Mycorrhiza (2006) 16: 137142

DOI 10.1007/s00572-005-0026-5
ORIGINAL PAPER
Lu-Min Vaario
.
Shu-Tang Xing
.
Zong-Qiang Xie
.
Zhi-Ming Lun
.
Xue Sun
.
Yu Hua Li
In situ and in vitro colonization of Cathaya argyrophylla
(Pinaceae) by ectomycorrhizal fungi
Received: 14 September 2004 / Accepted: 7 September 2005 / Published online: 16 November 2005
# Springer-Verlag 2005
Abstract Cathaya argyrophylla, a critically endangered
conifer, is found to grow at four isolated areas located in
subtropical mountains of China. To examine the involve-
ment and usefulness of mycorrhizas for sustaining the
population of this tree, we compared the root system,
morphology, and structure of mycorrhizal roots of C.
argyrophylla, which were collected from a natural stand
and an artificial stand, each grown at a different location.
More mycorrhizal roots were found for trees from an
artificial stand. The presence of extramatrical mycelium,
mantle, and Hartig net revealed that C. argyrophylla
formed an ectomycorrhizal association in both sampling
sites. Starch granules were found in mycorrhizal roots
collected only from a natural stand. The aseptic synthesis
of C. argyrophylla and Cenococcum geophilum was
established for the first time in vitro. Typical ectomycor-
rhizas formed on seedlings on RM medium containing
0.1 g/l glucose, 5 weeks after inoculation. By light mi-
croscopy, the synthesized mycorrhizas showed a thin
mantle from which emanated extramatrical hyphae and
highly branched Hartig net. A simple, rapid, and con-
venient mycorrhiza synthesis system was developed,
which facilitates further studies on ectomycorrhizal de-
velopment of C. argyrophylla.
Keywords Cathaya argyrophylla
.
Ectomycorrhizal
fungi
.
In vitro
.
In situ
.
Hartig net
Introduction
Cathaya argyrophylla, which is a relic species of a mono-
typic genus endemic to China (Silba 1986; Vidakovic
1991), is distributed in provinces of Guangxi, Hunan,
Sichuan, and Guizhou (Fig. 1; Wang 1990). It grows at
narrow mountain ridges with sharp slopes, at the top of
solitary rocky mountains, or in the crevice of sheer prec-
ipices and overhanging rocks at altitudes of 9401,870 m.
Pollen fossils of this plant have been found in the Asia
Europe continental deposits of the Tertiary period; thus,
Cathaya is honored to be called as Panda of the plant
kingdom. The development of the embryo in Cathaya is
similar to that of Pinus (Hu et al. 1976; Hu and Wang 1984;
Wang 1990). C. argyrophylla, which is native to the
subtropical mountain regions of the northern hemisphere,
has great ecological importance within natural forests (Xie
1995). However, this tree is now facing problems in natural
propagation, which include seed loss due to predispersal
predation and the decreased seed germination rates, and
competition with shade-forming broad-leaved trees. Thus,
it is threatened by habitat loss (Ying et al. 1983). The
population is decreasing year by year. It is reported that
there are less than 5,000 trees in China (Xie et al. 1999).
This species is listed as endangered by the State En-
vironmental Protection Administration of China (1992).
As mycorrhizas can improve fitness of plants, knowl-
edge of mycorrhizas is particularly important for ecological
restoration, preservation, and maintenance of endangered
species (Smith and Read 1997). In particular, ectomycor-
rhizal fungi provide a major link between carbon fixed by
primary producers and other trophic levels in plant eco-
systems. Information on the mycorrhizal status of C.
argyrophylla can be crucial for the implementation of
successful conservation strategies; however, it is little
available so far.
Our present study had two objectives. The first one was
to verify whether C. argyrophylla is a mycorrhizal species
and to compare the mycorrhizal status of these trees from
both a natural stand and an artificial stand. The second was to
characterize in vitro the interaction between C. argyrophylla
L.-M. Vaario
.
S.-T. Xing
.
Z.-M. Lun
.
X. Sun
.
Y. H. Li (*)
Research Institute of Flower Biotechnology,
Northeast Forestry University,
Harbin 150040, China
e-mail: [email protected]
Tel.: +86-451-82191738
Fax: +86-451-82191738
Z.-Q. Xie
The Laboratory of Quantitative Vegetation Ecology,
The Institute of Botany, CAS,
Beijing 100093, China
and several ectomycorrhizal fungi with broad host ranges
and to establish a mycorrhizal seedling culture system in C.
argyrophylla.
Materials and methods
Study sites and sampling of soil
C. argyrophylla trees were investigated at two locations.
First site is a small plantation of 30 transplanted C.
argyrophylla trees at 2328 years old, located at Guangxi
Institute of Botany, Guangxi Zhuangzu Autonomous
Region, and The Chinese Academy of Sciences, Yanshan,
Guilin, China (elevation of 195 m, 250410N, 11017
59). Second site is a natural forest, where there were 219
C. argyrophylla trees, located at Dayaoshan, Jinxiu,
Guangxi, China (elevation of 1,100 m, 2492424N,
10927E). The oldest tree of them is more than 500 years
old. Five soil samples were randomly collected from each
site and stored at 4C until analysis. Soil pH (Thomas
1996), the contents of organic carbon, total nitrogen (The
Standards of Forest Systems, P. R. China, 2000), and
nitrate-nitrogen and ammonium-nitrogen (a Skalar SAN
Plus segmented flow analyzer, Skalar, Inc., Norcross, GA,
USA) were measured.
Collection and classification of root samples
Soil samples with five replicates for each site were
collected by digging out a soil core of 101010 cm near
a C. argyrophylla tree. They were put in paper bags, taken
to a laboratory, and kept at 4C. The soil core with roots of
C. argyrophylla was soaked in tap water of a beaker. Root
tips were observed under a stereomicroscope and catego-
rized by color as black, brown, white, or other color ac-
cording to Agerer (19872002). Percentage of colonization
by ectomycorrhizal fungi was measured using the gridline
intersection method (Brundrett et al. 1996). Only fine roots
with less than 2-mm diameter were counted in quantifica-
tion because wider roots were not colonized.
Fungal culture
Cenococcum geophilum and Pisolithus tinctorius, both of
which were deposited in the culture collection of Labora-
tory of Forest Botany, University of Tokyo, Japan, as
strains FBCg1 and Pt2, were kindly provided by Prof.
Suzuki. Boletus edulis was kindly provided by Prof. X-F
Yan, Northeast Forestry University, China. These three
isolates were maintained at 232C in darkness on mod-
ified MelinNorkran medium (MMN; Marx 1969), which
contained 10 g/l glucose instead of sucrose and was
solidified with 1.5% agar.
Preparation of plant material
Seeds of C. argyrophylla were collected in a natural forest
in Guangxi Province, China, in 2002. They were air-dried
and stored in a polyethylene bag in darkness at 4C. The
seeds were surface-sterilized in 70% ethanol for 1 min and
then in 30% H
2
O
2
solution for 30 min. Following air dry-
ing, they were placed on agar medium containing 5 g/l
glucose for 7 days, and then, the seed capsule was removed
with a knife and forceps. After germination, the seedlings
were transplanted on modified MS media (Murashige and
Skoog 1962), which contained 0.2 mg/l 6-benzylamino-
purine (6-BAP) and 0.02 mg/l -naphthaleneacetic acid
(NAA) with one fourth strength of macroelements, a
strength of microelements, and organic elements prescribed
for MS medium, 20 g/l sucrose, and 8 g/l agar.
Aseptic synthesis of ectomycorrhizas
Clear plastic culture plates (2009010 mm) were filled
with 80 ml RM medium (Vaario et al. 2002). For each
culture plate, one sterile seedling was laid directly on the
agar surface and covered with a sheet of autoclaved filter
paper (Hangzhou Xinhua Paper Ltd.) to maintain root
surface moisture. The plates were then incubated for 1 week,
after which the seedlings were well attached to agar me-
dium. The cover paper was then aseptically removed, and
Fig. 1 Distribution map of
Cathaya argyrophylla (from
http://www.plant.csdb.cn/sdb/
teyou/ty.htm)
138
subsequently, three plugs of fungal mycelium of 6-mm
diameter were placed on the medium adjacent to lateral
roots of a seedling. The plates were sealed with Parafilm
(American Can Company, Detroit, MI, USA). The lower
part of the plate, where both the host root system and
ectomycorrhizal (ECM) fungus were developing, was
covered with aluminum foil. Fifty-eight seedlings were
selected for ectomycorrhiza synthesis including 20 for
control, 20inoculatedwithC. geophilum, 10inoculatedwith
P. tinctorius, and 8 with B. edulis.
All incubations were carried out at 3,000 lx by diffuse
fluorescent light at 232C at a 16-h photoperiod.
Preparation for studying ECM structure
Mycorrhizal and control root samples collected at both
field and laboratory conditions were immersed in 2.5%
glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) in
0.1 M phosphate buffer (pH 7.2) overnight at 4C. They
were then rinsed three times in 0.1 M phosphate buffer
(pH 7.2), postfixed for 90 min in 2% OsO
4
(SPI-CHEM
TM, West Chester, PA, USA) in the same buffer, and
dehydrated in an ascending acetone series in 20% in-
crements, each for 20 min, followed by three changes, each
for 15 min, of 100% propylene oxide. The mycorrhizal
roots were finally embedded in Epon 812 resin (Heidel-
berg, New York). Thin sections were cut with a glass knife
on an ultramicrotome (Leica Ultraunt R) and gently heat-
fixed to glass microscope slides. The sections were then
stained in 0.1% toluidine blue in 1% acetate buffer for
10 min, kept in tap water for 15 min, air-dried, then
mounted in 100% glycerin beneath a cover slip, and ex-
amined with an Olympus BX51 light microscope.
Results
Site and soil investigation
Investigation of pedological soil showed a difference
between soils collected in a natural stand at Dayaoshan
and an artificial stand at Yanshan (Table 1). Dayaoshan soil
had a higher percentage of organic C and N as well as a
higher concentration of NO
3

-N than Yanshan soil. There

was no significant difference in NH
4
+
-N concentration of
soil between both sites. The pH of both site soils was lower
than 3.8, especially Dayaoshan soil, being strongly acidic
at pH 2.7.
Ectomycorrhizal roots were found in both sites. There
were four main types of mycorrhizal roots, which had such
characteristics in color of fungal mantle, type of ramifica-
tion, and features of mantle surface as shown in Table 2.
Two types were found in both sites. Many mycorrhizal
roots of white, glossy type were found at the Yanshan site
but not at Dayaoshan. Also, a much higher percentage of
mycorrhization was observed at the Yanshan site (Table 1).
Microscopic features of excavated roots
Microscopic observation revealed detailed characteristics
of mycorrhizal roots from both sites. A yellowish and
unramified type was selected from both sites for micro-
scopic observation (Fig. 2a). In root samples from Daya-
oshan, thick mantles and big tannin cells were found
(Fig. 2b,c). Intercellular space between cortical cells are
colonized by fungal hyphae (Fig. 2b,c). The highly
branched, distinctively multilobed, and fan-shaped Hartig
net was confirmed in plan view (Fig. 2d). In the root
samples from Yanshan, distinct mantles and Hartig net
were also found (Fig. 2e). In contrast, nonmycorrhizal roots
from both sites had root hairs but no Hartig net structure
(data not shown).
Development of mycorrhizal roots in vitro
C. argyrophylla + C. geophilum Three weeks after
inoculation, a taproot produced short lateral roots, some
of which came into contact with hyphae arising from the
fungal inocula. Lateral root formation occurred in all
Table 2 The macroscopic characteristics of ectomycorrhizas
collected from both a natural stand and an artificial stand
Type of mycorrhizas Dayaoshan Yanshan
Black, unramified, smooth mantle ++
Light brown, dichotomous, smooth mantle + +
Yellow, unramified or dichotomous, reticulate + +
White with gloss, monopodial pyramidal,
smooth
+++
*-, absent;
+, less than 25% tested samples
++, 2550% tested samples
+++, more than 75% tested samples
Table 1 Comparison of two sampling sites of Cathaya argyr-
ophylla
Site Dayaoshan Yanshan
Type of stand Natural stand Artificial stand
Altitude (m) 1,100 190200
No. of C. argyrophylla trees 219 30
Soil type Red soil Yellow soil
Humus type Fulvic acid type Fulvic acid type
Soil pH 2.70.3 3.80.2
C (%) 6.42.9 1.20.2
N (%) 0.50.0 0.20.0
NO
3

-N (ppm) 33.85.9 18.47.0

NH
4
+
-N (ppm) 24.116.8 9.21.3
Mycorrhizal colonization (%) 34.911.5 54.18.9
a
a
Using the gridline intersection method (Brundrett et al. 1996)
139
Fig. 2 External morphology and light micrographs of C. argy-
rophylla mycorrhizal roots and nonmycorrhizal roots. a External
morphology of C. argyrophylla mycorrhizal roots showing taproot
(tr), mycorrhizal root (mr), and external mycelium (em). Bar, 500 m.
b Light micrograph of a mycorrhiza collected at Dayaoshan. The
thick mantle (m) overlies the epidermis, which is filled with tannins
(t). A number of starch granules (double arrowheads) are present in
the cortical cells. The intercellular Hartig net (arrowheads) colo-
nized the epidermis and cortex. Bar, 100 m. c Magnification of
b showing thick mantle (m), tannin cells (t), and Hartig net structure
(arrowheads). Bar, 20 m. d Longitudinal section of mycorrhizal
roots collected at Dayaoshan showing the distinctively multi-
branched pseudoparenchymatous structure of the Hartig net in plan
view (arrowheads). Bar, 10 m. e Light micrograph of a mycorrhiza
collected at Yanshan. Mantle (m) overlies the epidermis. The
intercellular Hartig net (arrowheads) colonized the epidermis and
cortex. No starch granule is present. Bar, 20 m. f The view of an in
vitro mycorrhization between C. argyrophylla and Cenococcum
geophilum showing unramified short mycorrhizal root (arrowheads)
and elongated roots with a hyphal mantle at their base (double
arrowheads). Bar, 1 cm. g Magnification of f showing typical C.
geophilum mycorrhiza with black mantle. Bar, 2 mm. h Light
micrograph of in vitro uninfected root showing distinctive thick cell
wall (cw) of cortical cells and a number of starch granules (s) in
cortical cells. Bar, 30 m. i Light micrograph of in vitro mycorrhizal
roots. A loose and thin mantle (arrowheads) lies on roots. Hartig net
(double arrowheads) colonizes epidermis and cortex with no starch
granules present in the cell. Bar, 50 m. j Longitudinal section
through the distal cortex of a mycorrhiza showing the distinctively
multibranched pseudoparenchymatous structure of the Hartig net
(arrowheads). Bar, 10 m
140
seedlings inoculated with ectomycorrhizal fungi and also
occurred in 14 out of 20 control seedlings. After 5 weeks,
a network of black extraradical hyphae was formed around
the bases of short lateral roots, and some roots stopped
elongating. Some still elongated but failed to form root
hairs in C. argyrophylla + C.geophilum plates (Fig. 2f).
After 67 weeks, C. geophilum had completely enveloped
the entire lateral root and formed black mycorrhizas with a
rough mantle surface (Fig. 2g). However, some lateral
roots did not stop elongating and were only enveloped by
a mantle at the basal portion (Fig. 2f). C. argyrophylla +
C. geophilum mycorrhizas were straight and unramified
(Fig. 2f,g) and consisted of a thin, loose fungal mantle.
Mantle hyphae gave rise to profuse hyphae, which invaded
and colonized the epidermal and cortical cell walls, and
developed a partially thick Hartig net (Fig. 2i). Hartig net
hyphae invaded host cortex and enveloped host cells with
a uniseriate or multiseriate layer of fungal cells but did not
penetrate them (Fig. 2i,j). In cross section of an un-
inoculated control C. argyrophylla root grown in the same
culture plate, thicker cell walls of cortex were observed.
Furthermore, there were starch granules in cortical cells
(Fig. 2h). However, these starch granules were not found
in mycorrhizal roots (Fig. 2i).
C. argyrophylla + P. tinctorius Fungal hyphae contacted
lateral roots 3 weeks after inoculation but did not envelop
roots. Later, roots still elongated and formed root hairs. No
mycorrhization was confirmed by microscopy.
C. argyrophylla + B. edulis Fungal hyphae grew quickly
and covered the whole roots 3 weeks after inoculation.
Finally, hyphae overgrew the whole of a seedling, the
needles turned yellow, and then the seedling died. No
mycorrhization was found between these two partners.
Discussion
It was proved that C. argyrophylla was a typical ectomy-
corrhizal tree according to both studies in fields and
laboratories. Although mycorrhizas of C. argyrophylla
were reported previously in a Chinese language publication
(Wang 1990), this was the first time that ectomycorrhizas
were described in situ and in vitro. It was shown that C.
argyrophylla formed typical ectomycorrhizas similar to
other pine trees.
According to investigation carried out at two sites, C.
argyrophylla growing in an artificial stand had exuberant
foliage with high branching, fresh green needles, and a
sturdy trunk (Fig. 3). In contrast, C. argyrophylla in a
natural stand had sparse foliage, especially in young trees,
although they gained vigor once they grew over forest
canopy. With regard to growth behavior, some studies
suggested that Cathaya trees grew suitably in nutrient-poor
soils and that they were not shade-tolerant (Xie et al. 1999).
C. argyrophylla trees grew well in gaps formed in the
natural forests; however, adult trees were stressed by
inadequate light (Xie 1995). In an artificial stand, sun light
was provided enough for growth, and the contents of C and
N in soil were lower with a higher frequency of mycorrhiza
formation (Table 1). These results suggested that environ-
mental conditions in both stands of C. argyrophylla in-
fluenced tree growth and also mycorrhizal formation.
In the present study, there was a noticeable difference in
amount of starch in cortical cells between mycorrhizal
roots collected from two sites. Starch granules were fre-
quently observed in cortical cells of nonmycorrhizal roots
formed by trees in fields (data not shown) and in vitro
seedlings (Fig. 2h). There were starch granules in mycor-
rhizal roots collected in a natural stand at Danyaoshan
(Fig. 2b,c). However, no starch granules were found in
mycorrhizal roots in an artificial stand at Yanshan (Fig. 2e)
and in those of C. argyrophylla + C.geophilum in vitro
(Fig. 2h). Root starch is a kind of energy storage for trees
so that the presence of starch may imply relatively low
metabolism in roots. Once a mycorrhizal root forms, fungal
hyphae will utilize a carbon source provided by a host tree
for their growth and functions in roots and soil. One
possibility for the presence of many starch granules at
the Dayaoshan site might be that mycorrhizas were not
functional anymore when sampled, or mycorrhizas at the
Yanshan site might have been more active than those at
the Dayaoshan site. Jordy et al. (1998) reported that the
density of starch grains increased in plant cells of Paxillus
involutusBetulapendula ectomycorrhizas, especially at
the early infection stage.
The long-term goal of our study is to understand the
physiological and biochemical events that control ectomy-
corrhization of Cathaya trees. Therefore, it is necessary to
establish a simple, rapid, and convenient mycorrhiza syn-
thesis system. In this study, C. geophilum was selected as a
candidate for in vitro ectomycorrhizal synthesis in Cathaya
since C. geophilum has frequently been described in
Fig. 3 A mature tree of C. argyrophylla at approximately 26 years
old, growing at the artificial stand at Yanshan, Guangxi, China
141
ectomycorrhizal association with economically important
tree families such as Myrtaceae, Salicaeae, and Pinaceae
(Trappe 1962; Heslin and Douglas 1986; Danielson and
Pruden 1989). Furthermore, C. geophilum is considered an
excellent model for the isolation and regeneration of pro-
toplasts from ascomycetous ectomycorrhizal fungi (Stlten
et al. 1995). Mycorrhizas synthesized by our seedling
culture system showed typical characteristics of ectomy-
corrhizas. The presence of a mantle and a Hartig net in-
dicated that the mycorrhizal roots examined were typical
ectomycorrhizal associations between C. argyrophylla and
C. geophilum under the applied culture conditions.
Although both P. tinctorius and B. edulis are broad-host-
range fungi (Carroll 1992; Hall et al. 1998), they failed to
produce mycorrhizas in C. argyrophylla. Recently, Martin
et al. (2002) focused on host-fungus specificity of Pisolithus
species to showthat each species of Pisolithus was confined to
hosts originated from a single biogeographical area. This
suggests a need to test more isolates of mycorrhizal fungi or
isolates collected much closer to Cathaya trees.
An established in vitro system ensures that inoculum is
produced in laboratory conditions enough to apply prac-
tically to the forests. Furthermore, this system will enable
us to follow the morphological, physiological, molecular,
and biochemical changes during development of C.
argyrophylla + C. geophilum mycorrhizas and other host-
fungus ectomycorrhizas. Such a variety of application of
this synthesis protocol ensures that it continued to be used
further in ectomycorrhizal studies in vitro.
In conclusion, an endangered tree, C. argyrophylla, was
a typical ectomycorrhizal tree. However, whether mycor-
rhization substantially helps to reestablish the endangered
trees is still a question. At present, it is necessary to make a
longer-term comparison between mycorrhizal and non-
mycorrhizal seedlings under controlled conditions.
Acknowledgements The field investigations were supported by
State Key Basic Research and Development Plan (G2000046805) and
the laboratory studies by a grant from Northeast Forestry University
(010-602024). The first author also wishes to thank Mr. Shi-Jiang
Ning (Guangxi Institute of Botany, Guangxi Zhuangzu Autonomous
Region, and The Chinese Academy of Sciences) for help in the field
investigations.
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