全 文 ：Received 27 Feb. 2003 Accepted 10 May 2003
Supported by the National Natural Science Foundation of China (39970894).
* Author for correspondence. Tel: +86 (0)10 62899729. E-mail: .
http://www.chineseplantscience.com
Characterization of Sclerotial Formation from Hyphae of Grifola umbellata
WANG Qiu-Ying*, GUO Shun-Xing, FAN Jin-Yan, XUE Mei
(Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing 100094, China)
Abstract: Grifola umbellata (Pers.) Pilat is a fungus whose sclerotium has potent pharmaceutical values.
The present study shows that its sclerotia can be reproduced from the hyphae, in addition to its traditional
mode of reproduction from sclerotia. On a culture medium, the hyphae first become swollen and enlarged
followed by differentiation to form a highly organized structure, sclerotia. We demonstrate that at a
microscopic level, sclerotia derived from hyphae and sclerotia obtained in the field are similar, both having
cortex and medullary substances.
Key words: sclerotia of Grifola umbellata ; hyphae of Grifola umbellata ; microstructure; fungus marrow
Grifola umbellata is one of the commonly used and
precious medicinal fungi, with sclerotia being widely used
in Chinese medicine as a diuretic agent as well as an antidote.
Unlike other sclerotium-forming fungi, such as Omphalia
lapidescens, Poria cocos and Claviceps purpurea whose
sclerotia can be formed from hyphae, the sclerotia of G.
umbellata has been known to be able to reproduce only
from sclerotia, not hyphae. Much of efforts, both in China
and in the international research communities, have been
given to the alternative, reproducing G. umbellata sclero-
tia from hyphae, with little success however reported to
date.
One report showed the formation of cortex-less “scle-
rotia” by means of suspension culture of Grifola umbel-
late hyphae. Like many other fungi, such a structure is
often formed in a static suspension culture by agglomera-
tion of hyphae, and is completely different from the true
sclerotium of G. umbellata, which is a dormant structure
with distinct cortex and medulla.
In the present report, we describe our method of pro-
ducing G. umbellata sclerotia from hyphae on a nutrient
medium under a culture condition, show the microscopic
structure of the hyphae, and compare microscopically the
sclerotia resulted from the culture to that obtained from
sclerotia in the field. We refer to the sclerotia produced
from hyphae on the culture medium as artificial sclerotia,
and those from the field in the mountain as wild-type
sclerotia.
1 Materials and Methods
1.1 Materials
Artificial sclerotia were produced from hyphae of Grifola
umbellate (Pers.) Pilat on a culture medium. Two types of
sclerotia were obtained, one with dark colored cortex and
the other colorless cortex. Wild-type sclerotia were collected
from the mountainous area in Beipingzheng, Gubei County,
Shanxi Province. The mycelium was grown on the wheat
bran-glucose agar medium.
1.2 Methods
Artificial sclerotia of less than 10 mm in diameter on the
culture medium were collected from the medium surface,
and cuboids of 3 to 7 mm3 (length×width×height) wild-
type sclerotia were taken from the field. Mycelium samples
were taken as cubes similar to a soybean grain in size from
the white and vigorously growing mycelium. All samples
were fixed in a fixing solution, sectioned by conventional
paraffin method (Yu, 1989), stained by Safraine-Fast green
double staining, and observed and photographed under a
microscope. Sections were sliced in 5 mm thickness.
2 Results
2.1 Development and reproduction of artificial sclerotia
The hyphae of Grifola umbellata grew very slowly on
the medium, taking its appearance as brownish keratoids.
After growth about one to two weeks, the hyphae broke
up to release conidia which germinated rapidly and cov-
ered the whole medium plate.
Two developmental pathways were observed following
germination of the conidia. The first pathway, the germi-
nated conidia gave rise to mycelia, which became differen-
tiated to form cortex followed by sclerotium formation (Fig.
1). Alternatively, in the presence of light, the mycelium
grew into a shell-shaped brownish structure. Under the
microscope, the shell-shaped structure was similar to the
Acta Botanica Sinica
植 物 学 报 2004, 46 (3): 328-331
WANG Qiu-Ying et al.: Characterization of Sclerotial Formation from Hyphae of Grifola umbellata 329
Figs. 1-10. 1. Direct formation of the sclerotium from the way of conidiospore to hyphae, to cortex, and to sclerotium, ×500. 2.
Hyphae germinated from conidospore turning brown under light and becoming shell-shaped. The microstructure of shell-shaped myce-
lium is similar to that of a sclerotium, ×2 000. 3. Newly formed sclerotia in an Elemyer flask, ×0.5. 4. Thin and hairy hyphae growing
out of cuticula, ×1 000. 5. Thin and hairy hyphae originating directly from cuticula, ×2 000. 6. Hyphal clusters protruding out of
cuticula. 7. Thin-walled and swollen hyphal cells, ×2 000. 8. Differentiation of cortex before sclerotium formation, ×1 000. 9. Two
types of hyphae coexisting within the newly developed sclerotium, × 2 000. 10. Cavity within medulla of a new sclerotium. Abbreviations:
S, sclerotia of Grifola umbellata; V, cavity of G. umbellata sclerotia.
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004330
cortex of sclerotium, and stained red with Safraine (Fig.2).
The second pathway, when the growth condition was met,
the hyphae beneath the shell-shaped structure began to
grow rapidly, protruded out of the mycelium, and became
interweaved tightly to form a sclerotium. Under the favor-
able growth conditions (i.e., no infection and no light), this
type of sclerotia spread all over the culture medium. Ap-
pearing in white and with the characteristic mushroom
aroma, the sclerotia grew as large as 7 cm×3 cm×2 cm in
size (Fig.3) and had a similar elasticity to the wild-type
sclerotia.
After about two months, the new sclerotium formed a
cortex. Meanwhile, the hyphae within the sclerotium started
a new cycle of growth. They became in contact with the
nutrient on the medium. In this case, some of the hyphae
grew rapidly by cell division and then broke up from the
cortex. Different morphological characteristics were ob-
served among hyphae growing out of the cortex. Some
hyphae were hairy and originated either from the inside or
from the surface of the sclerotium, while others were in
cluster and were derived from the interior of the sclerotium.
The latter were composed of cells with large cavity, thin-
walled, and capable of developing new sclerotia. When
contaminated, the developing sclerotium secreted onto its
surface a large amount of brownish droplets which dried
out as dark spots on the sclerotium. When there was light,
on the other hand, the sclerotium ceased growth and
changed its surface color from white to gray.
2.2 Morphological characteristics of the hyphae in the
artificial sclerotium and on the culture medium
The artificial sclerotium was derived from the cultured
hyphae and was made of the hyphae in a highly differenti-
ated and organized state.
Within an artificial sclerotium, the hyphae were highly
packed and specialized, irregular in cell shapes, and quite
different from those on a culture medium. The latter were
colorless, hyaline, with a similar diameter of 2-5 mm,
branched, made up of elongate thick-walled cells, and have
septa and clamp connections. Development of the sclero-
tium started with differentiation of vegetative hyphae. Swol-
len cells with thin walls were first developed from these
hyphae. The hyphal cells were colorless, hyaline, branched,
and have clamp connections and septa. Each of these cells
was short and wide, different in size sometimes diameter as
large as 10 mm (Fig.7). These hyphae gave rise to the cortex
of the sclerotium. Microscopic examination showed that
the color of the cortex was darker than that of the inner part
of the sclerotium (Fig.8). The rapid growth of hyphae in the
cortex and their conglomeration eventually led to the
formation of the sclerotium. There were two types of hy-
phae in the sclerotium. One had thin walls, and was called
structural hyphae, and the other was sparse hyphae or con-
necting hyphae. The connecting hyphae were very thin-
walled with a diameter of only 1 mm, and did not have any
septa, branching and clamp connections, spread across
the artificial sclerotium. The thin-walled hyphae were simi-
lar to those of cultured hyphae, having branching and clamp
connections. The thin-walled hyphae within the sclerotium
had more branching and were irregular in shape than those
found in the cultured hyphae.
2.3 Microstructural comparison between artificial and
wild-type sclerotia
Artificial sclerotia and its wild-type counterparts were
similar in their highly differentiated structures. There were
two different types of hyphae in a sclerotium: thin, long
hyphae, and wide, short hyphae (Fig.9). Both types can be
either thin-walled or thick-walled (Guo and Xu, 1991). Fol-
lowing the differentiation, different structures were observed
in a sclerotium. Arranged from the outer layer to the core,
there are cuticla, subcuticla, sparse layer and structural layer.
The new sclerotium was derived from a cluster of hyphae
from an old sclerotium. These hyphae grew rapidly and
eventually led to form a new sclerotium. An abscission layer
might be observed between the old sclerotium and the new
one. Since the newborn of the wild type was growing
underground, it was subjected to the pressure exerted by
surrounding soil and stones, while the artificial sclerotium
was differentiated from hyphae growing on the medium.
This may explain why artificial sclerotium shared some simi-
larities to the wild-type sclerotium but differed in other
aspects. The growth period of artificial sclerotium was about
one to two months, which was similar to that of wild type.
Sparse hyphae and structural hyphae could be observed in
artificial sclerotia shortly after its formation, but the similar
structures could be seen only in those wild sclerotia older
than one year. A new sclerotium in the field was mainly
made up of swollen hyphal cells that were similar to those
found in the artificial sclerotium on the medium. Those hy-
phae close to cortex became thinner, and hyphae wall be-
came thicker. The hyphae in a newly formed wild-type scle-
rotium were more tightly packed than that in an artificial
new sclerotium. The other characteristics, such as cavity in
medulla (Fig.10) and hyphae growing on the surface of cor-
tex were similar both in the field and on artificial medium.
3 Discussion
Sclerotia of Grifola umbellata derived from the culture
have not been reported so far, although one article claimed
WANG Qiu-Ying et al.: Characterization of Sclerotial Formation from Hyphae of Grifola umbellata 331
that a “sclerotium” without cortex had been formed in a
suspension culture. Like any other fungi, hyphae agglom-
erate can be formed during a static suspension culture of
Grifola umbellata. The sclerotium is, however, a dormant
structure consisting of cortex and medulla (Guo and Wang,
2002). The sclerotium obtained in our laboratory possesses
both cortex and medulla. Compared with the wild-type
sclerotium, the artificial sclerotium had unique
characteristics. On the one hand, the artificial sclerotium
was formed by tight interweaving of the swollen and differ-
entiated hyphae from the medium culture. Some qualitative
changes have happened. Because of specialization and in-
teractions between them, sclerotium-forming hyphae were
quite different from the vegetative hyphae on the culture
medium.
Our newly formed artificial sclerotia were grown in flasks
containing the culture medium, while wild-type sclerotia
were formed in the soil. The different growth conditions
were responsible for the differences observed between the
two types. For example, beside interactions between hy-
phae within a sclerotium, the wild-type sclerotium had to
suffer the pressure from the surrounding soil. Despite some
differences in the morphology of sparse hyphae and in the
compactness of hyphae within the sclerotia, both the artifi-
cial and wild-type sclerotia were similar in their basic
structures. Further studies at the ultrastructural level are
needed in order to elucidate the difference among the hy-
phae in newly formed artificial sclerotium, the wild-type
sclerotium, and the vegetative hyphae on the culture
medium.
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Guo S-X , Xu J-T. 1991. Origin and development of crystal and
thick-walled cells in sclerotia of Grifola umbellata. Acta Mycol
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(Managing editor: WANG Wei)