全 文 ：Successful Mycorrhizal Synthesis of Tuber indicum with Two
Indigenous Hosts and Their Recognition
?
GENG Li-Ying1 , WANG Xiang-Hua1 , YU Fu-Qiang1 , DENG Xiao-Juan1 , 2 ,
SHI Xiao-Fei
1 , 2
, XIE Xue-Dan
1 , 2
, LIU Pei-Gui
1??
(1 Key Laboratory of Biodiversity & Biogeography, Kunming Instituteof Botany, Chinese Academy of Sciences,
Kunming 650204 , China; 2 Graduate University of Chinese Academy of Sciences, Beijing 100049 , China)
Abstract : Tuber indicumis oneof themost renownedcommercializedfungi inChina . Mycorrhizal investigations, however,
have been carried out mainlywith exotic trees . Up to nowthere is nodetailed description of morphologyof themycorrhizae
formed with the indigenous hosts of T. indicum . Containerized seedlings of two indigenous hosts of thefungusin southwest-
ern China, Pinus armandii and Castanea mollissima were inoculated with aqueous spore suspension of T. indicum in two
kinds of substrates . Mycorrhizae beganto form4 months after inoculationandwereharvestedat 9 months . The contributing
fungus of the mycorrhizae was confirmed to be T. indicumby morphological and ITS-rDNA sequence analyses . The mor-
phology of emanatinghyphaeandepidermoid-likemantleappearancewassimilar to themycorrhizae obtainedwith someEu-
ropean trees . The highmorphological variation and the similarity to that of T. melanosporummakes it difficult to distinguish
the mycorrhizaeof thetwo speciesbymorphology alone . Thesynthesisandtheir recognitionof mycorrhizaeof T. indicumwith
its indigenous hostswill be of great significance for planned cultivation of the Asian black truffles .
Key words: Asian black truffle; Ectomycorrhizal fungi ; ITS-rDNA ; Compare inmorphology
CLC number : Q 934 Document Code : A Article ID : 0253 - 2700 (2009 ) Suppl .ⅩⅥ - 029 - 08
Tuber indicumCooke& Masseeis oneof themost
renowned commercialized fungi in southwestern China .
Though the Asian black truffle Tuber indicumCooke &
Massee was described early in 1892 ( Cooke and Mas-
see, 1892 ) , it drew little attention until it was export-
ed to Europe and North America in the 1990′s . Espe-
cially up to now there is no detailed descriptionof mor-
phology of the mycorrhizae formed with the indigenous
hosts of T. indicum . The high similarity of T. indicum
to the Périgord black truffle, T. melanosporumVittad .
and the potential confusion between the two species,
leads to a series of intensive confusion in taxonomy,
commercial and mycorrhizal recognition . With the
Périgord black truffle threatened by importation of the
Asian black truffle to Europe, some European mycolo-
gists have been trying to seek feasible and reliable
methods to distinguish the two truffles . This prompted
various works on the mycorrhizal synthesis of T. in-
dicumwith several European trees, including Corylus
avellana L ., Quercus cerris L ., Q. pubescens Willd .,
Q. ilex L ., and Pinus pinea L . (Di Massimo et al. ,
1996; Comandini and Pacioni , 1997; Gandeboeuf et
al. , 1997; Zambonelli et al. , 1997; Mabru et al. ,
2001) . Since theaimof thesesyntheses was to identify
of T . indicummycorrhizae, no detailed information on
the factors affecting the formation of mycorrhizae were
published . The most recent data on the mycorrhizae
and the substrates of T. indicum given by García-
Montero et al . ( 2008) , also is based on mycorrhizae
synthesized on European trees . In China attempts to
云 南 植 物 研 究 2009 , Suppl . ⅩⅥ : 29～36
Acta Botanica Yunnanica
?
?? ?Author for correspondence; E-mail : pgliu@ mail. kib. ac. cn
Foun ?dation items: Thework was supported by“West Light”Program of ChineseAcademy of Sciences (2004) , National Natural ScienceFoundation of
China (No . 30470011 , 30770007 ) , the Joint Founds of the National Science Foundation of China and Yunnan Province Government ( No .
U0836604) , the Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences ( No . KBB-
200804) , the president fund of the ChineseAcademyof Sciences (1022 , 1035) , thesupportive poverty program of ChineseAcademy of Sciences,
Natural Science Foundation of Yunnan Province (No . 2004C0050M ) and theKnowledge Innovation Program of the ChineseAcademy of Sciences
(No . KSCX2-YW-G-025)
cultivate Asian black truffle by breeding mycorrhizal
seedlings have also been made . Mycorrhizal synthesis
of T. indicum with its possible indigenous hosts in
southwestern China, Castanea mollissima BL ., P . -
armandii Franch ., P . massonia Lamb ., and P . y-
unnanensis Franch . using spores as inocula were re-
cently reported by Chen (2003) and Hu et al . (2004 ,
2006) . Hu et al . ( 2005 ) declared that ascocarps of
T. formosanum [ = T. indicum, according to Chen
(2007) ] were successfully harvested 8 years after pla-
nting the truffle-infected seedlings of Cyclobalanopsis
glauca (Thunb .) Oerst . to the field (10 years after in-
oculation) . Yamanaka et al . (2000 , 2001) show that
T. indicumvar . yunnanenseYamanaka formed dichot-
omous ectomycorrhizae with P . armandii , and gave a
general description and provided photographs of these
mycorrhizae . However, up to now, there has been no
detailed morphological data of the mycorrhizae on the
indigenous trees .
As a result of excessive andundue exploration and
utilization, the natural resource of T. indicum in
southwestern China is seriously threatened ( Wang and
Hall , 2001; Wang et al . , 2007) . It is urgent to con-
serve the Asian black truffle, either by protecting the
habitats, limitinghuntingactivities, or by producing as-
cocarps with methods of planting truffle-infected seed-
lings to meet the market demands . The limited knowl-
edgeof themorphology of mycorrhizae and the influence
of biological and non-biological factors that ensure suc-
cess, such as substrates, host plants, inocula, and in-
oculating methods necessitates the research on the es-
tablishment of mycorrhizaeof T. indicumon its indige-
nous hosts .
Asian black truffles were reported in association
with a variety of species, including Alnus nepalensis
D . Don ., Cy. glauca, P . armandii , P . yunnanensis,
P . taiwanensis Hayata ., Quercus spp . Keteleeria eve-
lyniana Mast . (Zhang and Minter, 1988; Zhang and
Wang, 1990; Hu, 1992; Zang et al. , 1992; Chen et
al. , 1998; Fukiharu et al. , 2006; Chen, 2007;
Wang et al. , 2007 ) . The symbiotic relationships of
T. indicumwith someof these trees, however, arestill
to beconfirmed by morphological , molecular, or exper-
imental proofs . Among the indigenous hosts confirmed
(Hu, 1992; Hu et al. , 2006; Chen, 2007 ) , P . -
armandii and C . mollissima are two main trees that are
economically important and popularly planted in the
south and north of China . Combining the cultivation of
mycorrhizal edible fungi with afforestation of economic
plants will have a practical significance in the moun-
tainous area . The successful cultivation of valuable
truffles and quality indigenous trees could have great
economic potentialities for the local people in marginal
regions . Sincemorphologic identificationof mycorrhizae
will be an important criteria and feasible target for
judgingthesuccessof synthesis, through 6 years exper-
iment, mycorrhizal synthesis of Tuber indicumwith two
indigenous hosts of Castanea mollosima and Pinus ar-
mandii was obtained great successful . Important steps
for the mycorrhizal synthesis was shortly provided as
following, the more detail information was provided in
Mycorrhiza (Geng et al. , 2009) .
Materials and methods
Mycorrhizal synthesis
Ascocarps of T. indicum were stored at - 20℃
until use and then sterilized with 75% alcohol . Aque-
ous spore suspension was prepared by blending the
chopped ascocarps with a blender, until the spores
were released . The spore suspension was diluted with
sterilized water into 106 spores?ml .
Seeds of C. mollissima and P . armandii were
surface sterilized with 30% H2 O2 for 1 hour and 4
hours respectively . After washing with distilled water,
seedswere sown in asquareplastic container filledwith
sterilized perlite?vermiculite (1?1 , v?v) . Theseedlings
of 3-month-old were transplanted in container (1 .5 l )
with 1 000 ml transplant substrate, consistingof humus?
vermiculite?peat moss ( 1?1?1 , v?v) and humus?soil?
limestone (4?4?1 , v?v) , previously steamsterilized for 3
hours . The final pH of both substrates was adjusted to 7
by adding slaked lime . All pots were maintained in the
greenhouse condition . After 9 months, 17 seedlings ran-
domly sampled in the treatment humus?vermiculate?peat
and 5 in humus?soil?limestonewereexamined for theob-
servation of ectomycorrhizae for both C . mollissima and
03 云 南 植 物 研 究 增刊ⅩⅥ
P . armandii . Molecular analysis was also performedon
these T. indicum-likemycorrhizae .
Morphological observation
Mycorrhizae were photographed under a Nikon
SMZ1500 stereoscope and all microscopical drawings
were made under a drawing tube installed in a Nikon
E400 microscope . All anatomical sections were pre-
pared fromfresh rootmaterial by hand . Sectionof outer
mantle layers wasmadeby peeling themycorrhiza along
longitudinal axes and then putting the mantle part on
the slide with its outer surface upwards . Inner mantle
layers wereobtained in thesameway but with the inner
surface upwards . A mycorrhizawere sandwiched in fil-
ter paper for 3 - 4 min for dehydrating and then cut it
into thin slides for transverse section preparation . For
all slides 5% KOH then Congo Rid aqueous solution
were used for observing and illustrating . The typing of
mantle in table 2 followed Agerer ( 1987 - 2002;
1991) .
Molecular analysis
DNA was extracted from fresh mycorrhizae using
the protocol of Doyle ( 1987 ) , Douet et al. ( 2004 )
and White et al. ( 1990 ) . DNA sequence data were
obtained from ITS region for rDNA . DNA sequences
were initially aligned using Clustal X ( Thompson et
al. , 1997) , followed by manual alignment in the data
editor of BioEdit ver . 7 . 0 . 1 (Hall , 1999 ) . Maximum
parsimony analyses were conducted with PAUP
*
4 .0b10 (Swofford, 2002 ) with the heuristic search op-
tion (TBR and MULTREES on) and 1000 replicates of
random addition sequence .
Twenty complete ITS sequencesof threeblack Tu-
ber species ( T. indicum, T. melanosporum, T. br-
umale) belonging to theMelanosporum group were used
for analysis, among which 6 were obtained from fresh
mycorrhizae, and 14 were downloaded from NCBI web-
site ( http:??www.ncbi .nlm.nih.gov?) . The T. br-
umalewas selected as outgroup (Fig. 2) . The phyloge-
netic tree revealed threemajor, well supported clades .
The Clade I and II correspond to the two T. indicum
clades was already suggested by Zhang et al . ( 2005)
and Wang et al . ( 2006) , and Clade III was basal to
Clade I and Clade II . Our 6 T. indicumsequences ob-
tained from mycorrhizae of P . armandii and C. moll-
issima truffle inoculated seedlings were identical and
clustered in the T. indicum ( Clade II ) . This clade
comprises most samples fromHuili (Sichuan Province) ,
Chuxiong and Kunming ( Yunnan Province) ( Zhang et
al. , 2005; Wang et al. , 2006) . As a result of thehigh
phylogeographic structureof T. indicum, it is suggested
that the ascocarps used for seedlings inoculation were
probably harvested fromthese regions of China .
Results and Discussion
Mycorrhizae formed 5 months after inoculation for
T. indicumwith C. mollissima and 4 months with P . -
armandii respectively . All seedlings were formed well
mycorrhizal systems with T. indicum without contami-
nating of other fungi (Fig. 1 : a, b, g) . Table 1 and 2
show the recognition characteristics of morphology and
anatomy of these ectomycorrhizae .
The mycorrhizae produced by P . armandii and
C. mollissima with T. indicum are identical in the
puzzle-like on the outer mantle layers the type H inner
mantle layers and the thin-walled filamentous emanat-
ing hyphae with right-angle branching . The mycorrhi-
zaeof P . armandii and C . mollissimawith T. indicum
are typical truffle mycorrhizae ( Zambonelli et al. ,
1997; Comandini and Pacioni , 1997; Smith and
Read, 2008 ) . By comparison, mycorrhizae on P . -
armandii are more dark-colored and stouter than those
on C . mollissima . One distinctive character of the my-
corrhizaeon P . armandii is the stripe-like color pattern
on the surface, which aremorevisible at thebasal part
or well-developed individuals ( Fig. 1 : g-i ) . There
were rich variations in dimensions of epidermal cells,
degrees of interlocking between adjacent cells and col-
ors found between different individuals of mycorrhizae
or even different parts of the samemycorrhiza .
A simple description of the mycorrhizae formed
between T. formosanum ( = T. indicum) and Cy. g-
lauca in Taiwan, China (Hu, 1992) is similar to the
ones produced by C . mollissima except thegolden yel-
lowish color of emanating hyphae in T. formosanum
( under stereoscope) . The mantle in the former de-
scription is thinner than that described here .
13增刊ⅩⅥ GENG Li-Ying et al. : Successful Mycorrhizal Synthesis of Tuber indicumwith Two Indigenous . . .
Table 1 Morphological characteristics comparison on the ectomycorrhizae of Tuber indicum
with Castanea mollissima & Pinus armandii
Combination T ?. indicumwith C. mollissima T g. indicumwith P y. armandii
Mycorrhizae
systems
Unramified tomonopodial-pyramidal , with 0 ?- 1 order of
ramification, single branch fingerlike, up to 6 ?. 5 mm
long ( Fig ?. 1a-c)
Dichotomous or irregularly dichotomous- like, sometimes coral-
loid or between the above two types, with 1 e- 4 orders of ramifi-
cation, sometimes ramifications with 3 - 4 orders densely clus-
tered along root rarely unramified , singlebranch pestle-like, 2
- 4 ?. 6 mm long ( Fig. 1g- i)
Main axes 0 ?. 2 - 0 .4 mm in diam 0 S. 25 - 0 ?. 35 mm in diam
Unramified ends
Straight, rarely slightly bent, usually cylindrical or
slightly tapering, up to 1 ?. 9 mm long, 0 . 2 - 0 K. 3 mm in
diam .
Straight, cylindrical , 0 ?. 8 - 3 .9 mm long, ( 0 . 22 ) 0 .35 - 0 z. 5
mm in diam
Surface of unrami-
fied ends
Smooth or woolly with whitish emanating hyphae, very
tips yellowish-white to paleochraceous, older part con-
colorous to darker, thevery base concolorous to brown;
mantledistinct, not transparent, no transversal stripe-
like color patterns, epidermal cells visible through
mantle; cortical cells not visible
Almost smooth or loosely woolly with yellowing emanating
hyphae, very tips yellowish to whitish, rarely yellowish brown
to reddish brown, becoming darker towards base, often forming
transversal stripe- like, dark color and ring-like patterns, whole
mycorrhiza becoming darker with age; mantle distinct, not
transparent; cortical cells not visible
Emanating hyphae
Whitish, long, distinct, numerous, distributed from
middle part to apex
Whitish , long, distinct, scattered or numerous, notspecifically
distributed
Table 2 Anatomical characteristics comparison on the ectomycorrhizae of
Tuber indicumwith Castanea mollissima & Pinus armandii
Combination T ?. indicumwith C. mollissima T g. indicumwith P y. armandii
Mantle
( 20 E) 25 - 40 (50 ) μmthick in transversesection, com-
posed of ( 5) 6 - 9 layers of hyphal cells, cells round to
elliptical from transverse section, 2 - 12 ( 14 ) ×2 - 6
μm in diam
20 x- 30 (35) μmthick in transversesection, composed of (6 ) 7
- 10 layers of hyphal cells; cells round to elliptical from trans-
verse section, 2 - 20 ( 25 ) ×2 - 7 ( 8) μm in diam . ( Fig ?. 1j)
Outer mantle
layers
Pseudoparenchymatous with polygonal and interlocking
cells arranged in a puzzle- like pattern in plan views
( mantle type M ) ; cells 10 ?- 35 ( 42) ×5 - 20μm, 6 -
10 cells in a square of 20 - 20μm, cell wall yellowish
brown and slightly thick-walled, 0 ?. 5 - 1μm thick, not
gelatinous ( Fig ?. 1e)
Pseudoparenchymatous with polygonal and interlocking cells ar-
ranged in apuzzle- likepattern in plan views ( mantle type M) ,
cells10 - 25 × (5 ) 8 - 11μm, 7 - 10 cells in a square of 20×
20μm; cell wall thin or slightly thicken, yellowish brown often
locally transversal lightly dark-colored, surface smooth and not
gelatinous ( Fig. 1 l)
Inner mantle
Pseudoparenchymatous or a transitional type between
plectenchymatous to pseudoparenchymatous in plan
views ( close to type H ) , partially Hartig net- like;
cells 5 ?- 30 (35 ) ×4 - 10 ( 15 ) μm, 9 - 14 cells in a
square of 20 - 20μm, often thin-walled ( Fig &. 1f)
Pseudoparenchymatous or a transitional type between plecten-
chymatous to pseudoparenchymatous in plan views ( close to
type H ) , partially Hartig net- like; cells 6 X- 30 ( 35 ) ×4 - 14
μm, 9 - 13 cells in a square of 20 - 20μm, often thin-walled
( Fig ?. 1m)
Emanating hyphae
Emerging from the outer mantle, scattered or abun-
dant, 100 ?- 300 (400) μm long, 2 - 3 μm in diam . at
the middle, 3 |. 5 - 5μm in diam at the base, septate,
sections between two septa 20 - 55 μm long, terminal
section (17) 25 - 50μm long, cylindrical , not inflated,
often branching in approx 90°angle near base, thin-
walled, yellowish brown, long, surface smooth, tips
cylindrical or tapering ( Fig ?. 1d)
Emerging from the outer mantle, scattered, 100 %- 300 ( 400 )
μm long, 2 - 2 .5μm in diamat the middle, 3 - 5μm in diam at
the base, septate, sections between two septa (14 ) 20 - 45μm
long, terminal sections ( 12) 15 - 40 ( 60 ) μm long, cylindri-
cal , not inflated, sometimes branching in approx . 90°angle
near base, slightly thick-walled at the base, elsewhere thin-
walled with yellowish brown walls, short, surface smooth, tips
cylindrical or tapering ( Fig t. 1k)
Very tip
With mantle structure as in other parts of the mycorrhi-
za but with small-sized cells
Idem
23 云 南 植 物 研 究 增刊ⅩⅥ
Fig . 1 Morphological-anatomical characteristicsof mycorrhizaeof Tuber indicumsynthesized with Castanea mollissima ( a-f) and Pi-
nus armandii ( g-m) . a . Monopodial-pyramidal ECM system; b . Simple ECM system; c . ECM tips withwhitish flocculent emanating
hyphae; d . emanating hyphae; e . puzzle-like outer mantle layers with epidermoid cells; f . inner mantle lays with some hyphae in a
Hartig net pattern; g . Dichotomous and irregularly pinnate ECM systems; h . ECM tips without emanating hyphae; i . ECM tips with
emanating hyphae; j . transverse section of ECM; k . emanating hyphae; l . puzzle-likeouter mantle layerswith epidermoid cells; m .
inner mantle lays with some hyphae in a Hartig net pattern . a, b, g bars = 2 mm; c, h- i bars= 600μm; d-f , j-m bars= 25μm
Morphologically, the mycorrhizae of C. moll-
issima with T. indicum are not remarkable different
from the ones produced by T. indicum with European
species, such as Q. pubescens ( Comandini and Pacio-
ni , 1997 ) , Q. cerris ( Zambonelli et al. , 1997 ) and
Q. ilex (Garcia-Montero et al. , 2008) except that the
cells in outer mantle layers of our mycorrhizae seem
bigger . The mycorrhizae of T . indicum reported by
33增刊ⅩⅥ GENG Li-Ying et al. : Successful Mycorrhizal Synthesis of Tuber indicumwith Two Indigenous . . .
Fig . 2 The parsimony phylogenetic tree obtained from maximum parsimony analysis of ITS sequences . Numbers above the branches
indicate bootstrap value ( * refers to sequences which obtained from this research, Pa, Cm and Qf refer to fungi sequences obtained
from ectomycorrhizae synthesized on Pinus armandii , Castanea mollissima and Quercus franchetii respectively)
Garcia-Montero et al . (2008) aremuchstouter (diam-
eter of unramified ends500 - 680μm) than another re-
port ( Comandini and Pacioni , 1997) .
The anatomical characters of mycorrhizae of T. -
indicum are very similar to those of T. melanosporum
( Comandini and Pacioni , 1997; Zambonelli , 1997;
García-Montero et al. , 2008 ) . Even compared with
the description and illustrations of T. melanosporumon
Nothofagus spp . ( Pérez et al. , 2007) , the mycorrhi-
zaeobtained here don′t show significant difference . It
is not surprising that the mycorrhizae produced by
T. melanosporum and T . indicum are very similar be-
cause these two truffle species are quite closely related
to each other phylogenetically ( Janex-Favre et al. ,
1996; Paolocci et al. , 1997 , 1999; Roux et al. ,
1999; Mabru et al. , 2001; Douet et al. , 2004;
Zhang et al. , 2005; Wang et al. , 2006) .
Zambonelli et al . (1997) and García-Montero et
al . ( 2008 ) found the mycorrhizae of T. indicum had
smaller but less lobed polygonal pseudocells than those
of T. melanosporum . Our result, however, shows that
the cells in theouter layers are always bigger than those
43 云 南 植 物 研 究 增刊ⅩⅥ
in T. melanosporum . Since the interlocking degree,
namely more lobate or not is very variable, depending
on different individuals of mycorrhizae and different
parts of the samemycorrhiza, it is, therefore, not reli-
able enough to distinguish the mycorrhizae of the two
black truffles by this difference alone . Molecular meth-
ods should be employed when tracingoriginal species .
Mabru et al . (2001 ) reported that the formation
of mycorrhizae of T. indicum was earlier and more
abundant compared with T. melanosporum . According
to García-Montero et al . ( 2008 ) , T. indicum and
T. melanosporum were able to form mycorrhizae 2 .5
months after inoculation . In our work, however, the
earliest mycorrhizae of P . armandii occurred 4 months
after inoculation and thoseof C. mollissima even later .
Moreover, inoculation of Cy. glauca with T. for-
mosanum ( = T. indicum) needed 5 - 6 months to form
ectomycorrhizae ( Hu, 1992 ) . By comparison, mycor-
rhization of T. melanosporum with fagaceous trees
( Nothofagus, Quercus) over 40% of the seedlings and
over 8% of root tips were colonized 6 months after in-
oculation ( Pérez et al. , 2007 ) . 50 - 88% seedlings
were infected 5 months after inoculation with T. macu-
latumVittad . ( Parladé et al. , 1996 ) . These results
indicate that infecting time of T. indicumseems not to
be as short as other known truffle mycorrhization using
spores as inocula .
The spores rate of T. indicumused inour experi-
ment was much higher than compared with 1.2×105
for T. formosanum ( = T. indicum) in Hu et al .
(2005) , 102 - 104 for T. maculatum in Parladé et al .
(1996 ) , 5×106 for T. aestivumVittad . (syn . T . un-
cinatumChatin) recommended by Wedén ( 2004 ) . In
our experiment, on average one seedling received
around 3g fresh ascocarps .
To our knowledge, this is the first time that de-
tailed morphological descriptions of the mycorrhizae of
T. indicumon its indigenous hosts . This will help to
identify mycorrhizae of Asian black truffle with its in-
digenous hosts . Basedon this, further experiments and
statistical analyses should be stressed to promote our
understanding on the optimized conditions in mycorrhi-
zation of the species T. indicum, and to synthesize
more mycorrhizal seedling by applying modified techn-
ology and to establish truffle plantations to meet con-
sumption demand .
Acknowledgements : The authors are verygrateful to Dr . Chris-
tine FISCHER , Dr . Carlos COLINAS and Dr . Alexis GUERIN
for their kind helpongathering literatures; Dr . Pierre SOURZAT
and Dr . Yun WANG are highly appreciated for helping to check
the mycorrhizae formed; and Dr . Yun WANG, Dr . Christine
FISCHER, Dr . Alessandra ZAMBONELLI and Dr . Randy MO-
LINA critically reviewed the manuscript and kindly offered in-
valuable suggestions .
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