全 文 ：菌 物 学 报 23（4）：563~567, 2004
Mycosystema
ABILITY OF ECTOMYCORRHIZAL FUNGUS TRICHOLOMA
MATSUTAKE TO UTILIZE CELLOBIOSE
LUN Zhi-Ming LI Yu-Hua VAARIO Lu-Min*
(Institute of Flower Biotechnology, Northeast Forestry University, Harbin 150040)
ABSTRACT: Six strains of an ectomycorrhizal fungus, Tricholoma matsutake, were investigated
for carbon nutrition and production of β-glucosi ase in liquid RM media. Cellobiose can serve
as a good carbon source comparing with glucose for all six strains. Xylan is also utilized
by all tested strains, though the utilization is limited in contrast with glucose or cellobiose.
The results provide an indirect proof for the potentiality of T. ma sutake in utilizing natural
organic materials. Data reported here may contribute to formulate new culture substrates for
co-cultivation of T. matsutakeand its host plant under controlled conditions.
KEY WORDS: Ergosterol assay, glucose xylan, carbon source
1 INTRODUCTION
Tricholoma matsutake(S. I o t Imai) Sing. (Matsutake), one of the most precious
edible mushrooms in the world, is an ectomycorrhizal fungus. However, there has been
conflicting debate ov r many years regarding the trophic status of T. matsutake. The
degree of uncertainty is well illustrated by Wang et al. (1997) who, in conclusion, wrote
it appears that T. matsutake occupies zones within the triangle defined by pathogen,
saprobe and symbiont and gravitates towards one or another depending on the time of year.
It has been reported that some ectomycorrhizal fungi possessed significant ability
to hydrolyze enzymatically various components of the plant cell wall in vitro (Cao &
Crawford, 1993). Terashita et al (1995) found that T. matsutake exhibite high levels
of both CM-cellulose and avicelase activities in potato dextrose medium. Vaario et al.
(2003) also reported that some strains of T. matsutake were capable of utilizing Japanese
Red Pine bark and beech sawdust in aseptic culture.
As concerning the carbon metabolism characterizes of T. mat utake, Ogawa (1978)
concluded that T. matsutake was capable of utilizing such as glucose monosaccharide,
yet was unable to utilize cellobiose and manntose.
We are interested in characterizing the carbon nutrition of T. matsutake to
understand more about its ability to grow saprophytically as well as in symbiotic
mycorrhizal associations. Toward this goal, in the present study, we investigated the
utilization of various carbon sources and measured the activities of β-glucosid e,
one of the cellulolytic enzymes by using six isolates of the T. matsutake.
2 MATERIALS AND METHODS
2.1 Isolates and culture conditions
Tricholoma matsutakeisola es W3, F1, F2, Y1, A and T945 were used in this study.
W3 was isolated from mature fruit bodies growing on beneath Pinus densifl ra S eb.
et Zucc. roots from Wangqing (43° 23′ 31″ N, 130° 1′ 57″ E, 650m altitude)
and, F1and F2 were isolated from Fuyu (42° 27′ 45″N, 129°34′ 14″E,

*Corresponding author
Received:2004-05-21, accepted: 2004-06-09
DOI:10.13346/j.mycosystema.2004.04.018
564 菌 物 学 报 23卷
500m altitude) in Jilin province of China; isolate A was kindly provided by Prof. W-J. Fu (Horticulture
Department, Yanbian University, China), T945 by Dr. K. Iwase (Biological Environment Institute, Kansai
Environmental Engineering Center Co. Ltd., Japan) and Y1 by Ms. K-M. Su (Forestry Research Insititute of
Chuxiong Prefecture, Yunann Province). Stock cultures were maintained on Ohta medium (Ohta, 1990)
containing 1.4% agar at 23oC in darkness and were subcultured bimonthly.
Five different liquid media were prepared: a modified RM medium (Vaario et al., 2002) without glucose
(N); a RM medium containing glucose 10g/l (G); a modified RM medium containing D-(+)-cellobiose
(Shanghai Lizhu, 500075) 10g/l (B); a modified RM medium containing xylan (Sigma X-0627) 10g/l (X); a
modified RM medium containing cellulose (Sigma C6288-250G) 10g/l (C). The final pH of all these media
was adjusted to 5.1 with 1N HCl solution before autoclaving at 121oC for 20min.
2.2 Growth in different carbon sources
As inocula, 8-mm-diameter mycelial plugs were cut from the margin of an actively growing colony on
Ohta agar medium. Two of them were cultured in 50 ml Erlenmyer flasks containing 5 ml aseptic liquid
medium. Four replicates were performed for each medium. The cultures were incubated at 23oC in darkness,
in stationary culture.
2.3 Ergosterol assay of mycelia
After 5 weeks of incubation, the mycelium of each culture was collected over a nylon mash Filter
(24×30µm) and freeze-dried. Ergosterol, an estimate of mycelial quantity, was then extracted in absolute
ethanol and assayed by HPLC (Martin et al., 1990). The control, which was inocula, also was done for
ergosterol assay. The culture filtrates of G and B were collected for estimation of ß-glucosidase activities.
2.4 ß-glucosidase activity measurement of culture filtrates
The 1, 4-ß-glucosidase activities were determined in filtrates, which were from medium G and B,
collected as described above, by monitoring the release of p-nitrophenol from p-nitrophenol-ß-D-
glucopyranoside, according to Habu et al. (1997), except that the incubation time was extended to 30min.
One unit (U) of ß-glucosidase was defined as the amount of enzyme required releasing 1 µmol of
p-nitrophenol per minute.
2.5 Statistical analysis
Multiple comparisons of fungal ergosterol content were made using one-way ANOVA LSD-Dunnetts C
test (P<0.05) and the ß-glucosidase activities were subjected to the Independent-simples T test (P<0.05) of
SPSS 10.0 (SPSS Inc.) for Windows.
3 RESULTS
3.1 Utilization of carbon nutrition by ergosterol assay
In the medium lacking carbon (N), the growth of all six isolates couldnt be observed with naked eyes.
For the modified medium containing cellulose (C), the result was similar. However, after one week of
incubation slight mycelia growth was noted in the liquid medium containing glucose (G) and cellobiose (B).
After 2 weeks, the growth was more obvious, especially for Y1 and T945. In the fifth week, two large
spherical colonies were formed in the medium G and B. When using xylan as sole carbon source, the
discernible growth couldnt be observed, nevertheless some extending peripheral areas of the plugs were
found after 5 weeks of incubation.
As shown in Fig. 1, the quantities of ergosterol produced by all six isolates were significantly higher in
4期 伦志明等：松口蘑对纤维二糖的利用能力 565
566 菌 物 学 报 23卷
4 DISSCUSSION
Though there has been conflicting debate over many years regarding the trophic status of T. matsutake
(Wang et al., 1997), some works have demonstrated that T. matsutake was capable of forming a typical
ectomycorrhizal association (i.e. Gill et al., 1999). Still, these demonstrations do not exclude the possibility
that T. matsutake also has some saprotrophic potentialities. The saprotrophic growth of T. matsutake in soil
has been reported recently (Guerin-Laguette et al., 2003). Vaario et al (2002) also noted that some isolates of
T. matsutake were able to grow over Pinus densiflora bark fragments either on soil or on agar media. In
addition, Vaario et al. (2003) observed that T. matsutake isolate T945 was capable of degrading plant cell
wall in medium lacking an exogenous carbon source thus primarily satisfying its carbon requirement from
beech sawdust. Meanwhile, they quantified cellulase activities by p-nitrophenyl-ß-D-lactopyranosidase
(p-NPL) assay (p-NPL mainly releases cellobiose from cellulose).
In the present study, the results suggest that the cellobiose can serve as good carbon source to support
the hypha growth of T. matsutake in the liquid medium and is comparable with the glucose. We also found
that some isolates of T. matsutake were able to utilize the xylan as sole carbon source to sustain their limited
growth, on the contrary, no any growth was found on cellulose-containing medium except for T945. While
there have been a lot of studies about nutrients media for hypha growth of T. matsutake, most of them
suggested that T. matsutake was not able to utilize cellobiose as a good carbon source (Ogawa, 1978) or very
limited to grow on cellobiose-containing medium (Yang et al., 2000). Here, our finding doesnt agree with
these previous reports. As we known, cellobiose isnt a simple carbon source that could be provided by plants
directly, but it could be released from complex carbon source with cellulolytic enzymes by soil organisms.
Also T. matsutake showed some activities of cellulase by p-nitrophenyl-ß-D-lactopyranosidase (Vaario et al.,
2003). Terashita et al. (1995) reported that T. matsutake produced relatively high levels of CM-cellulase and
avicelase, though they were lower than those of Lyophyllum shimeji. It could be presumed that T. matsutake
was able to degrade and/or utilize some complex carbon sources.
The growth of T. matsutake isolates on cellobiose/xylan-containing media evinces that T. matsutake has
a potentiality of utilization of a complex carbon source. This potentiality may play an important role during
fruit bodies forming periods. Indeed, both symbiotic and saprotrophic carbon sources may be required for the
development the Shiro of Matsutake. However, it is very difficult to culture large amounts of mycelia using
monosaccharides in a pure culture because of the osmotic pressure in the medium (Ohta, 1997). Recently,
although a number of researchers have described the aseptic in vitro synthesis of Matsutake mycorrhizas
(Vaario et al., 2000), the maintenance and further development of these artificial associations present
difficulties. The carbon supply is regarded as being an influential factor in the establishment of
ectomycorrhizas (Duddridge, 1986). Maybe the potentiality of utilization of complex carbon source by T.
matsutake will provide new insights into establishing new cultivating substrates for T. matsutake in nature.
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[附中文参考文献]
杨民和, 杨新美, 陈立国, 2000. 松茸的营养生理及培养基的筛选. 菌物系统, 19: 272~277

松口蘑对纤维二糖的利用能力
伦志明 李玉花 傅禄敏*
(东北林业大学花卉生物工程研究所 哈尔滨 150040)
摘 要：本文采用液体培养的方法，对六个松口蘑菌种的碳素营养生理及培养液中的 ß-葡萄糖苷酶的
活性进行了分析。结果表明，纤维二糖能很好的支持所有菌株的生长，其效果不逊色于葡萄糖；木聚
糖能够一定程度地维持各个菌种的生长，但效果不及葡萄糖和纤维二糖。这些数据为松口蘑具有一定
的利用天然有机碳源的潜在能力提供了间接的证据，也为松口蘑人工栽培基质的调制提供了新的思路。
关键词：纤维二糖，麦角固醇，松口蘑
中图分类号：Q939.96 文献标识码：A 文章编号：1672-6472（2004）04-0563-0567