . Ecological stud-
ies on theEEMM havetended to concentrateon thesoil
and climatic characteristics of the production areaswith
generally little attention paid to the associated micro-
biota which a priori can be assumed tostrongly interact
with them (Hall et al. , 2003) .
In the forests of the Northern Hemisphere, that
are dominated by ectomycorrhizal trees, it is not un-
usual to find many different ectomycorrhizal fungi on
the roots of a single host plant . Among these fungi
there is an intense competition for space on the roots
and for nutrients arising a dynamic equilibrium condi-
tioned by thehost plant, biotic factors and environmen-
tal conditions ( Jumpponen and Egerton-Warburton,
2005) . The soil also include numerous other micro-
organisms such as pathogenic and saprobic fungi , and
bacteria which arewell known to interact with the ecto-
mycorrhizal fungi ( Paulitz and Linderman, 1991; Cair-
ney and Meharg, 2002; Frey-Klett et al. , 2007 ) but
their relationship with EEMM was poorly investigated
aforetime .
Competition between ectomycorrhizal fungi
The first studies conducted by our research group
were conducted in the greenhouse, where we investi-
gated competition between T. borchii , Hebeloma si-
napizans and Laccaria bicolor on Pinus pinea seedlings
(Zambonelli and Branzanti , 1990 ) . In these experim-
ents we showed that the competitiveness of T. borchii
云 南 植 物 研 究 2009 , Suppl . ⅩⅥ : 81～85
Acta Botanica Yunnanica
? ?Author for correspondence; E-mail : zambonel@agrsci . unibo. it, phone + 39 0512096579 , fax + 30 0512096565
Fig . 1 Ectomycorrhiza of Tuber borchii invaded by Hebeloma sinapizans
With arrow is indicated a T . borchii cystidium bended by Hebeloma sinapizans ( from Zambonelli et al. , 1995)
varied with the substrate . In peat moss and vermiculite
both H. sinapizans and Laccaria bicolor out competed
T. borchii whereas in calcareous soil only H. sina-
pizans was able to form abundant mycorrhizae when
inoculated together with T. borchii . Similarly, in the
greenhouse plants previously colonised by T. borchii
were invaded and colonised by H. sinapizans after it
was introduced (Zambonelli et al. , 1995) .
However, when the seedlings were transplanted
into the field in an area particularly favourable to
T. borchii , it became the most competitive fungus and
mycorrhizae formed by H. sinapizans, L . bicolor and
the common greenhouse contaminant Spharosporella
brunnea completely disappeared after four years ( Zam-
bonelli et al. , 2000) . This experiment showed that the
soil characteristics could completely change competition
between EEMM and other fungi .
In a subsequent field experiment in ayoung T .
aestivum truffière, we modified the soil conditions by
mulching with different materials ( Zambonelli et al. ,
2005) . Mulching is a traditional practice in truffle cul-
tivation where a layer of straw, leaves, branches, or
plastic film is laid on the soil surface in the rooting
zones of the host plants to limit the development of
grasses, maintain soil moisture duringdrought, and in-
crease truffle production ( Fioc, 1987; Ciani et al. ,
1988; Etayo and De Miguel , 2001) . It is well known
that mulching increase soil moisture and equilibrate soil
temperature but can also have long-term responses such
as an increase in soil mineral nutrients, soil aggregation
and drainage characteristics, microbial activity, and
cellulase activity (Downer et al. , 2002) . As a conse-
quencemulching can influence the development of dis-
eases, insects, nematodes and soil borne fungi as well
as plant growth and truffleyields ( Elder et al. , 2002) .
The experimental truffière was located in a fresh
and moist part of the Appennino mountains ideal for
T. aestivumdevelopment . Themulchingmaterialswere
wheat straw, an aluminised cloth, a black mulching
cloth and a synthetic grey tissue . Threeyears after the
start of the experiment the mulches significantly influ-
enced the degree of ectomycorrhizal colonization in the
upper 0 - 15 cm of soil and also differentially affected
the competitiveness of T. aestivum against other soil
fungi . Both straw and theblack mulchdepressed T. a-
estivum infection, probably because increasing the soil
moisture favoured thedevelopment of contaminant ecto-
mycorrhizal fungi . In contrast, the tissue stimulated
T. aestivum colonization and depressed the develop-
ment of the contaminating ectomycorrhizal fungi proba-
28 云 南 植 物 研 究 增刊ⅩⅥ
bly because it slightly increased soil temperatures . So
the outcomes of competition are not solely affected by
the competitive abilitiesof the component fungi in ade-
termined soil but also other environmental factors such
as temperature and water can influence competition
among ectomycorrhizal fungi . These factors can be
modified by mulchingwhich can have an impact on the
development of EEMM fungi .
EEMM fungal communities
In the cultivation of EEMM competition fromresi-
dent ectomycorrhizal fungi is one of the biggest prob-
lems particularly in the first few years after planting
when the introduced species can be replaced . In cont-
rast, naturally theEEMM survivein adynamic equilib-
rium with other ectomycorrhizal fungal species some-
times coexisting on thesame root systemor evenon the
same root tip . For example, Hall and collaborators
consistently found Boletus edulis fruiting bodies in the
same locations and at thesame timeof year as Amanita
muscaria or Amanita excelsa in Austria, England, Ita-
ly, New Zealand, Sweden and USA ( Hall et al. ,
2003) . Moreover A. muscaria or A. excelsa and B. e-
dulis hyphae and rhizomorphs were often closely inter-
woven .
This emphasises the need to better understand the
consistence and the dynamics of soil ectomycorrhizal
communities .
Until recently studies on ectomycorrhizal fungal
communities were only conducted using morphological
and cultural techniques that were ableto identify only a
limited number of microbial species underestimating
most of the autochthonous species that are uncultiva-
ble . The application of molecular techniques for taxo-
nomic affiliation has dramatically changed the situation
on microbial ecology, so that it is now possible to in-
vestigate the composition of microbial communities in
soil, mycorrhizas and fruiting bodies and provide a
more complete and precise scenario of their structural
composition and dynamics (Dahlberg, 2001; Selosse,
2001) .
The internal transcribed spacers ( ITS region) of
rDNA are the most frequently used DNA region for the
identification of ectomycorrhizal fungi . The comparison
between the ITS sequence obtained fromthose deposited
in sequence data base often allows us to identify the
fungal species atgenusor even at thespecies level . It is
also useful to compare the obtained ITS sequences with
thoseof the fruiting bodies present in the studied areas
using the entire ITS sequences or their RFLP profiles
(Dahlberg, 2001) .
Inorder to reduce thetime and the costsof molec-
ular analyses of mycorrhizae our research group uses a
method for direct PCR of the mycorrhizal mantle so
avoiding DNA extraction ( Iotti et al. , 2006 ) . This
method is moreprecise than the conventional technique
because examiningonly a small piece of fungal mantle
reduces the risk of PCR contamination fromother fungal
species often present as mycelium on the surface of a
mycorrhiza .
Ectomycorrhizal communities have been studied
usingmolecular tools in natural T. magnatumtruffières
where it was shown that T. magnatummycorrhizae are
quite rare ( less than 5% even in productive areas) and
that themost common ectomycorrhizal fungi present are
other species of Tuber such as T. rufum ( Bertini et
al. , 2006; Murat et al. , 2005) . In contrast, a study
we made of natural T. borchii truffières in the highly
productive littoral zone near Ferrara, Italy, we found
abundant T. borchii mycorrhizae with often more than
30% of the root tips infected by the fungus ( unpub-
lished data) .
In a survey of all ectomycorrhizae, saprobic and
pathogenic fungi in a Boletus edulis sensu lato ( por-
cini ) productive area using ( Peintner et al. , 2007 )
morphological and molecular identification 39 ectomy-
corrhizal fungi were identified on root tips whereas 40
fungal species were found in the soil using the cloning
techniques . However, the overlap between above- and
below-ground fungal communities was very low with
Boletus mycelia rare and scattered compared to other
soil fungi whereas their fruiting bodies dominated the
aboveground fungal community . Only B. aestivalis ec-
tomycorrhizas and soil mycelia were relatively abun-
dant . In thestudied areas nospecific fungus-fungus as-
sociation as suggested by Hall and collaborators ( Hall
et al. , 2003 ) was found .
38增刊ⅩⅥ Zambonelli Alessandra et al. : The Microbial Communities and Fruiting of Edible Ectomycorrhizal . . .
These studies emphasise that some EEMM such as
Tuber magnatumand Boletus edulis are able to produce
large quantities of fruitingbodies supported by only rare
mycorrhizas and mycelia in the soil whereas other spe-
cies such as T. borchii are supported by a largenumber
of infected tips . Evidently there is some unknown eco-
logical component that is involved in fruiting body for-
mation and development .
EEMM bacterial communities
It is now well established that some bacteria asso-
ciated with mycorrhizal fungi in thesoil and in the rhiz-
osphere are able to promote the establishment of the
symbiosis and very few studies were devoted on the mi-
crobe host association in EEMM productive areas . These
so-called mycorrhiza helper bacteria ( MHB ) can be
found with ecto- or endomycorrhizas and belong to vari-
ous taxonomic groups but particularly fluorescent pseud-
omonades have been studied within these association
(Frey-Klett et al. , 2007) .
Of particular importance has been the discovery
that the bacterium Staphylococcus pasteuri , found on
the roots of vitroplants of Populus alba could inhibit
the growth of T. borchii but not Hebeloma radicosum,
another ectomycorrhizal fungus ( Barbieri et al. ,
2005b) . Whether S. pasteuri commonly occurs in soil
is largely irrelevant what is important is that an organ-
ism apparently unrelated to either symbiotic partner
might influence the composition of the ectomycorrhizal
flora in a soil . This andother interactions between EE-
MM and associated microrganisms, could be an expla-
nation of specific chemiotactic behaviour and spreading
of certain ectomycorrhizal fungi .
Molecular techniques present also the opportuni-
ties for visualizing how organisms interact in the soil .
The use fluorescent markers that can reveal the location
of the (DNA) nucleic acids of specific organisms can
provide important additional information on microbial
soil interactions . For example, using culture-indepen-
dent and dependent 16S rRNA gene based approaches
we were able to increase our taxonomic knowledge of
Tuber-associated bacteria occurring in the T. borchii
and T. magnatum fruiting bodies ( Barbieri et al. ,
2005a, c, 2007 ) . From this work we have been able
to identify new bacterial species belonging to at least
six divisions: alpha-proteobacteria, beta-proteobacte-
ria, gamma-proteobacteria, Bacillaceae, Firmicutes e
Bacteroidetes . Of particular interest was the constant
and significant presenceof alpha-proteobacteria, main-
ly represented by membersof the Sinorhizobium?Ensifer
and Rhizobium?Agrobacterium groups, and Bradyrhi-
zobiumspp ., well known for their ability to fix nitro-
gen . Moreover the use of specific oligonucleotide
probes for the alpha-proteobacteria, have allowed to
quantify the incidence of theRhizobi-like in T. borchii
and T. magnatum ascocarps, estimated to account for
approximately 60% - 80% of the entire Eubacterial
population present in situ .
This percentage appears to be independent of the
degreeof ascoma maturation . These bacterial species,
probably underestimated in thepast becauseof the lim-
itations of classical cultural techniques, constitute the
dominant fractionof the bacterial communities associat-
edwith the ascomaof prizedwhite trufflesyet their role
has been completely ignored .
Conclusion
Althouth our knowledge of the composition of soil
microbial communities has increased considerably in re-
cent years, their influenceon theEEMM biology is still
poor understood . However, it is nowobvious that fungi
and bacteria living in association with the myceliumof
EEMM , mycorrhizas and?or fruiting bodies do have a
direct or indirect effect on competition for space or nu-
trients or via molecules that may trigger morphogenetic
changes .
In addition the research so far completed raise two
important questions about EEMM biology: i ) can the
composition of microbial soil communities has infuence
on the production of fruting bodies, and ii ) can nitro-
gen fixing bacteriaplay a role in thenutritionof fruiting
bodies in particular truffles ?
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