全 文 ：Mycosystema
菌 物 学 报 15 July 2008, 27(4): 554-558

jwxt@im.ac.cn
ISSN1672-6472 CN11-5180Q
©2008 Institute of Microbiology, CAS, all rights reserved.






Electron microscopic study of conidia produced by the
mycelium of Polyporus umbellatus

XING Xiao-Ke GUO Shun-Xing*
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing
100094, China


Abstract: The ultrastructure of conidia produced by the mycelia of Polyporus umbellatus was studied by use of
scanning and transmission electron microscopy. The results suggested that only clamped (dikaryotic) mycelia
produced aerial conidiophores with ovoid to rod-shaped conidia. There was a distinctive bulge near the top of the
conidium. The contents of the conidia were dense. This type of conidium could be regarded as arthroconidia.
Key words: asexual spores, ultrastructure


1 INTRODUCTION
Polyporus umbellatus (Pers.) Pilát, one of the rear medicinal fungi, belongs to the Polyporaceae
of the Basidiomycetes. The sclerotia of P. umbellatus is a traditional Chinese medicine known for
curing edema and for promoting a diuretic process. It was also used as herb medicine in other oriental
countries, such as Korea and Japan. Although various studies of ultrastructure and morphogenesis
have been done on sclerotia of P. umbellatus (Guo & Xu 1991a, b), the ultrastructural aspects of
conidium formed by mycelium have not been investigated. The objective of the present paper was to
illuminate the process of conidia formation, and provide information for better understanding the life
cycle of P. umbellatus.

2 MATERIALS AND METHODS

Supported by the Chinese National Science Fund for Outstanding Youths (No. 30325047)
*Corresponding author. E-mail: sxguo@implad.ac.cn
Received: 12-03-2008, accepted: 18-06-2008
DOI:10.13346/j.mycosystema.2008.04.025
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2.1 Fungal isolates and growth conditions
Fruitbody used in this study were collected from Shanxi Province of China. 0.2% malt agar in an
open Petri dish was exposed to an overhead fresh sporophore of P. umbellatus for a few minutes,
which was subsequently covered and incubated at 22 to 24 und℃ er dark condition. Several days later,
a colony was derived from a single basidiospore. A piece of agar with mycelia in enlarged colony was
then picked out at interval of 2 days. The agar block was examined under light microscope for hyphal
growth and conidiogenesis. As soon as the single conidium was observed, the materials were
prepared for transmission and scanning electron microscopy.
2.2 Transmission electron microscopy
The cultures were cut into blocks (2 × 2mm) and fixed in 0.1mol/L sodium cacodylate buffer
containing 2.5% glutaraldehyde, pH 7.6, for 4h at 4 , and postfixed in the same buffer℃ containing
2% osmium tetroxide for 2h at room temperature. Samples were dehydrated in an ethanol series,
transferred to propylene oxide, and infiltrated with mixtures of propylene oxide and araldite. After
transfer to fresh resin, specimens were polymerized at 40 and 60 . Radial℃ transverse sections were
cut by using a diatome diamond knife on a Leica UC6b Ultracut microtome, mounted on uncoated
200-mesh copper grids, stained with lead citrate uranyl acetate (Daddow 1983), and examined in a
JEOL 1230 transmission electron microscope.
2.3 Scanning electron microscopy
Mycelia of the colony and underlying agar were cut and fixed with 2% (wt/vol) aqueous osmium
tetroxide for 20h at room temperature, air-dried, and sputter-coated with gold palladium in a Hitachi
IB-5 sputter coater. The sclerotia were kept in desiccators before examination with a Hitachi S-570
scanning electron microscope.
3 RESULTS
Basidiospores of Polyporus umbellatus were allowed to fall for a few minutes on 0.2% malt
agar in an open Petri dish to give a dark deposit. The dish was then covered and kept at room
temperature. It was observed that 1% of the spores germinated in 5 days. In 10 days germination rose
to 10% and in 15 days 50%. In 20 days no fusions of hyphae were observed. After a further 10d
incubation conspicuous clamp connections formed. These were presumably dikaryotic. The dikaryon
grew slowly on PDA, and a colony from a central inoculum reached a diameter of 2cm in 20d at 20 . ℃
Initially the colony consisted of little branched hyphae on the surface of and submerged in the agar.
When it reached the margin of plate, conidiophores were still not produced. Further 10 days later,
aerial conidiophores were produced. The conidiophores were not different in diameter from
vegetative hyphae. However, the conidiophores (Fig. 1-A) were of more dense content and the
clamp-connections were more aggregate as compared with the vegetative hyphae. The initial process
of conidium formation was quite similar to that of clamp connection formation (Fig. 1-A). However,
the hyphal branch did not form a bridge connecting two adjacent cells at a septum but formed a
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conidiophore from which the conidium formed (Fig. 1-C, D). Sometimes, the septum was typical
dolipore septum with parenthesome (Fig. 1-B).
The clamped dikaryon derived from the mating of monokaryons formed a vigorous colony with
leading hyphae having clamps spaced 200-250μm, while in growing margin of the colony numerous
hyphal branches formed clamps spaced only 20-30μm. It was clear that the conidiophore was a
hyphal branch of limited growth. The apical cell of hyphal branch became swollen and then broke
away from mother hypha. The hyphal wall fragments were often attached to the conidia but they
disappeared gradually (Fig. 1-D, Fig. 2-A). This kind of conidium could probably be regarded as
arthroconidia. No retraction septa were involved in the conidial formation.


Fig. 1 Transmission electron micrographs of conidial formation of Polyporus umbellatus. A: Conidiophores of P. umbellatus.
The bulge (arrow) like initial clamp connection; B: Dolipore septum with parenthesome (arrow); C: Evident septum (arrow)
was formed at the apex of conidiophore from which a conidium broke away; D: Single conidium was breaking away from the
conidiophore. The conidium with attached hyphal wall fragments of its conidiophore (mother hypha) (arrow); E, F: Sectioned
conidium in longitudinal (E) and transverse (F) views. Compared to the conidiophores, the content of conidia were dense and
of more electron opaque content. Bar=2μm.


The conidia varied inconsiderably in size and form, and rod-shaped conidia measured 7-10 ×
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3-4.2μm. There was a distinctive bulge near the top of the conidium (Fig. 2-B). In contrast to the
vegetative hyphae, the contents of the conidia were dense, being initially finely granular, with
droplets appearing later which increased in size and decreased in number as the conidia matured (Fig.
1-E, F).


Fig. 2 Scanning electron micrographs of conidial formation of Polyporus umbellatus. A: Conidia round off one by one from the
conidiophores. The conidium with attached hyphal wall fragments (arrow) which disappeared gradually. Bar=15μm; B: Single
conidium of Polyporus umbellatus, with a distinctive bulge near the top of the conidium (arrow). Bar=12μm.

4 DISCUSSION
Buller (1931) stated that in most Hymenomycetes conidia were produced by monokaryon.
However, a number of species in which conidia are formed in the dikaryon as well, e.g. Flammulina
velutipes (Ingold 1980). In some species of Polyporus, asexual spores on both monokaryon and
dikaryon were formed (Ingold 1986). It has not been reported that whether asexual spores of
Polyporus umbellatus were produced by monokaryotic or dikaryotic mycelia or both of them. Our
observation revealed that the conidia of P. umbellatus could be produced by dikaryotic mycelia.
When the conidium just broke away from the conidiophore, remnant portions of the
mother-hyphal wall still attached to the conidium. This characteristic was also described in other
fungus, such as Paracoccidioides brasiliensis (Splend.) F.P. Almeida (Mercedes et al. 1991). Asexual
spores of P. umbellatus were all aerial and there were distinct conidiophores, and that was the case
with P. varius (Pers.) Fr. In P. squamosus (Huds.) Fr. retraction septa (septa formed for cause of
cytoplasm retraction) are conspicuously involved in the formation of conidia, while in P. umbellatus
no such septa are formed. Conidia of P. varius varied considerably in size and form, and only in a few
of them there was a distinctive bulge near the top of the conidium. In P. umbellatus the conidia varied
558 Mycosystema
little and most of them had a distinctive bulge near the top.
Conidia of P. umbellatus were not easy to be observed because of their few quantity produced at
the later phase of culture. During thriving growth of mycelia, no conidiophores and conidia were
observed. The conidia always formed 10 or more days later after the colony took up the whole
surface of medium. In fact, the conidial production has relation to age of mycelium and nutrition
exhaustion.

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