全 文 ：Received 17 Apr. 2003 Accepted 17 Sept. 2003
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Ectomycorrhizal Synthesis Between Abies firma Roots/Callus and
Laccaria bicolor Strain
Lu-Min VAARIO1, 2, 3*, Kazuo SUZUK3
(1. College of Life Sciences, Northeast Forestry University, Harbin 150040, China;
2. Laboratory of Quantitative Vegetation Ecology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China;
3. Laboratory of Forest Botany, Graduate School of Agricultural and Life Sciences,
University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan)
Abstract: A simple in vitro system was employed for ectomycorrhizal synthesis of Abies firma Sieb. et
Zucc. with Laccaria bicolor (Maire) Orton. The aim of this present study was to study whether the symbio-
sis of A. firma broad occurred with L. bicolor and whether the symbiosis of A. firma only occurred at the
whole plant level. The results of the study show that the typical ectomycorrhizal structures, i.e. thick
mantle and intracortical Hartig net, were observed in the lateral roots of A. firma after incubation of 10
weeks. In case of callus, three weeks following incubation, fungal hyphae were visible within the
intercellular spaces of the callus cells and Hartig net-like structures were observed in transverse section.
This was the first report of aseptic ectomycorrhization of A. firma seedlings, and ectomycorrhizal
colonization on A. firma callus by L. bicolor. These results suggested that the callus system might be a
useful tissue system for mycorrhiza synthesis in the present study. This model system may facilitate
detailed studies on ectomycorrhizal development of Abies species.
Key words: Abies firma ; callus; ectomycorrhizas; Laccaria bicolor
Mycorrhizal associations have increasingly been rec-
ognized to have significant influences on forest ecosys-
tems by enhancing the nutrient uptake and improving the
adaptation to various environmental conditions of host trees
(Smith and Read, 1997).
The genus Abies is complex by comparison with other
genera of the family Pinaceae, with growing around the
world and including about 50 species (China National For-
est Bureau, 2001). All these taxa are native to the cool tem-
perate and boreal regions of the Northern Hemisphere, and
all of which form obligate mycorrhizas (Meyer, 1973). In
China, there are 23 species and three varieties (Liu et al.,
2002). Consequently, each Abies species has great ecologi-
cal importance within natural forests. Abies species are sen-
sitive to environmental stresses such as drought, low po-
tassium levels and air pollution (Freer-Smith, 1996), however,
it is difficult to grow in vitro (Saravitz and Blazich, 1996).
They are considered as indicators of sound forest vegeta-
tion in the warm temperate zone. Abies firma Sieb. et Zucc.
is a species endemic to the warmer regions of Japan
(Iwatsuki, 1995), its timber is used for various purposes
such as construction, packing crates, paper pulp and coffin
(Japan Forest Technical Association, 1964). A. firma is also
introduced into North and East of China (China National
Forest Bureau, 2001). In a few studies of naturally occurring
ectomycorrhizas (ECM) of Abies species other than A. firma
have been shown to host a number of naturally occurring
ECM fungi (Alvarez and Cobb, 1977; Acsai and Largent,
1983; Harley and Harley, 1987; Kernaghan et al., 1997). Within
Japan, a number of naturally occurring ECM fungi on A.
firma have been recorded (Masui, 1926; Matsuda and Hijii,
1998).
Laccaria bicolor (Maire) Orton, a broad host-range ECM
fungus (Le Tacon et al., 1992; Parlade et al., 1999), is very
frequently used in applied studies on ectomycorrhizas (Kropp
and Langlois, 1990) and also has been recorded in mycor-
rhizal association with some host trees (Molina, 1982; Perrin
et al., 1996). However, L. bicolor has not been recorded on
A. firma.
The studies on mycorrhizal development, function and
molecular biology within this genus Abies are not well
documented. The previous reports on the establishment of
an in vitro synthesis system between A. firma and Pisolithus
tinctorius (Pers.) Coker et Couch and Cenococcum
geophilum Fr. (Vaario et al., 1999; 2000) showed that the
typical ectomycorrhizal structures occurred in a simple in
vitro system. However, whether the symbiosis of A. firma
broad occurred with ectomycorrhizal fungi and whether the
Acta Botanica Sinica
植 物 学 报 2004, 46 (1): 63-68
Acta Botanica Sinica 植物学报 Vol.46 No.1 200464
symbiosis of A. firma only occurred at the whole plant level
are still unclear.
The objective of this study was to establish a simple,
easily manipulated model system for the in vitro synthesis
of ECM between A. firma and L. bicolor using seedling
and culture tissue, which may be applied to any number of
mycorrhizal fungus-host associations.
1 Materials and Methods
1.1 Fungal culture and plant material
Laccaria bicolor (Maire) Orton was kindly provided by
Dr. Lapeyrie FREDERIC (INRA, France) (Di Battista et al.,
1996). The isolate was stored on MMN (Marx, 1969) in dark-
ness at (23±2) ℃.
Seeds of A. firma were collected in a warm-temperate
natural forest at Chiba, University of Tokyo, which were
air-dried and stored in a polyethylene bag in darkness at 4
℃ until use. Seeds were sown in vermiculite, following im-
mersed in 1/2000 Benlate (Dupont Co. Ltd., USA) for 1 d
and germinated at room temperature under diffused fluo-
rescent illumination. Once germinated, the seedlings were
surface-sterilized in 70% ethanol for 1 min and then in so-
dium hypochlorite solution containing 1% (W/V) active
chlorine for 10 min. After three rinses in sterile deionized
water, they were soaked in 0.05% (W/V) mercuric chloride
for 6 min and finally rinsed four times with sterile deionized
water.
Hypocotyl segments about 1 cm long were cut from
young sterilized seedlings and placed on SH medium
(Schenk and Hildebrandt, 1972) containing cytokinin (BAP)
0.5 µmol/L and auxin (NAA) 0.05 µmol/L on a petri dish.
After four weeks, the callus was induced from each segment.
All incubations were carried out at 3 000 lx diffuse fluo-
rescent light at (23±2) ℃ with a 16 h photoperiod.
1.2 Aseptic synthesis of ectomycorrhizas
One mycelia-plug (6 mm diameter) cut from the margin
of a one-month-old fungal colony was placed on the center
of the Fungus-Host (FH) medium containing KNO3, 2 500
mg; (NH4)2HPO4, 300 mg; MgSO4·7H2O, 400 mg;
CaCl2·2H2O, 200 mg; FeSO4·7H2O, 15 mg; Na2EDTA, 20
mg; H3BO3, 0.5 mg; ZnSO4·7H2O, 0.1 mg; MnSO4·H2O, 1
mg; Na2MoO4·2H2O, 0.01 mg; KI, 0.1 mg; CuSO4·5H2O,
0.02 mg; CoCl2·6H2O, 0.01 mg; myoinositol, 100 mg; thia-
mine HCl, 5 mg; nicotinic acid, 5 mg; pyridoxine HCl, 0.5
mg; NAA, 0.05 µmol/L; BAP, 0.5 µmol/L; glucose, 0.1 g;
agar, 15 g; and distilled water to 1 000 mL. The pH was
adjusted to 5.6 with 0.1 mol/L NaOH before being auto-
claved (121 ℃, 20 min). After two weeks of incubation, two
segments of the callus were placed on FM medium,
adjacent to the margin of the fungal colony. Sterile cotton
rolls (10 mm ´ 5 mm) were placed along the bottom edge of
the plates which were sealed with Parafilmâ (American Can
Company, Detroit) and the lower portion of the plates, con-
taining both the developing host root system and the ECM
fungus, were covered with aluminum foil. Forty sterilized
seedlings were selected for ectomycorrhiza synthesis in-
cluding 20 for control.
Rectangular clear plastic culture plates (200 mm´ 90 mm
´ 10 mm) were filled with 80 mL FH medium. For each cul-
ture plate, two sterilized seedlings were put directly on the
agar surface and covered with a sheet of autoclaved
Advantec No.2 filter paper (Toyo Roshi Kaisha Ltd.) to
maintain root surface moisture. The plates were then incu-
bated for one week, after which the seedlings fixed well
with agar medium. Then the cover paper was aseptically
removed and five 6-mm diameter plugs of L. bicolor myce-
lia were placed on the medium, adjacent to the lateral roots.
Sterile cotton rolls (10 mm ´ 5 mm) were placed along the
bottom edge of the plates which were sealed with Parafilmâ
(American Can Company, Detroit) and the lower portion of
the plates, containing both the developing host root sys-
tem and the ECM fungus, were covered with aluminum foil.
Forty sterilized seedlings were selected for ectomycorrhiza
synthesis including 20 for control.
The cultures were incubated at (23±2) ℃, 3 000 lx fluo-
rescent light, 16 h photoperiod.
1.3 Preparation for ECM structure
Both mycorrhizal/control roots and colonized/control
callus were removed from the culture plates. The segments
of root 1-2 mm in length and callus tissue (2 mm3) were
fixed in 2.5% glutaraldehyde in 0.1 mmol/L sodium cacody-
late buffer (pH 7.2) containing 1% acrolein for 2 h under
vacuum at room temperature. The samples were washed
twice in 0.1 mmol/L sodium cacodylate buffer and were then
postfixed for 90 min in 2% OsO4 in 0.1 mmol/L sodium ca-
codylate buffer (pH 7.2) at room temperature, washed in
three changes of distilled water and dehydrated in an as-
cending acetone series in 20% increments followed by three
changes of 100% propylene oxide. The root segments were
subsequently infiltrated with Spurr’s resin (Spurr, 1969) prior
to polymerization at 70 ℃ for 12 h. Sections of 4-6 mm
thickness were cut with glass knives on a Super Nova
Ultratome (Reichert Jung, Vienna, Austria) and gently heat
fixed to glass microscope slides. Sections were bleached in
1% hydrogen peroxide for 45 min, washed in tap water and
then stained with 0.05% toluidine blue O in 1% sodium
tetraborate for 3 min. After three tap water washes, the sec-
tions were destained in tap water for 20 min, air-dried,
65Lu-Min VAARIO et al.: Ectomycorrhizal Synthesis Between Abies firma Roots/Callus and Laccaria bicolor Strain
mounted in DPX (Fluka BioChemika) and examined under
an Olympus microscope (BH2).
2 Results
2.1 Mycorrhizal colonization in callus
After 2-week incubation, the margin of the colony of L.
bicolor reached the callus (Fig.1a), then hyphae colonized
on the surface of the callus, but did not overgrow the callus.
A small amount of browning (17.5%) occurred. After 3-week
incubation, the colonized callus was covered with a thin,
loose, discontinuous fungal mantle. Fungal hyphae oc-
curred within the intercellular spaces of the callus (Fig.1b).
A multibranched, fan-shaped Hartig net-like structure was
also observed in the callus (Fig.1c).
2.2 Mycorrhizal development in roots
After the inoculation of eight weeks, short lateral roots
were induced from the taproots, some of which came into
contact with hyphae arising from the fungal inocula. Lat-
eral root formation occurred in all inoculated seedlings with
L. bicolor, but occurred in only three control seedlings. A
network of purple extraradical hyphae contacted with the
short lateral roots, and the roots stopped elongating and
failed to form root hairs. After 10-week inoculation, the fun-
gus had completely enveloped the entire lateral root and
formed purple mycorrhizas with a rough mantle surface. A.
firma - L. bicolor mycorrhizas, were straight and unramified
Figs.1, 2. Callus colonization (1) and root colonization (2) in Abies firma colonization by Laccaria bicolor in vitro. 1a. The hypha was
colonized on the surface of the callus after two weeks of inoculation. Bar = 15 mm. 1b. Light micrograph of an inoculated A. firma cal-
lus demonstrating extraradicle mycelium (em) near the surface of the callus cell (c), and the branched hyphae (h) were able to are
distinguished between the callus cells (c), Bar = 18 mm. 1c. A Hartig net-like structure (hn-l) was observed between the callus cells (c), Bar
= 18 mm. 2a. External mycorrhiza morphology. Abies firma-L.bicolor mycorrhizal root formed on the FH medium after 10 weeks of
inoculation. The lateral root (lr) was colonized by the hyphae forming a purple, denseness mantle. The hypha was able to be distinguished
from the main root (mr). Bar = 1 mm. 2b. Light micrograph of an inoculated A. firma transverse section demonstrating the thick mantle
(m). Bar = 50 mm. 2c. In longitudinal section, the multibranched, nonseptate fan-shaped Hartig net “palmetti” (hn) were observed. Bar
=18 mm.
Acta Botanica Sinica 植物学报 Vol.46 No.1 200466
(Fig.2a), and consisted of a thick, continuous fungal mantle
which ensheathed the single lateral root entirely. The main
root was also sheathed by hyphae (Fig.2a). Mantle hyphae
gave rise to profuse cortical hyphae, which invaded and
colonized the epidermal and cortical intercellular spaces,
appearing to separate and isolate host cells (Fig.2b). The
Hartig net formed a typical labyrinthine “palmetti” struc-
ture (Fig.2c). No intracellular penetration of host tissue by
the fungus was observed.
3 Discussion
In the Abies firma - Laccaria bicolor mycorrhizas syn-
thesized by the seedling culture system in this study, typi-
cal characteristics of ectomycorrhizas were obtained: the
hairless short root tips, along with possessing well-devel-
oped mantles; the thick continuous mantle supports hyphae,
which penetrate and colonize the intercellular spaces of
root cortex. These invasive hyphae undergo change in their
growth morphology to form a highly branched Hartig net.
The modified hyphae envelop the root cortical cells (Kottke
and Oberwinkler, 1986; Gianinazzi-Pearson and Smith, 1993),
increasing the contact surface area between the two sym-
bionts and maximizing nutrient transfer (Wiemken, 1995).
The presence of these defining features in the mycorrhizal
roots examined indicated that an ectomycorrhizal associa-
tion was formed between A. firma and L. bicolor under the
culture conditions. This method had been used in previous
studies (Vaario et al., 1999) and other reports (Wiemken,
1995) and similar results were obtained in the present study.
Meanwhile, L. bicolor was also able to form a mycor-
rhiza-like structure, which was multibranched, “palmetti”
fan-shaped Hartig net, in callus culture of A. firma, although
the frequency of mycorrhiza-like structures was lower than
that in seedling culture system. This result is in agreement
with that of Sirrenberg and co-workers (Sirrenberg et al.,
1995) who obtained mycorrhiza-like structures in an experi-
ment with ECM fungi and callus cells derived from radicles
of Norway spruce. Similarly, mycorrhiza-like structures be-
tween embryogenic cultures of Scots pine and
ectomycorrhizal fungi have been reported (Niemi et al.,
1998). These results suggested that the callus system might
be a useful tissue system for mycorrhiza synthesis in the
present study.
The callus system is artificial, however, it allows
nondestructive, morphological and physiological investi-
gations of the symbiosis. Callus tissue has been used for
in vitro screening for resistance to fungal pathogens in
forest trees (Ragazzi et al., 1995; Kvaalen and Solheim, 2000).
Moreover, it is possible to increase its similarity to a natu-
ral system by providing the extrametrical mycelium with a
nutrient environment closer to that of the mineral soil (Fortin
et al., 2001).
Laccaria bicolor, a broad host range ectomycorrhizal
fungal species, exhibits properties such as potential use in
increasing wood volume (Selosse et al., 2000), applied stud-
ies on ectomycorrhizas (Kropp and Langlois, 1990). These
features and the different patterns of colonization between
gymnosperms and angiosperms such as Abies spp. make it
an important fungus species for further study in the con-
text of biochemical and genetical responses between host
and fungus.
The clearly defined in vitro system ensures that suffi-
cient inoculums can be produced in the laboratory to be
applied to practical forestry situation. Furthermore, by us-
ing this system, it will be possible to follow the
morphological, physiological, molecular and biochemical
changes which occur during development of A. firma - L.
bicolor mycorrhizas and in other host-fungus
ectomycorrhizal associations. Such widespread applicabil-
ity will ensure that this synthesis protocol will continue to
be used further in vitro ectomycorrhizal studies.
Acknowledgements: This research was supported by a
grant from the Bio-oriented Technology Research Advance-
ment Institution (BRAIN) in Japan. We want to thank Dr. W.
M. GILL (Tasmanian Institute of Agricultural Research,
Australia) for his useful discussion. The first author was
also supported by a grant from Northeast Forestry Univer-
sity (010-602024) when this manuscript was prepared.
References:
Acsai J, Largent D L. 1983. Ectomycorrhizae of selected conifers
growing in sites which support dense growth of bracken fern.
Mycotaxon, 16:509-518.
Alvarez I F, Cobb F W Jr. 1977. Mycorrhizae of Abies concolor
in California. Can J Bot, 55:1345-1350.
China National Forest Bureau. 2001. Seeds of Woody Plants in
China. Beijing: Chinese Forestry Press. 10-17. (in Chinese)
Di Battista C, Selosse M, Bouchard D, Stenström E, Le Tacon F.
1996. Variations in symbiotic efficiency, phenotypic charac-
ters and ploidy level among different isolates of the
ectomycorrhizal basidiomycete Laccaria bicolor strain S 238.
Mycol Res, 100:1315-1324.
Freer-Smith P H. 1996. Forest growth and decline: what is the
role of air pollutants? Yunus M, Iqbal M. Plant Response to
Air Pollution. Chichester: Wiley. 437-447.
Fortin J A, Bécard G, Declerck S, Dalpé Y, St-Arnaud M, Coughlan
A P, Piché Y. 2001. Arbuscular mycorrhiza on root-organ
cultures. Can J Bot, 80:1-20.
67Lu-Min VAARIO et al.: Ectomycorrhizal Synthesis Between Abies firma Roots/Callus and Laccaria bicolor Strain
Gianinazzi-Pearson V, Smith S E. 1993. Physiology of mycor-
rhizal mycelia. Tommerup I C. Mycorrhiza Synthesis. Ad-
vances in Plant Pathology. Vol. 9. London: Academic Press.
55-82.
Harley J L, Harley E L. 1987. A check-list of mycorrhiza in the
British flora. New Phytol, 105 (Suppl.):1-102.
Iwatsuki K, Yamazaki T, Boufford D E, Ohba H. 1995. Flora of
Japan. Vol. Ⅰ. Pteridophyta and Gymnospermae. Tokyo:
Kodansha.
Japan Forest Technical Association. 1964. Illustrated Important
Forest Trees of Japan. Tokyo: Chikyu Shuppan.
Kernaghan G, Currah R S, Bayer R J. 1997. Russulaceous
ectomycorrhizae of Abies lasiocarpa and Picea engelmannii.
Can J Bot, 75:1843-1850.
Kottke I, Oberwinkler F. 1986. Mycorrhiza of forest trees —
structure and function. Trees, 1:1-24.
Kropp B R, Langlois C D. 1990. Ectomycorrhizae in reforestation.
Can J For Res, 20:438-451.
Kvaalen H, Solheim H. 2000. Co-inoculation of Ceratocystis and
Heterobasidion annosum with callus of two Norway spruce
clones with different in vivo susceptibility. Plant Cell Tiss
Org, 60:221-228.
Le Tacon F, Alvarez I F, Bouchard D, Henrion B, Jackson R M,
Luff S, Parladé J, Pera J, Stentröm E, Villeneuve N, Walker
C. 1992. Variations in field response of forest trees to nursery
ectomycorrhizal inoculation in Europe. Read D J, Lewis D H,
Fitter A H, Alexander I J. Mycorrhizas in Ecosystems.
Wallingford: CAB. 119-134.
Liu Z-L , Fang J-Y, Piao S-L . 2002. Geographical distribution of
species in genera Abies, Picea and Larix in China. Acta Geograph
Sin , 57:577-586. (in Chinese with English abstract)
Marx D H. 1969. The influence of ectotrophic mycorrhizal fungi
on the resistance of pine roots to pathogenic infections. Ⅰ.
Antagonism of mycorrhizal fungi to root pathogenic fungi and
soil bacteria. Phytopathology, 59:153–163.
Masui K A. 1926. Study of the mycorrhiza of Abies firma S. et
Z., with special reference to its mycorrhizal fungus,
Cantharellus floccosus, SCHW. Mem Coll Sci Kyoto Imp Univ
Ser B, 2:15-84.
Matsuda Y, Hijii N. 1998. Spatiotemporal distribution of
fruitbodies of ectomycorrhizal fungi in an Abies firma forest.
Mycorrhiza, 8:131-138.
Meyer F H. 1973. Mycorrhizas in native and man-made forests.
Marks G C, Kozlowski T T. Ectomycorrhizae, Their Ecol-
ogy and Physiology. New York: Academic Press. 79-101.
Molina R. 1982. Use of the ectomycorrhizal fungus Laccaria
laccata in forestry. Ⅰ. Consistency between isolates in effec-
tive colonization of containerized conifer seedlings. Can J For
Res, 12:469-473.
Niemi K, Krajnakova J, Häggman H. 1998. Interaction between
embryogenic cultures of Scots pine and ectomycorrhizal fungi.
Mycorrhiza, 8:101-107.
Parladé J, Alvarez I F, Pera J. 1999. Co-inoculation of container-
ized Douglas-fir (Pseudotsuga menziesii) and maritime pine (Pinus
pinaster) seedlings with the ectomycorrhizal fungi Laccaria bi-
color and Rhizopogon spp. Mycorrhiza, 8:189-195.
Perrin E, Parlade X, Pera J. 1996. Receptiveness of forest soils to
ectomycorrhizal association. Ⅰ. Concept and method as ap-
plied to the symbiosis between Laccaria bicolor (Maire) Orton
and Pinus pinaster Art or Pseudotsuga menziesii (Mirb.)
Franco. Mycorrhiza, 6:469-476.
Ragazzi A, Morrica S, Dellavalle I. 1995. Growth of axenic callus
of Cronartium flaccidum on callus tissue from Pinus nigra
var. laricio and Pinus sylvertris. Eur J For Pathol, 25:31-37.
Saravitz C H, Blazich F A. 1996. Abies fraseri (Pursh) Poir.
(Fraser Fir). Bajaj Y P S. Biotechnology in Agriculture and
Forestry. Berlin: Springer-Verlag. 345-358.
Schenk R U, Hildebrandt A C. 1972. Medium and techniques for
induction and growth of monocotyledonous and dicotyledon-
ous plant cell cultures. Can J Bot, 50:199-204.
Sirrenberg A, Salzer P, Hager A. 1995. Induction of mycorrhiza-
like structures and defence reactions in dual cultures of spruce
callus and ectomycorrhizal fungi. New Phytol, 130:149-156.
Selosse M A, Bouchard D, Martin F, Le Tacon F. 2000. Survival
after outplanting and effect of two Laccaria bicolor strains
inoculated on Douglas-fir (Pseudotsuga menziesii (Mir.)
Franco). Can J For Res, 30:360-371.
Smith S E, Read D J. 1997. Mycorrhizal Symbiosis. 2nd ed. New
York: Academic Press.
Spurr A R. 1969. A low-viscosity epoxy resin embedding medium
for electron microscopy. J Ultr Res, 26:31-34.
Vaario L M, Tanaka M, Ide Y, Warwick W M, Suzuki K. 1999. In
vitro ectomycorrhizal formation between Abies firma and
Pisolithus tinctorius. Mycorrhiza, 9:177-183.
Vaario L M, Warwick W M, Tanaka M, Ide Y, Suzuki K. 2000.
Aseptic ectomycorrhizal synthesis between Abies firma and
Cenococcum geophilum in artificial culture. Mycoscience, 41:
395-399.
Wiemken V. 1995. Contributions of studies with in vitro culture
systems to the understanding of the ectomycorrhizal
symbiosis. Varma A, Hock B. Mycorrhiza. New York:
Springer-Verlag. 411-425.
(Managing editor: WANG Wei)