全 文 ：Progress Toward Truffle Cultivation in the Central USA
?
Bruhn J . N.
??
, Pruett G.E. , Mihail J . D.
( Division of Plant Sciences, 110 Waters Hall , University of Missouri , Columbia, MO 65211 USA )
Abstract : We havefound that mycorrhizaformation by somestrains of T. aestivumis suppressed by theuse of high-Capel-
letized lime to raise the pH of greenhouse potting medium, as compared with the use of natural crushed dolomitic lime-
stone . We have also found that seedlings grown under a novel process ( RPM? ) for producing very large root systems in
bark-based media failed to becomeaswell colonized by T. aestivumas did seedlings grown under amoretypical systemin
a peat-basedmediumin book planters . We havefollowedectomycorrhizacommunity development on both RPM? andmore
typically produced seedlings for two years in the field . Competing ectomycorrhizal species dominated RPM seedlings two
years after outplanting, whereas T. aestivumremained dominant on moretypically produced seedlings . Mycorrhizaeof four
Tuber spp . have been detected in this plantation ( i. e ., T. whetstonense, T. maculatum, T. rufum, and T. lyonii ) .
These arethefirst recordsof T. whetstonense, T. maculatum, and T. rufumoccurrence in MO . Mycorrhizae andascocarps
of T. lyonii had been found in nearbyplantings of Corylusseedlings in2005 , and again in 2007 . A third seedling produc-
tion process (Missouri Gravel Bed) shows promise in a pilot study with T. aestivum and warrants further study .
Selection of T. melanosporumvs . T. aestivumasthetarget truffle species for a specific location is complicated by the
uncertainty of future climatic conditions . Different mulching practices modify soil temperature and moisture, andmay pro-
vide clues to the environmental tolerances of T. aestivumand T. melanosporum . Weare comparingthe effects on soil tem-
perature and soil moisture of water-permeable weed barrier fabric vs . athin layer of calcareous gravel , as compared to no
soil surface treatment .
Key words : Truffle cultivation; Tuber aestivumsyn; T. uncinatum; Tuber melanosporum; Seedling production; Mulch-
ing; Ectomycorrhizal community
CLC number : Q 646 Document Code : A Article ID : 0253 - 2700 (2009 ) Suppl .ⅩⅥ - 052 - 03
Introduction
We are studying the biology and ecology of Tuber
aestivumsyn . T. uncinatumand T. melanosporumfrom
theperspectiveof their eventual cultivation in the cen-
tral United States . Our efforts toward truffle cultivation
focus on: 1 ) development of seedling production sys-
tems that improve plantation occupation through aggres-
sive root colonization and emphasis on lateral root de-
velopment; and 2) analysisof climatic andmanagement
factors influencing mycorrhizal community development
following plantation establishment . We present here an
overview of our first two studies established to evaluate
aspects of T. aestivum-infected seedling production and
plantation truffière establishment .
Materials and Methods
Our first study was designed to evaluate the effects
on Quercus robur root system development of two types
of lime, three root dip inoculation techniques, two soil
infestation methods, and two sources of T. aestivum
truffle inoculum . Seedlings were grown in the green-
house in a 2∶1∶1 mixture of air-dried field soil , ver-
miculite, and perlite ( v∶v∶v) . High-Ca, pelletized
quick release Pel-Lime
?
was compared with a natural
crushed dolomitic lime . Onesource eachof T. aestivum
was provided by Dr . Gérard Chevalier and Dr . Eric
云 南 植 物 研 究 2009 , Suppl . ⅩⅥ : 52～54
Acta Botanica Yunnanica
?
?? ?Author for correspondence; E-mail : bruhnj@ missouri . edu; Tel .: 1 573 882 1471; Fax .: 1 573 882 0588
Foun ?dation items: This work was funded through theUniversity of Missouri Center for Agroforestry under cooperative agreements 58 - 6227 - 1 - 004 ,
58 - 6227 - 2 - 008 and 58 - 6227 - 5 - 029 with the ARS and C R 826704 - 01 - 2 with the US EPA . The results presented are the sole responsi-
bility of the authors and?or theUniversity of Missouri and may not represent thepolicies or positions of the EPA . Any opinions, findings, con-
clusions or recommendations expressed in this publication are thoseof the author (s) and do not necessarily reflect theview of the U .S . Dep-
artment of Agriculture . The University of Missouri LifeSciences Predoctoral Fellowship Program provided stipend funding for GEP
Danell . Soil infestation was accomplished by mixing
aqueous slurry of ascospores into the potting mix, but
the slurry used to infest half of the pots was amended
with the gel-based compound Stockosorb
?
. Each seed-
ling also received a root dip inoculation: either water;
water+ ascospores; or water + ascospores + Stockosor-
b? . Details of themethods and materials are published
elsewhere (Pruett et al. , 2008a) .
Our second study compared the effects of two very
different seedlingproduction systems: the RPM
? ( Ro-
ot Production Method) system, and a more typical
seedling production system . To summarize, the RPM
?
system is based on aground bark potting medium, and
involves three potting events culminating in 8-l pots at
outplanting . In contrast, the more typical system in-
volves growing seedlings in book planters containing a
peat-based medium . Tuber aestivum colonization levels
on hybrid Q. robur x Q. bicolor seedlings were com-
pared for all four possible combinations of the two pot-
ting media and the two container methods . Methods and
materials for this first stageof the study are detailed in
Pruett et al . ( 2008b) . We then evaluated the fate of
T. aestivum in relationship with thedevelopment of ec-
tomycorrhizal communities for two years following out-
planting . Developing root systems were sampled in the
field by soil coringone and two years following outpla-
nting, and ectomycorrhizal communities were compared
with those that had developed in thegreenhouse . Ecto-
mycorrhizal community development was evaluated on
thebasis of species identity, species richness, Shannon
diversity index, and non-metric multidimensional scal-
ing (Pruett et al. , 2008c) .
Results
The typeof lime used to raise the potting mix pH
affected Q. robur seedling colonization by T. aestivum
strains differentially . Overall , thedolomite limeprovid-
ed better results than did Pel-lime? . Also, root dip in-
oculation did not increase infection levels beyond those
achieved by pottingmix infestationwith ascospores . Fi-
nally, the hygroscopic polymer was ineffective ( Pruett
et al. , 2008a) .
Seedlings grown in the more typical system were
much smaller yet much more densely colonized by
T. aestivum than were seedlings produced by the
RPM
?
system . Seedlings grown in book planters devel-
oped more root tips l - 1 than did seedlings grown in
RPM? containers regardless of the potting medium
used, and the peat-based medium supported develop-
ment of more T . aestivum root tips than did the bark-
based RPM? mediumregardlessof thecontainer method
used ( Pruett et al. , 2008b) . At the time of outplant-
ing, seedlings grown under the more typical system
contained a higher percentage of T. aestivum ectomy-
corrhizae and a lower percentage of competitor ectomy-
corrhizae than did seedlings grown under the RPM
?
system (Pruett et al. , 2008c) .
Only two competitor ectomycorrhiza species devel-
oped in the greenhouse, and one of these ( unidenti-
fied) could no longer be detected two years after outpla-
nting . Over the threeyears of sampling, twice as many
competitor species were detectedon RPM
?
seedlings as
were detected on‘ typical’seedlings ( 34 vs . 15 ) .
Among these species were three Tuber spp . with affini-
ties to T . whetstonense, T. maculatum, T. rufum, and
T. lyonii ( in order of decreasing frequency of detec-
tion) . Ectomycorrhizal species diversity has increased
more quickly on RPM? seedlings than on typical seed-
lings, thoughdiversity increased dramatically on typical
seedlingsduring their second year in the field ( Pruett
et al. , 2008c) .
Discussion
It′s not surprising that different forms of lime can
affect the levels of seedling infection by some strains of
T. aestivum, but it was useful to note that both strains
performed well with our dolomite lime source, even
though its Ca and Mg contents were lower and higher,
respectively, than those of Pel-lime
? ( Pruett et al. ,
2008a) . Becauseof both the less refined nature of do-
lomite lime and its physical proximity to many green-
houses and plantation sites in thecentral USA , both the
retail and transportation costs of dolomitic lime argue in
favor of its use .
A comparison of our plantation soil properties with
their ranges throughout the European habitat of T. aes-
35增刊ⅩⅥ Bruhn J .N. et al. : Progress Toward Truffle Cultivation in theCentral USA
tivum seems favorable to our endeavor ( Pruett et al. ,
2008c) . Mid-Missouri generally receives more precipi-
tation May-August, and less duringDecember, than do
Burgundy, Parma, and Gotland ( Pruett et al. ,
2008c) . Mid-Missouri temperatures are generally more
similar to those in Burgundy and Parma than those of
Gotland, except in December-February ( Pruett et al. ,
2008c) .
The sudden increase in ectomycorrhizal community
diversity that took place during the second year in the
field on seedlings produced using the more typical sys-
temwas intriguing ( Pruett et al. , 2008c) . We suggest
two possible explanations . First, removal of the water-
permeable mulch fabric weed barrier fromseedlings af-
ter their first year in the field may have favored devel-
opment of indigenous ectomycorrizal species (Zambonel-
li et al. , 2005 ) . Second, the prolonged heavy freeze
that took place after a warmwinter in early April 2007
may have influenced the ectomycorrhizal populations
sampled 1 .5 months later after two years in the field .
In a small pilot experiment located beside our
T. aestivum plantation ( Bruhn & Mihail , unpub-
lished) , we have observed that a thin layer of gravel
has had a slight drying and warming effect on soil tem-
perature, whereas water-permeable mulch fabric has
had asummer cooling effect and a slight winter warming
effect without influencing soil moisture . Of course, the
mulch fabric provides nearly complete prevention of
weed development, whereas the gravel reduces weed
development only slightly .
The detection of ectomycorrhizae representing
T. whetstonense (Frank et al. , 2006) , T. maculatum,
T. rufum, and T. lyonii inour plantation was interest-
ing, both becauseof theold-field natureof the planta-
tion site, and because there were no records of truffle
collections fromMissouri until 2005 , when T. lyonii was
found fruiting under a hybrid Corylus shrub planted
about 40 maway in 1999 (Bruhn, 2005; Bruhn et al. ,
2006) . We hypothesize that various mammals have been
depositing inoculaof these Tuber spp . in our plantation
siteover avery long period of time (Ashkannejhad and
Horton, 2006) . Native Tuber spp . weremuch less fre-
quently encountered on seedlings produced using the
more typical systemthanon RPM
?
seedlings .
The‘Missouri gravel bed’methodof producing trees
and shrubs with very large and densely branched fine root
systems (Bohnert and Starbuck, 2008) shows some prom-
ise as amethod for producing largeseedlings well-infected
by T. aestivum . We continue to look for ways to produce
seedlings with highly developed lateral root systems that
could rapidly and efficiently occupy a plantation site .
Acknowledgements: We thank Amanda Williams and Jamila
Batchelder for their field and laboratory assistance, and Forrest-
Keeling Nursery for their expertise and materials .
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