全 文 ：菌 物 系 统 22（3）：369~373, 2003
Mycosystema

INTERACTION BETWEEN ROOT AND CALLUS OF PINUS DENSIFLORA
AND AN ECTOMYCORRHIZAL FUNGUS, TRICHOLOMA MATSUTAKE*
Lu-Min VAARIO1, 2* Kazuo SUZUKI2
(1The Laboratory of Quantitative Vegetation Ecology，Institute of Botany， The Chinese Academy of Sciences, Beijing 100093, China)
2 The laboratory of Forest Botany, Graduate School of Agricultural and Life Sciences,The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku,
Tokyo 113-8657, Japan)
ABSTRACT: A simple in vitro system is described for interaction between tissues of Pinus densiflora and
an ectomycorrhiza fungus, Tricholoma matustake. The callus and roots, which were initiated from
hypocotyl segments, were inoculated with T. matsutake in artificial condition. After two-week incubation,
fungal hyphae had colonized the root surface. Fungal hyphae occurred within the root cortex, and Hartig
net was formed after three-week. A few fungal hyphae and Hartig net-like structures were also visible
within the callus after three-week, while they were rather less abundant in callus as compared with roots.
KEY WORDS: Hartig net, in vitro, mycorrhization, pine tree, tissue
1 INTRODUCTION
Tricholoma matsutake (S. Ito et Imai) Sing., Matsutake, is one of the most renowned edible mushrooms in the
world (Ogawa, 1978; Wang et al., 1997). The rapid decrease in natural production and high economic value has
raised interest in developing an artificial cultivation system. The artificial cultivation of this mushroom has so far
proved elusive (Iwase, 1997). Our previous works have established a system for mycorrhiza formation in vitro
(Guerin-Laguette et al., 2000; Vaario et al., 2000), however, less is known about the biochemical and physical signals
of recognition in this ectomycorrhiza (ECM) and during fruiting. To gain knowledge about these fundamental views
simplified experimental techniques were often needed.
Dual cultures of fungi and plant cells can provide useful models for the study of host-fungus interaction, since such
a simplified system allows control of nutritional and environmental factors. There is a little information on the
application of in vitro culture methods on investigating the reactions between fungi and host tissue (Hendry et al., 1993;
Sirrenberg et al., 1995; Niemi, et al., 1998; Kvaalen & Solheim, 2000). However, there is little report about T. matsutake
in this area.
The aim of the present study was to establish in vitro root-organ and callus culture system for studying the
interactions between T. matsutake and Pinus densiflora.
2 MATERIALS AND METHODS
2.1 Plant material
Seeds of Pinus densiflora Sieb. et Zucc.were collected in 1996 in a natural forest at Tanashi, The University of
Tokyo. They were air-dried and stored in darkness at 4°C. Seeds were sterilized (H2O2 30% for 30 min), dried on
sterilized paper and placed on water agar (1%) medium containing 2 g/l glucose. After 7-10d of incubation (3000lux
fluorescent light, 23±2°C, 16h photoperiod), the small seedlings with 1-2 cm hypocotyls and 3-4 needles were used

*E-mail: luminfu@ns.ibcas.ac.cn
Received:2002-12-27, accepted: 2003-01-27
DOI:10.13346/j.mycosystema.2003.03.008
370 菌 物 系 统 22卷

for initiation of callus and roots formation respectively.
Hypocotyl segments about 1 cm long and needles were cut from young seedlings and placed on modified SH
medium (Schenk & Hildebrandt, 1972) containing cytokinin 0.5 µM (BAP, Zea, 2ip, or 4PU respectively) and 0.05
µM auxin (NAA). Thirty explants were used for each treatment.
2.2 Fungal isolates
Tricholoma matsutake (deposited as strain T3 in the culture collection of the Laboratory of Forest Botany, The
University of Tokyo) was isolated from fruit-body growing beneath P. densiflora in Nagano Prefecture. Stock
cultures were maintained on agar medium (Ohta, 1990) in darkness at 23°C.
Interaction between T. matsutake and roots and callus of P. densiflora
Each three segments of root or callus was transferred onto a modified rinse medium (RM) containing 0.5 µM
BAP, 0.05 µM NAA and 0.1g/l glucose in one petri dishes (90 mm) (Vaario et al., 2002). Then, three mycelial plugs
(6 mm diameter) cut from the margin of 1-month-old fungal colony were also placed into the same petri dish, about 1
cm from each explant. The controls were cultured on the same medium without fungal mycelia. The cultures were
incubated at 23±2°C (3000lux fluorescent light, 16h photoperiod).
2.3 Microscopy observation
Interaction between T. matsutake and adventitious roots and callus of P. densiflora was examined with light
microscopy. After 3-week incubation, both explants, which were from RM solid medium were sampled and cleared
in 10% H2O2 at 90°C for 120 min, bleached in 10% H2O2 in 10% KOH for 10 min, acidified with 0.1 N HCl for 5
min and then stained with Chlorazol black E for 90 min at 90°C (Gill et al., 1999). Following destaining for 8 h in
glycerol, the roots and callus were mounted in glycerol beneath a coverslip and sealed to give a semi-permanent
preparation. Mounted whole roots and callus were examined with an Olympus microscope BH2 fitted with standard
brightfield optics. Photographs were taken on Fuji 100ASA Provia color reversal film.
3 RESULTS
The adventitious roots occurred on more than 80% hypocotyl segments induced by 0.5µM BAP and 0.05 µM
NAA, but they also occurred on less than 10% hypocotyl segments induced by 0.5µM Zea and 0.05 µM NAA.
Callus occurred on all tested hypocotyls, but few from needles.
Interaction between adventitious roots and callus and T. matsutake:
After Two-week incubation, the margin of colony reached the adventitious roots. The adventitious root surface was
colonized by fine discrete hyphae (Fig.1) after three-week incubation. Fine individual hyphae formed connections
between root explants and elongating adventitious roots, which lacked root hair. Fungal hyphae occurred in the root
cortical intercellular spaces, and definite well-developed Hartig net structures were confirmed in adventitious root (Fig.2).
The margin of colony of T. matsutake reached callus, but most of the hyphae colonized on the bottom of the callus and
they did not overgrow the callus. No browning occurred in the tested callus (Fig.3). Multibranched fungal hyphae within
the intercellular spaces became visible (Fig. 4). However, well-developed Hartig net ‘palmetti’ was not observed in the
callus tissue.
4 DISCUSSION
Ectomycorrhiza formation between T. matsutake and P. densiflora under controlled conditions has been reported
in previous studies (Yokoyama & Yamada, 1987; Eto, 1990; Yamada et al., 1999; Guerin-Laguette et al., 2000;
Vaario et al., 2000). However, we demonstrated to achieve rapid infection between T. matsutake and P. densiflora
using non-intact host.
3期 傅禄敏等：赤松不定根和愈伤组织与松口蘑外生菌根菌的相互作用 371



In the present study, the adventitious roots were rapidly infected by fungal hyphae of T. matsutake, the evidence
of mycorrhizal infection was provided by well-developed Hartig net, i.e. a distinctive, multibranched Hartig net
structure. These Hartig net structures did not differ significantly from those described in situ by Gill et al. (1999),
which was inside P. densiflora lateral roots collected from beneath T. matsutake basidiomata, and also similar to those
described in vitro ectomycorrhizal infection on seedling roots (Guerin-Laguette et al., 2000; Vaario et al., 2000). This
indicated that adventitious roots of P. densiflora produced signals, which influenced fungal morphology to generate
mycorrhiza-like structures and those compatible mycorrhizal fungi specifically recognized these signals. Meanwhile,
during early stages of mycorrhiza formation, wall to wall contacting also seems to be crucial evidence. Bailey and
Fig. 1, 2. Pinus densiflora adventitious root from hypocotyl segment colonized by T. matsutake in vitro after 3-week
inoculation.
1. Hypocotyl segment (hs) and adventitious root (ar) are colonized by fungal mycelium that, in places, is highlighted by air
trapped within the fungal layer (arrow). Bar=1 mm.; 2. Whole adventitious root tip cleared, bleached and stained with
Chlorazol black E was mounted in glycerol beneath a coverslip. The intensified branching (arrow) of intercellular hyphal
apices could be observed in the center of root. Bar=12µm.
Fig. 3, 4. Pinus densiflora callus from hypocotyls segment colonization by T. matsutake in vitro after three weeks inoculation.
3. Fungal mycelium was not found on the surface of Hypocotyl segment (hs) and callus (c). Bar= 1mm; 4. Callus cleared,
bleached and stained with Chlorazol black E was mounted in glycerol beneath a coverslip. The early infection structures,
branched (b) hyphal apex colonized cortical intercellular spaces, could be observed. Bar= 12µm
372 菌 物 系 统 22卷

Peterson (1988) reported that Pisolithus tinctorius formed typical ectomycorrhizas on root cultures of Eucalyptus
pilularis, which showed that signals for fungus-host recognition were independent from plant shoots.
In case of the callus, Hartig net-like structures were also visible after three-week although no ‘palmetti’ Hartig
net was observed. These Hartig net-like structures were very similar to those described by Guerin-Laguette et al.
(2000) inside the root of P. densiflora infected by T. matsutake in vitro in the early stages (after 2- week inoculation).
Here, the hyphal apices began to undergo extensive branching, showing greater invaginations. This also proves that
host-fungus recognition does not require an intact host plant (Hendry et al., 1993; Sirrenberg et al., 1995).
There have been a few studies concerning host-ectomycorrhiza interaction by using of tissue culture. Sirrenberg
et al. (1995) observed mycorrhiza-like structures in an experiment with ECM fungi and callus cells derived from
radicles of Norway spruce. Other interesting results showed that early embryogenic stage Scots pine cultures were
able to interact with ECM fungi; and some fungi produced a positive reaction or even an increase in proliferation
(Niemi et al., 1998). Therefore, the presence of intercellular hyphae in callus infected by fungi is suggestive of a
close relationship between host and colonizing fungus (Hendry et al., 1993).
The validity of using culture tissues for studying some of the most challenging questions regarding biochemical,
genetical, and physiological relationships between fungi (arbuscular mycorrhiza, ectomycorrhiza) and their hosts is
supported by the fact that these tissues show similar mycorrhizal characteristics as the plants from which they were
developed (Bécard & Piché 1990; Schreiner & Koide 1993). Our demonstration in this experiment also agrees with
these results.
In vitro culture system, which allow control of most parameters and provides root or callus and fungal material
at various interactive stages, should permit more in-depth cellular, biochemical and molecular investigations into this
aspect (Wiemken, 1999; Fortin et al., 2001), and fulfill most of the requirements for selecting candidate fungal strains,
monitoring host responses and other physiological, biochemical questions.
ACKNOWLEDGEMENT This research was supported by the Bio-oriented Technology Research Advancement
Institution (BRAIN) in Japan. The authors thank Dr. Frederic Lapeyric (INRA, France), Dr. Warwick M.Gill
(Tasmanian Institute of Agricultural Research, Australia) and Dr. Alexis Guerin-Laguette (The University of Tokyo,
Japan) for their useful discussion on this work. The first author was also supported by an innovation fund from the
Chinese Academy of Sciences KSCX2-SW-104-04 and a grant from Ministry of Science and Technology project
G2000046805 when she prepared this manuscript.
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赤松不定根和愈伤组织与松口蘑外生菌根菌的相互作用
傅禄敏 1，2 铃木和夫 2
（1中国科学院植物研究所植被数量生态开放室， 北京 100093； 2. 日本东京大学农学与生命科学研究科， 东京 113-8657， 日本）
摘 要：本实验报道了以简单的离体培养方式来诱导赤松不定根和愈伤组织与松口蘑的菌根反应。不
定根和愈伤组织均起源于无菌苗的下胚轴，接种 2周后，菌丝体开始包围不定根。接种3周后，菌
丝体出现在不定根皮层细胞间，哈蒂氏网型的形成也同时被确认。在愈伤组织培养物中，细胞间也
能观察到菌丝体及拟-哈蒂氏网结构。这是第一个离体条件下成功地诱导赤松培养组织与松口蘑形成
外生菌根的报道。
关键词：哈蒂氏网，离体培养，菌根化，松树，组织
中图分类号：Q939.96 文献标识码：A 文章编号：1007-3515（2003）03-0369-0373