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Neiu Phytol. (1994),
126,
677-690
Biology
of
the
ectomycorrhizal genus,
Rhizopogon
II.
Patterns
of
host-fungus specificity following spore
inoculation
of
diverse hosts grown
in
monoculture
and
dual culture
BY
HUGUES
B.
MASSICOTTE^*, RANDY MOLINA^f,
DANIEL L. LUOMA^
AND
JANE E. SMITH'
^Department
of
Forest Science, Oregon State University, Corvallis,
OR
97331,
USA
^Department
of
Agriculture, Forest Service, Pacific Northwest Research Station, Forestry
Sciences Laboratory, 3200 Jefferson
Way,
Corvallis,
OR
97331,
USA
{Received 27 January
1993;
accepted
30
September
1993)
SliMMARY
Seedlings
of
Abies grandis, Alnus rubra, Pinus ponderosa, Picea sitchensis, Pseudotsuga menziesii
and
Tsuga
heterophylla were grown
in
monoculture
and
dual culture
in
the
greenhouse
and
inoculated with spore slurries
of
20 isolates representing
15
species
of
ectomycorrhizal hypogeous fungi
{11
Rhizopogon species, Alpoz'a
diplophloeus, Truncocolumella citrina, Melanogaster euryspermus and Zetleromyces gilkeyae). The primary objectives
were
to
assess
and
compare
the
pattern
of
bost specificity between symbionts
and
to
study
the
influence
of
neighbouring plants
on
ectomycorrhiza development. None
of
the fungai species
had
broad host range affinities.
A variety
of
specificity responses were exhibited
by
the
different fungal taxa, rangmg from genus-restricted
to
intermediate host range.
In
monoculture, nine species
oi
Rhizopogon
(R.
arctostaphyli,
R.
ellenae,
R.
flavofibrillosus,
R.
occidentalis,
R.
rubescens,
R.
smithii,
R.
suhcaerulescens,
R.
truncatus
and
R.
vulgaris) formed ectomycorrhizas
on
Pinu.'!
ponderosa whevess three Rhizopogon species
{R.
parksii,
R.
vinicolor
and
R.
suhcaerulescens) formed
ectomycorrbizas
on
Pseudotsuga menziesii. Truncocolumella citrina associated with Pseudotsuga menziesii
and
Alpozia diplophloeus with Alnus ruhra. Melanogaster euryspermus and
Z.
gilkeyae did
not
form ectomycorrhizas with
any hosts. None
of
the
fungi tested developed ectomycorrhizas
on
Ahies grandis, Tsuga heterophylla
or
Picea
sitchensis in monoculture.
In
dual culture, the same nine Rhizopogon species that formed abundant ectomycorrhizas
on Pinus ponderosa formed some ectomycorrhizas
on
secondary hosts such
as
Ahies
grandis.,
Tsuga heterophylla,
Pseudotsuga menziesii and Picea sitchensis. Similarly, Truncocolumella citrina formed abundant ectomycorrhizas
on
Pseudotsuga menziesii
and low
levels
on the
secondary hosts Abies grandis, Tsuga heterophylla
and
Picea sitchensis.
Rhizopogon parksii and
R.
vinicolor only formed ectomycorrhizas
on
Pseudotsuga menziesii, and Alpova diplophloeus
only formed ectomycorrhizas
on
Alnus rubra. The specificity pattern obtained
by
using this dual-culture approach
is contrasted with previous pure-culture synthesis data
and
is
discussed
in
terms
of
potential interplant linkages
and community dynamics.
Key words; Rhizopogon, ectomycorrhizas, specificity, spore, inoculation.
where Pinaceae
are
widespread dominants,
par-
INTRODUCTION
i i n ..j ^ • ,i\/f u \ c j
ttcularly Pseudotsuga menziesn {Mirb.) rranco
and
The genus Rhizopogon comprises
a
diverse assem- Pinus
spp.
A
few
Rhizopogon
spp. are
found
in
blage
of
hypogeous ectomycorrhizal fungi that
are
Europe (Smith
&
Zeller,
1966) and
Asia (Bakshi,
mostly restricted
to
the Pinaceae. Most species occur
1974;
Khan, 1980; Hosford
&
Trappe, 1988),
and
in
the
coniferous forests
of
Western North America several species
are
found
in
exotic pine plantations
worldwide (Molina
&
Trappe, 1994).
In a
pure-
*
Current address: University
of
British Columbia, Depart- culture synthesis study, MoHna
&
Trappe (1994)
ment
of
Forest Sciences, Faculty
of
Forestry, MacMillan tested
29
isolates from
20
Rkizopogon species
for
Building, 193-2357 Main Mall, Vancouver, B.C., Canada V6T ^j.^orrhizal formation wjth Pseudotsuga menziesii,
t To whom correspondence should be addressed. Tsuga heterophylla (Raf.) Sarg.,
and
Pinus contorta
678H. B. Massicotte and others
Laws.
They report three general responses: strong
specificity
to
Pseudotsuga menziesii, specificity
or
strongest development
on
Pinus contorta,
and an
intermediate response where mycorrhizas were
formed
on
two
or
three
of
the hosts. Some
of
the
synthesized ectomycorrhizas, however, were weakly
formed
or
present only
in
limited numbers;
the
presence
of
glucose
in the
substrate
may
have
contributed
to
these resu]ts (Duddridge
&
Read,
1984;
Duddridge, 1986a, 6). Molina
&
Trappe
(1994) stressed the need to confirm patterns of host
specificity
in
Rhizopogon by examining mycorrhiza
formation
in
natural soil conditions.
The concept
of
ecological specificity
was de-
veloped
to
emphasize that ectomycorrhizal fungus
host ranges seen
in
nature may differ from those
experimentally determined in pure-culture syntheses
(Harley & Smith, 1983). In
a
recent review, Molina,
Massicotte & Trappe (1992) point out that ecological
specificity probably involves
a
complex set of biotic
and environmental factors, many of which are poorly
studied. One such factor, the matrix of neighbouring
plants,
may
afFect
the
development
of
certain
mycorrhizal fungi on particular hosts. Understand-
ing vegetation matrix effects becomes important
when evaluating the potential for plants to be linked
by shared, compatible fungi
in
natural ecosystems
(Molina et al., 1992).
Several seedling bioassays demonstrate the wide-
spread occurrence
of
Rhizopogon spp.
in
disturbed
and undisturbed forest habitats
in
Oregon
(Schoenberger & Perry, 1982; Pilz & Perry, 1984;
Amaranthus & Perry, 1989a, b; Borchers & Perry,
1990;
Miller, Koo & Molina, 1992). These studies
indicate that some Rhizopogon t>'pes form only
on
Pseudotsuga menziesii and others
are
restricted
to
Pinus ponderosa Dougl. How^ever,
the
soils were
bioassayed with single plant species grown
in
monoculture. This tests the ability of host seedlings
to form ectomycorrhizas with fungal propagules
(spores or hyphal fragments)
in
the disturbed soils,
but does
not
test ectomycorrhiza formation
by
vegetative mycelium attached to other hosts.
To determine the potential
of
hosts
to be
con-
nected by connpatible mycorrhizal fungi, the host's
response
to
ectomycorrhiza] fungi already physio-
logically associated with other host species can
be
examined. The present study used spore inoculation
and
a
combination of monoculture and dual culture
host bioassays
to
examine
the
initiation
and es-
tablishment
of
ectomycorrhizal fungi from spore
propagules and the influence of neighbouring plants
and their associated fungi
on
mycorrhizal devel-
opment. Our specific objectives were
to
(1) assess
host-fungus specificity and compare
it
to the results
of previous pure culture syntheses with Rhizopogon;
(2) broaden the scope
of
Pinaceae tested
to
include
Picea and Abies (both are widespread forest trees that
often associate with Pseudotsuga, Pinus and Tsuga);
(3) determine
the
infiuence
of
primary hosts
on
development
on
neighbouring host plants; and
(4)
evaluate
the
potential
for
interplant linkages
via
compatible mycorrhizal fungi.
MATERIALS AND METHODS
Seedling preparation and growth conditions
Seeds
of
grand
fir
[Abies grandis (Dougl.) Lindl,],
red alder {Alrms rubra Bong.), Sitka spruce [Picea
sitchensis (Bongard) Carriere], ponderosa pine (Pinus
ponderosa), Douglas
fir
{Pseudotsuga menziesii) and
western hemlock
{Tsuga
heterophylla) were soaked
overnight in distilled water, spread to dry on paper
towels and cold stratified at 4
°Q.
for 37 d. Seeds were
then planted in either "Pine Cells' {60-mI capacity,
25 mm top diameter, 165 mm long) or
'
Super Cells'
(160-ml capacity, 38 mm
top
diameter, 210 mm
long) (Cone-Tainer Nursery, Canby,
OR,
USA)
containing
a
mixture
of
equal parts
by
volume
of
peat and vermiculite, filled to 2 5 cm frotn the top of
the container. The smaller containers were used for
single tree species (monoculture)
and the
larger
containers
for a
mixture
of
two tree species (dual
culture).
Seeds were planted in each container and covered
with
a
thin layer
of
w^hite quartz sand (8 grade)
to
reduce splash during watering. Most seeds
germinated within 10 d. Abies grandis
and
Tsuga
heterophylla germinated over a period of one month.
Seedlings were grown
in the
greenhouse under
a
combination of sunlight and artificial light (280 /(mol
m '^ s""^) provided
by
sodium-vapour lamps.
Air
temperature fluctuated from 21
to
32 °C. Seedlings
were watered
at
least twice weekly with tap water.
Each seedling was fertilized monthly with 5 ml
of
Peter's fertilizer (N-P-K/473-449-426 ppm plus
trace elements) applied
at
half strength. This
amounts
to
11-9 mgN, 113 mgP
and
10 7 mgK,
applied
per
seedling over
the
length
of the ex-
periment.
Inoculation and host combinations
Hypogeous sporocarps
of
the test fungi were col-
lected from different habitats
in the
Pacific
Northwest over
a 6
month period and stored in tap
water at 4 °C until used. Voucher specimens for each
fungus were deposited with
the
Oregon State
University Herbarium (OSC). Voucher numbers,
associated host trees
and
collection numbers
are
listed in Table 1. Sporocarp characteristics including
colour, potassium hydroxide (KOH) reaction, and
rhizomorph structure were noted
for
later com-
parison with ectomycorrhizal characters.
Overall, 2,440 containers (monocultures and dual
cultures
of
3,880 seedlings) were inoculated with
spores 18 and 21 wk after planting. Of these, 3,737
(96-3%) plants survived
for
analysis. Each spore
Biology of Rhizopogon //.
679
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680 H, B. Massicotte and others
Figures 1-8. For legend see opposite.
Biology
of Rhizopogon //.681
slurry used for inoculation was prepared according
to Castellano & Molina (1989) from 1-2 sporocarps.
Each sporocarp was gently brushed to remove soil
and organic matter, cut into pieces (1-3 cc) and
thoroughly blended in 200 ml of distilled water with
a blender at high speed for about 5 min. Spore
density was calculated via haematocytometry, and
the spore suspension was adjusted and divided so
that two inoculations of equal amount could be
performed (Table 1). No attempt was made to
standardize the inoculum concentration (number of
spores ml"') between the different fungi used. For
each inoculation, 10 ml of diluted spore suspension
was deposited with a pipette on the top of the
substrate in each container.
Depending on the availability of germinated
seedlings, six to 10 replicate containers were
inoculated with each fungus for each host mono-
culture and dual culture. In addition to the six
monocultures, nine dual cultures were tested (Table
2).
To reduce cross-contamination and facilitate
growth, seedlings inoculated with the same fungus
were distributed on four neighbouring racks in the
following fashion: one rack with all dual cultures
except those containing Alnus
rubra,
one rack with
dual cultures with A. rubra, one rack with A. rubra
monocultures and one rack with other monocultures.
Because A. rubra monocultures and dual cultures
required frequent watering, they were separated
from the other groups. Racks were rotated peri-
odically in the greenhouse over the length of the
experiment.
Spore viability test
Spore viability' was assessed with the vital stain
fluorescein diacetate (FDA) by using a modification
of methods by Ingham & KJein (1982, 1984) and
Soderstrom (1977). Fluorescein diacetate was dis-
solved in acetone (2 mg ml"') and stored at —20 °C.
One ml of FDA in acetone was diluted in 50 ml
potassium phosphate buffer (0-2
M,
pH 7-6). A 1/10
dilution of each spore slurry was prepared separately
in potassium phosphate buffer (0
2
M, pH 5). One ml
of the 1/10 spore dilution was added to
1
ml of the
final FDA dilution. The spores were examined for
fluorescence at 1000 x magnification in non-
Huorescent immersion oil with an epifluorescent
microscope after 4 to 16 h of incubation in the dark.
Illumination was provided by excitation filter blue
interference (455^90 nm) and chromatic beam
splitter green interference FT 510 and barrier filter
(520-560 nm). Percentage viability was calculated by
counting the number of fluorescing spores out of a
total of 500 (Table 1).
Ectomycorrhiza assessment
Roots were examined over a 4 month period when
the seedlings were 10 to 14 months old. Inoculated
seedlings were removed carefully from the substrate,
their roots washed with tap water and ectomycor-
rhizas examined with the aid of a dissecting micro-
scope. Ectomycorrhiza (and associated rhizomorphs)
were characterized for form, colour, hyphal dimen-
sions and morphology, mantle structure, presence of
incrustations and crystals, presence of septa and
clamps, and 10"o KOH reaction. Colours were
recorded under bright tungsten illumination and
designated according to ISCC-NBS standards
(Kelly & Judd, 1955; Kelly, 1965) except for
occasional use of CIC-RBGE standards (Royal
Botanic Garden, 1969). Photographs were on
Kodachrome 64 film with a camera n:iounted with a
bellows, 55-mm macro or reversed 28-mm lens,
and a ring fiash. For each seedling, the number of
ectomycorrhizal root tips of each fungus species was
estimated visually by the classes described in the
next section. The presence and abundance of
greenhouse ectomycorrhizal contaminants were
noted but not analyzed further.
Statistical
analyses
For each seedling and fungal type, the number of
tips was converted into abundance classes as follows:
1:
1-5; 2: 6-25; 3: 26-100; 4: 101-1000; and 5: >
1000 tips. This tip abundance scale approximates a
logarithmic transformation. Examination of the data
did not reveal any reasons to question the assump-
tions of normality and constant variance. Com-
parisons of mycorrhizal tip abundance among host
treatments were made for each inoculated fungus by
using one-way analysis of variance (ANOVA). Host
treatments with no ectomycorrhiza formation by
inoculated fungi were excluded. When the mode!
specified overall differences, between-treatment
differences were determined with Fisher's protected
least significant difference test at P ^ 0 05.
RESULTS
Descriptions of ectomycorrhizas
Alpova diplophloeus {Ad). On Alnus rubra, ecto-
Figures 1-8. Ectomycorrhizas synthesized on to ponderosa pine or Douglas fir following spore inoculation
with species of Rhizopogon or Truncocolumella citrina.
Figure 1. R. occidentalis+
pon<ieTOsa
pine. Figure 2. R. rubescens+ pondeTosB pine. Figure 3. R.
.tm/Y/in-I-ponderosa pine. Figure 4. R. truncatus-^ponderosa pine. Figure 5. i?. arctostaphyli
+
pondcrosa pine:
inset: a portion of the root system, after KOH treatment. Figure 6. R.ftavofibriltosus-^-pondtros^pine. Figure
7.
Truncocolumella citrina-h Douglas fir. Figure 8. R. vinicolor
+
Douglas fir.
H. B. Massicotte and others
mycorrhizas single to pinnate; mantle more or less
compact plectenchyma (50-62-5 /*m thick), light
orange yellow (70) to dark orange yellow (72) to light
yellowish hrown (76) with occasional greenish-hlue
(168,
169) hyphae interspersed within the mantle;
outer mantle hyphae (3'5-12 5/im in diameter),
either hyaline or greenish blue, the latter irregularly
swollen and with pigments dissolving in KOH;
emanating hyphae from outer mantle 3 5-8 fim in
diameter, septate, some with clamps; no
rhizomorphs observed.
Melanogaster euryspermus (Me). No ectomycor-
rhizas formed.
Rhizopogon arctostaphyli {Ra) On Pinus ponderosa,
ectomycorrhizas single to multiple-dichotomous to
clustered; mantle plectenchymatous (more compact
in inner mantle) white with occasional pinkish hue,
mantle hyphae surrounded with red, orange and
pale translucent crystals (up to 25 /(m diameter),
mantle often frosty-looking, due to crystal-coated
seta-like emanating hyphae protruding out, older
mantle with dusty appearance, becoming light
reddish hrown (42) to grayish reddish brown (46);
ramified fascicular rhizomorphs (up to 300 /tm wide)
with small (3-5 /<m) hyphae and crystal-coated,
frosty-looking, hyphae protruding outward; strong
hlue reaction to KOH on rhizomorphs and mantle
(more evident on root tip), older portions of mantle
react pink-magenta (Fig. 5, inset); emanating hyphae
(2-5-5 j«m wide) from rhizomorphs and mantle with
elongated incrustations along the walls, septate,
without clamps (Fig. 5).
Rhizopogon ellenae (Re2). On Pinus ponderosa,
ectomycorrhizas single to multiple-dichotomous to
clustered; mantle pJectenchyznatous (more compact
in inner mantle), white with red. orange and
amorphous crystals among the hyphae; ramified
fascicular rhizomorphs (up to 300 fira wide) with
thick-walled vessel hyphae (up to 17 5 //m wide) and
outward emanating hyphae with prismatic in-
crustations deposited longitudinally along or pro-
truding from the walls; strong blue reaction to KOH
on rhizomorph and mantle, with pink hue;
emanating hyphae (2-5-3 /<m wide) from rhizo-
morphs and mantle sometimes clavate at the tip,
septate, without clamps.
Rhizopogon ellenae (Rel). Identical to R. ellenae
(Re2) except for; raniified fascicular rhizomorphs
(up to 250 fim wide) with thick-walled vessel hyphae
(up to 15//m wide). .,
Rhizopogon flavofibrillosus (Rf) On Pinus ponderosa,
ectomycorrhizas single to multiple-dichotomous to
densely clustered; mantle plectenchymatous (more
compact in inner mantle), vivid yellow (82) to light
yellow (86) with olive hue, fibrillose, hydrophobic
and floating on water; loosely ramified rhizomorphs
(up to 50 ^m wide) with similar colours and no
distinct vessel hyphae; KOH reaction rapidly to red-
magenta on mantle and rhizomorphs; emanating
hyphae (2-5-5 //m) from rhizomorphs and mantle
sometimes clavate at the tip, septate, without clamps
(Fig. 6).
Rhizopogon occidentalis (Ro)). On Pinus
ponderosay
ectomycorrhizas single to multiple-dichotomous to
clustered; mantle plectenchymatous (more compact
in inner mantle), white to vivid orange (48) to dark
greyish-reddish brown (47); more or less
fasciculated, ramified rhizomorphs (up to 200 //m
wide) with vessel hyphae (up to 15/tm wide), pale
grey to white to orange; KOH-soiuble pigments
released from mantle but no reaction per se;
emanating hyphae (2-0-375 //m wide) from
rhizomorphs and mantle sometimes clavate at the
tip,
septate, without clamps (Fig. 1).
Rhizopogon occidentaJis (Ro2). Identical to R.
occidentalis (Rol) except that vessel hyphae are up to
20 fim wide.
Rhizopogon parksii (Rp2). On Pseudotsuga menziesii^
ectomycorrhizas single to pinnate to pinnate-dis-
torted; mantle plectenchymatous (more compact in
inner mantle), white with pink-purple hue, often
with brown spots (likely root cap cells) on the white
portion, two types of hyphae (i) hyaline and non-
tapering or (ii) moderate reddish brown (43) to
greyish-reddish brown (46) and usually tapering,
some with knee-like appendage, the latter developing
a fibrillose epicutis on the outer mantle of well-
developed ectomycorrhizas; branching system of
rhizomorphs (up to 200 /im wide) with similar brown
hyphae intermingled with hyaline ones; KOH
reaction on mantle brilliant greenish-yellow (98) to
light olive (106), becoming dark-orange to fuscous;
emanating hyphae (2-3-5 /im wide) from rhizo-
morphs and mantle, septate, without clamps.
Rhizopogon parksii {Rpl). Identical to R. parksii
{Rp2).
Rhizopogon rubescens (Rr2). On Pinus ponderosa,
ectomycorrhizas single to multiple-dichotomous to
clustered; mantle plectenchymatous (more compact
in inner mantle), cottony, white to vivid yellow (82)
to strong yellow (84) with faint purple hue (more so
on older roots); thick rhizomorphs (up to 500/im in
diameter), ramified, cottony and fasciculated with
vessel hyphae (up to 15/im wide), pale white to
yellow cream, hyaline crystals present (some poly-
hedral); weak KOH reaction on mantle, yellow
pigments dissolved in KOH and pink pigments
became more obvious; emanating hyphae (l-5-5'O
Biology of Rhizopogon IJ. 683
fim wide) from rhizomorphs and mantle sometimes
clavate at the tip, septate, without clamps {Fig. 2).
Rhizopogon rubescens (Rrl) Identical to R. rubescens
(Rr2) except for: thick rhizomorphs (up to 600 //m in
diameter); emanating hyphae {2-5-7-5 fvm wide).
Rhizopogon smithii (Rsm). On Pinus ponderosa,
ectomycorrhizas single to multiple-dichotomous to
clustered; mantle plectenchymatous (more compact
in inner mantle), white to white with brilliant yellow
(83) to strong yellow (84) hue; rhizomorphs (up to
125/(m in diameter) brilliant yellow (83) to light
yellow (86) to pale yellow (89) with numerous
yellow, pale orange and hyaline granular crystals,
vessel hyphae up to \5 fizn wide; KOH reaction on
mantle limited to a quick red-pigment release;
emanating hyphae (1-5-3-75/fm wide) from rhizo-
morphs and mantle coated with red-brown incrust-
ations, septate, without clamps (Fig. 3),
Rhizopogon subcaeruJescens (Rs7). On Pinus pon-
derosa, ectomycorrhizas single to dichotomous to
dense clusters of elongated roots; mantle plecten-
chymatous (more compact in inner mantle), white to
pale yellow-brown with emanating hyphae coated
with numerous prismatic incrustations; rhizomorphs
(up to 125//m in diameter) hyaline-greyish com-
posed of hyphae coated with numerous in-
crustations; KOH reaction turning rhizomorphs
greenish-bfue {168, J7J, 172), on mantJe mottled
greenish-blue and deep red (13) to dark red (16) to
deep reddish brown (41) and finally strong reddish
brown (40) to deep reddish orange (36); emanating
hyphae (2-5-5 ftm wide) from rhizomorphs and
mantle sometimes clavate at the tip, septate, without
clamps.
Rhizopogon subcaerulescens (Rs2). Identical to R.
subcaerulescens (RsJ) plus: on Pseudotsuga menziesii,
ectomycorrhizas single to pinnate to clustered-
pinnate.
R. truncatus {Rt). On Pinus ponderosa, ectomycor-
rhizas single to dichotomous to clustered-
tuberculated; mantle plectenchymatous (more com-
pact in inner mantle), lemon yellow^ (53, 54 CIC-
RBGE), older clusters deep orange (51) to brownish
orange (54); rhizomorphs (up to 125 //m in diameter)
lemon yellow, ramified, with vessel hyphae (up to
12-5/im) and localized hyphal aggregations; yellow-
pigments on mantle and rhizomorphs are KOH-
soluble; emanating hyphae (2-3-5//m wide) from
rhizomorphs and mantle, thin-walled, hydrophobic,
septate, without clamps, sometimes clavate at the tip
(Fig. 4).
R. vinicoJor (Rv). On Pseudotsuga menziesii, ecto-
mycorrhizas single to pinnate to pinnate-
tuberculated (often elongated clusters) with distorted
roots;
mantle plectenchymatous (more compact in
inner mantle), white with purple hue composed of
two hyphal types (i) hyaJine and nontapering or (ii)
moderate reddish (43) to greyish-reddish brown (46)
and usually tapering, some with knee-like append-
age,
the latter developing a fibrillose epicutis on the
outer mantle of well-developed ectomycorrhizas;
rhizomorphs (up to 800 fim wide) also made of two
hyphal types, one hyaline and the other fibrillar
brown; KOH reaction on mantle pinkish (no green,
as on R. parksii); emanating hyphae (2-35 /^m wide)
from rhizomorphs and mantle, septate, without
clamps (Fig. 8).
R. vulgaris {Rvu). On Pinus ponderosa, ectomycor-
rhizas single to dichotomous to clustered; mantle
piectenchymatous (more compact in inner mantle),
white to brilliant orange yellow (67) to light orange
yellow (70) to dark greyish-yellowish brown (81)
with a dark red tinge (16), numerous hyaline, red and
yelJow crystals within the mantJe; fascicuJar rhizo-
morphs (up to 50 lira wide) with vessel hyphae (up to
15 (lui wide), rhizomorphs with numerous granular
deposits attached to the hyphae; slow pink-purple
reaction on mantle and rhizomorphs with KOH,
crystals becotning more visible; emanating hyphae
(1 5—2-5//m wide) from rhizomorphs and mantle,
septate, without clamps.
TruncocolumeWa citrina
(Ti:).
On Pseudotsuga
menziesii, ectomycorrhizas single to pinnate to
pinnate-distorted; mantle plectenchymatous (more
compact in inner mantle), yellow (53, 54 CIC-
RBGE) with dark yellowish-brown patches (78);
brown to black ramified rhizomorphs (up to 150//m
wide) with convoluted surface hyphae and vessel
hyphae (up to 20//m wide) constricted at septa;
KOH-soluble brown pigments (also water-soluble
pigments) on mantle and rhizomorphs; emanating
hyphae (35-5 //m wide) from rhizomorphs and
mantle pale yellow to pale brown, verrucose, septate,
clamped (Fig. 7).
Zelleromyces gilkeyae [Zg). No ectomycorrhizas
formed.
Ectomycorrhiza development
Thirteen fungal species, representing 18 of the 20
spore slurries used, formed ectomycorrhizas to
varying degrees on at least one host (Table 2).
Greenhouse ectomycorrhizal contaminants such as
Thelephora terrestris were regularly seen. Other
ectomycorrhizal fungi, such as Mycelium radicis
atrovirens and Cenococcum geophilum were spor-
adically observed and probably originated from the
peat used.
In monocultures, nine species of Rhizopogon (two
684H.
B.
Massicotte and others
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Biology of Rhizopogon //.
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686H. B. Massicotte and others
Table 4. Mean mycorrhizal
abundance^
on
seedlings
for fungi that have primary associations with Pseudotsuga
menziesii or Alnus rubia. Host acronxms in parentheses indicate the alternate
species
present in dual host pots.
Fungal acronyms are given in Table 1 and host acronyms are given in Table 2
Host
PSME
PSME
PSME
PSME
PSME
PSME
PISI
THSE
ABGR
ALRU
ALRU
ALRU
Alternate
(PIPO)
{PISI)
(TSHE)
(ABGR)
(ALRU)
(PSME)
{PSME)
(PSME)
(PSME)
(PIPO)
Fungus
Rpl
3-56^
1-78"
2-78"'
l'5O*'
3-00'^
2-00"''
(0-44)^
(0-57)
•
(0-43)
(0-60)
(0-32)
{0-54)
Rp2
3-67"
2-67"
2'78"
3-25"
3-33"
2'50"
(0-37)
{0-71)
{0-60)
(0-41)
(0-33)
(0-62)
Rv
2'22-
2-50"
2'67''
2-33"
2-60"
2-00^
(0-36)
(0-42)
(0-44)
(0-47)
{0-25)
(0-58)
Tc
4-00"
2'29''
4-11"
4'13"
4-00"
2'43^
0-56"
0-25"
0-33"
(0-00)
(0-61)
{0-31)
(0-30)
{0-00)
(0-48)
(0-38)
(0-25)
(0-33)
Ad
—
—
—
—
—
—
—
—
—
2-40" (0'98)
3-00" (0-58)
2-00" (0-76)
' Abundance class: 1: 1-5. 2: 6-25, 3: 26-100, 4: 101-1000, 5; > 1000 root tips colonized.
^ Standard error in parentheses (based on MSEs from the AXOVAs). Values for individual fungi that do not share
a superscript letter are significantly difFerent by Fisher's Protected LSD [P ^ 0 05).
isolates of R. subcaerulescens, R. truncatus, R.
vulgaris, two isolates oiR. occidentalis, two isolates of
R.
ellenae. R. arctostaphyli, R. smithii, two isolates of
R.
rubescens, and R. flavofibrillosus) formed ecto-
mycorrhizas with pine, whereas three species [R.
vinicolor, two isolates of R. parksii and Rhizopogon
subcaerulescens (R.s2)] formed ectomycorrhizas with
Douglas fir {Table 2). Truncocolumella citrina formed
mycorrhizas with Douglas fir and Alpova
diplophloeus formed mycorrhizas with alder. None of
the fungi tested developed ectomycorrhizas with fir,
hemlock or spruce in monoculture. Melanogaster
euryspermus and Zelleromyces gilkeyae did not form
ectomycorrhizas with any host (Table 2).
In dual cultures, the same nine Rhizopogon species
continued to form ectomycorrhizas with pine grown
with other hosts (Table 2), although R. sub-
caerulescens (Rsl) was inconsistent in forming ecto-
mycorrhizas on pine. In several instances, some of
these Rhizopogon species also formed a few ecto-
mycorrhizas with other hosts {fir, hemlock, Douglas
fir and spruce), in contrast to the monoculture
situation. Rhizopogon vinicolor and R. parksii formed
ectomycorrhizas only with Douglas fir and never
with companion plants in dual cultures.
Truncocolumella citrina formed abundant ectomycor-
rhizas on Douglas fir and small numbers on fir,
hemlock or spruce. Truncocolumella citrina did not
form ectomycorrhizas with pine. Alpova diplophloeus
associated only with alder (Table 2).
Tables 3 and 4 indicate the mean abundance class
value of short roots colonized for all associations
recorded in Table 2.
Table 3 documents the degree of variation in
specificity and intensity of colonization among those
Rhizopogon species primarily associated with pine.
For example, in dual cultures, the colonization level
of R. ellenae {Re2) was uniformly high on pine but
significantly lower on all alternate hosts. Fungi such
as R. subcaerulescens {Rsl) and R. truncatus {Rt) in
dual cultures associated only with pine, w^hereas R.
subcaerulescens {Rs2) and R. flavofibrillosus {Rf)
associated with three or four other hosts, as well as
pine (Table 3). The above example also illustrates
the variation between isolates of the same species.
The isolate R. subcaerulescens (Rsl, originally found
around pine) was restricted to pine, but R. sub-
caerulescens (Rs2, originally found around Douglas
fir and other mixed conifers) had a wider host range,
developing ectomycorrhizas
w
ith fir, Douglas fir and
hemlock in dual cultures. A similar host range
variation was noted for the two R. ellenae isolates
(Table 3).
Table 4 indicates the degree of variation in
specificity and intensity of colonization among those
fungi primarily associated with Douglas fir or alder.
The colonization level of Douglas fir remained the
same for R. vinicolor and R. parksii {Rp2), whereas it
fluctuated significantly among the host dual cultures
for R. parksii {Rpl). Although R. parksii is placed in
section Villosuli and R. i-inicolor in section Fulvi-
glebae by Smith & Zeller (1966), evidence from
ectomycorrhizal syntheses (Molina & Trappe, ]994)
and examination of cultural characteristics and
numerous sporocarp collections (Moiina & Trappe,
unpublished data) suggests the placement of R.
vinicolor into section Villosuli. This placement is
reflected in the section designations in Table 1.
Truncocolumella citrina colonization on Douglas fir
was typically high; on secondary hosts, colonization
was low and developed only in the presence of
Douglas fir. Alpova diplophloeus colonized alder
only, always at moderate levels (Table 4).
Biology of Rhizopogon //.687
DISCUSSION
Using spore slurries, we succeeded in producing
ectomycorrhizas on various tree seedlings in mono-
culture and dual culture with 13 fungal species (11
Rhizopogon species as well as Alpova diphphloeus
and Truncoeolumella citrina). This work confirms
that spores of several Rhizopogon spp. germinate well
during inoculation trials (Castellano & Trappe,
1985;
Castellano, Trappe & Molina, 1985;
Castellano & Molina, 1989). Likewise, A.
diplophloeus readily formed ectomycorrhizas with
alder and T. citrina with Douglas fir. This ease of
germination provides further opportunities to study
the rhizosphere process of spore germination and
ectomycorrhiza establishment, as well as
mechanisms regulating specificity in early stages of
symbiosis. At present, events and signals involved in
triggering germination of Rhizopogon spores in the
rhizosphere are poorly known, particularly when
roots of different tree species are present (Theodorou
& Bowen, 1971, 1987; Fries & Birraux, 1980).
Because the level of colonization was usually low on
secondary hosts, the most probable explanation is
that spores germinated, rapidly established colonies
on the primary hosts, and then occasionally,
colonized the secondary" hosts. We cannot discount
the possibility, however, that spores might have
germinated first on roots of secondary hosts (in dual
culture) and failed to form ectomycorrhizas, or that
they gave rise to a few small colonies on the
secondary host that later spread to the primary host.
The pattern of specificity obtained in this study
with greenhouse-grown spore-inoculated seedlings
and dual host culture expands earlier observations
using mycelium cultures and single-host plants in
sterile test tubes (Molina & Trappe, 1994). Unlike
the conventional one host-one fungus trial, dual
cultures showed that several host species (fir,
hemlock, spruce and Douglas fir) became mycor-
rhizal when primary host plants were also present.
Repeatedly, several Rhizopogon species (as well as
Truncoeolumella citrina) showed mycorrhiza! coloniz-
ation on secondary hosts only in the presence of a
well-colonized primary host species.
This raises questions concerning the physiological
basis of, and compatibility processes involved in,
colonization of
a
secondary host. One could speculate
that a fungus fully compatible and mycorrhizal with
pine might colonize, and physiologically support, a
companion plant such as a true fir. Energy supplied
by the primary host may allow the companion plant
to form mycorrhizas with a fungus that would
otherwise be too costly in terms of energy to
maintain. Alternatively, a fully mycorrhizal host
may provide such a high inoculum potential that
some mycorrhizal rootlets may form on the sec-
ondary host that otherwise would not. Regardless of
the mechanism, this phenomenon may help to
establish hyphal links between host plants and may
be ecologically significant.
Previous workers have demonstrated examples of
interplant connections in vitro (Read, Francis &
Finlay, 1985; Finlay & Read, 1986a, 6; Finlay,
1989).
Of special relevance is a study (Finlay, 1989)
examining aspects of phosphorus uptake and carbon
translocation in ectomycorrhizal associations be-
tween Pinus sylvestris L. and two larch-specific
fungi, Suillus grevillei (Klotzsch) Sing, and Boletinus
cavipes (Opat) Kalchbr. in mixed cultures of larch
and pine, both infected by B. cavipes, ^^P-labelled
orthophosphate uptake and transtocation were
greater to larch than to pine seedlings, thereby
indicating a preferential allocation to the larch
(Finlay, 1989). In our study, most mycorrhizas that
formed on secondary 'incompatible' hosts seemed
healthy, had a mantle and some degree of Hartig net
formation and were probably functional, although
this was not tested. In field situations, it is not known
whether these less common ectomycorrhizas (formed
by 'specific' fungi on 'incompatible' hosts) will
persist under competition from more generalist or
specialist fungi or eventually be selected against.
Fungi restricted in their host range but with this
linkage potential may possess different functional
attributes than those of broad host range fungi.
Importantly, this study reveals variation in the
colonization potential of a fungus depending on
whether it is already linked to a compatible host.
Research is needed to determine the differences in
function {metabolite transfer) between primary in-
fection (hyphae newly established on a root system)
and secondary infection (hyphae already established
somewhere else on a root system) in monoculture
and polyculture situations. Chilvers & Gust (1982)
emphasized the importance of these two modes of
colonization in the development of ectomycorrhizal
populations on pot-grown seedlings of Eucalyptus st-
johrtii R. T. Bak. In our experiment, several fungi
such as Truncoeolumella citrina failed to establish
primary colonization on hosts such as fir but did
succeed in establishing secondary colonization on fir
after presumably first de\ eloping primary
colonization with a 'compatible' host (Douglas fir).
This variation of colonization potential is remi-
niscent of a phenomenon documented by Fleming
(1984),
in which seedlings of Betula pendula Roth.
formed ectomycorrhizas with different groups of
fungi when planted in trenched versus undisturbed
areas.
The difference
w as
attributed to the re-
quirement of some fungi for a continuous supply of
photosynthate from a mature tree in order to colonize
seedling roots. The physiological requirements (i.e.
carbohydrates) of some fungi may not be satisfied by
certain hosts and therefore must be modified or
supplemented by an established 'compatible' host
relationship before the fungi can form an association
with a 'less compatible' host.
688H. B. Massicotte and others
Different degrees of interplant linkages seem
possible among the so-called pine-specific fungal
associates. For instance, R. truncatus remained
strongly specific to pine (also noted by Molina &
Trappe, 19826) whereas R.
subcaerulescens
(Rs2) and
R.
fiavofibrillosus consistently formed limited
numbers of mycorrhizas on other hosts such as
Douglas fir and fir. R. truncatus, however, also forms
ectomycorrhizas with Tsuga mertensiana (Bong.)
Carr. in the field (Zak, 1973) so we must be careful
not to extrapolate beyond the host-fungus com-
binations tested.
This trial confirms that R. vinicolor and R. parksii
(two isolates) are strongly specific to Douglas fir.
Even in dual cultures, where Douglas fir seedlings
had well-developed ectomycorrhizal clusters and
rhizomorphs, no ectomycorrhizas were found on
companion hosts. Molina & Trappe (1994) noted
similar behaviour by other isolates of these species
although a few^ mycorrhizas also formed on T.
heterophylla. Generally, this group of fungi (section
Villosuli) is restricted to Douglas fir (Molina &
Trappe, 1994) and is detected routinely in field trials
in both young and old Douglas fir stands
(Schoenberger & Perry, 1982; Pilz & Perry, 1984;
Amaranthus & Perry, 1989a, 6; Borchers & Perry,
1990;
Roth, 1990; Miller et al., 1992; Massicotte,
unpubhshed data). Because of their restricted host
range, these Rhizopogon species are unlikely to form
intergeneric host linkages. Linkage may be sought
intraspecifically, such as between mature tree and
young seedling, a possibility discussed by Read
(1984),
Reade/a/. (1985) and Finlay & Read (1986o),
or intragenerically if host species of the same genus
should overlap in their geographic distribution, as
pointed out by Mohna et al. (1992). A demonstration
of the functional differences between intrageneric
linkages (via ectomycorrhizal fungi with a restricted
host range) and intergeneric hnkages (via fungi with
a broad host range) and the ecological imphcations of
these remains a challenge for the future.
Truncocolumella citrina associated consistently
with Douglas fir but also colonized a few secondary
hosts,
though never Pinus. This confirms the in vitro
studies of Molina & Trappe (1982 a, b) and correlates
with sporocarp collection data in the Pacific North-
west (Hunt & Trappe, 1987; Luoma, P'renkel &
Trappe, 1991; Trappe, unpublished data). Fogel &
Pacioni (1989), however, indicated that T. citrina
was associated with Pinus spp. in the Great Basin
region of the United States. This difference in host
specificity may be due to ecotypic variations where
the fungus behaves differently in the centre versus in
the limits of its geographical distribution. Regional
differences in fungal community structure and
associated competitive interactions also may con-
tribute to such a shift in primary host species.
Alpova diplophloeus was strongly alder-specific,
supporting previous reports (Molina, 1979, 1981;
Godbout & Fortin, 1983), This restriction to the
genus Alnus suggests a good potential for intra-
specific linkages. Interestingly, a recent bioassay
(Miller el al., 1992) indicates a widespread dis-
tribution of ^. diplophloeus propagules in a variety of
Pacific Northw'est forests, even when Alnus plants
are rare or absent. This observation raises questions
about the maintenance of viable propagules of genus-
restricted ectomycorrhizal fungi in situations where
the host plants are absent.
The siniilarities in patterns of specificity obtained,
both in pure culture synthesis with vegetative
mycelia (Molina & Trappe, 1994) and under dual-
culture synthesis with spores in greenhouse con-
ditions (present study), suggest that some common
mechanisms govern specificity. Emerging data on
specificity patterns for the genus Rhizopogon raise
several issues concerning isolate variation. For
example, in both monocultures and dual cultures,
Rhizopogon species belonging to the section
Amylopogon {R. arctostaphyli, R. ellenae and R.
subcaerulescens)
were confined mainly to pine, except
in a few dual cultures where occasional mycorrhizas
were noted on secondary hosts. In the pure culture
tests by Molina & Trappe (1994), the same species
formed abundant mycorrhizas on both pine and
Douglas fir and a few on Tsuga heterophylla. Sugar
amendment of the substrate may explain some
discrepancies between studies, as suggested by
Duddridge & Read (1984) and Duddridge (1986 a,
b),
but intraspecific isolate variation also might be a
factor.
The assumption that isolates from the same species
behave similarly with respect to host range needs to
be examined further. Fungal species consist of
populations under different selection pressures that
might result in differences in host or substrate
affinities. Evidence from the few available studies
suggests that most isolates from the same fungal
species behave similarly with regard to host
specificity (Molina, 1979; Molina & Trappe, 1994).
In the present study, the isolates for section Villosuli
consistently only colonized Douglas fir. For isolates
of R. subcaerulescens, R. rubescens and R. ellenae,
however, the spectrum of hosts colonized differed
within each species. A contributing factor may be the
origin of the isolates involved. For instance, R.
subcaerulescens (Rsi) was found in a habitat
dominated by pine and in the synthesis trials
associated only with pine. Rhizopogon
subcaerulescens
(Rs2),
on the other hand, was found with Douglas fir
and mixed-conifer species, and in the synthesis trials
associated with other hosts as well as pine. In
addition, R. subcaerulescens sporocarps are collected
regularly where pines are absent (James Trappe,
personal communication). It is therefore possible
that habitat differences might be reflected in trial
patterns of colonization and may indicate some
degree of specialization (and consequent host pre-
Biology
of
Rhizopogon
II.
&S9
ference) among isolates.
A
related phenomenon
was
also discussed
by
Malajczuk, Lapeyrie
&
Garbaye
(1990)
in
which the geographical origin
of
Pisolithus
tinctorius {Pers) Coker
&
Couch isolate resulted
in
differences
in
the speed of colonization on Eucalyptus
urophylla S.
T.
Blake roots. Experimental conditions
that differ from the original habitat
of
an isolate may
alter
the
growth
of
that fungus
and its
potential
for
mycorrhizal colonization.
In
field conditions, root
systems
of
neighbouring hosts may interact
to
create
diverse microhabitats
and
promote niche special-
ization
for the
fungus,
a
condition
not
encountered
in host monoculture situations.
The
Pacific North-
west region
is
rich
in
members
of
Pinaceae that have
developed genus level fungus specificity (Molina
et
al., 1992), perhaps
an
indication
of
microhabitat
richness.
It
is
difficult
to
relate spore density
of
inoculum
used
to the
level
and
consistency
of
colonization
obtained. Rhizopogon subcaerulescens (Rs2),
for in-
stance, was inoculated
at a
rate
of
152 million spores
per container compared with
92
million spores
per
container
for R.
subcaerulescens (Rsl). Consistent
colonization
on
pine
was
recorded
for the
former,
but inconsistency
was
seen with
the
latter.
The
viability of the spores used remains a critical question
in our understanding
of
the biology
of
this group
of
fungi. Inoculation success
may
decrease
if a
large
proportion
of
spores are inimature
or
senescent.
The
vitality assessment
of
the spore slurries, using FDA,
indicated
a
large variation
in
the percentage
of
FDA-
positive spores presumed viable and
did
not seem
to
relate
to
the level
of
mycorrhization obtained. Spore
dormancy
may
distort
the
results
of
viability tests
and subsequent interpretation (Miller, Torres
&
McClean, 1993).
The ectomycorrhizas descriptions given here agree
with those already published
for a few
species
of
Rhizopogon (Fontana & Centrella, 1967; Zak, 1971;
Pachlewski
&
Pachlewska,
1974; Uhl,
1988).
An
important feature
of the
peridium,
the
reaction
to
10 "o
KOH,
used
for
identification
of
Rhizopogon
species, also w"as found
to
be applicable to the mantle
of many ectomycorrhizas formed
by the
same
species.
For
example,
R.
arctostaphyli mycorrhizas
and rhizomorphs give
a
strong blue reaction
to
KOH,
a
feature also seen
on
fresh sporocarps
and
noted
by
Smith
&
Zeller (1966). Other members
of
the section Amylopogon
in the
genus Rhizopogon,
such as
R.
ellenae and
R.
subcaerulescens,
also reacted
to KOH
in a
fashion similar
to R.
arctostaphyli.
The
red-magenta
KOH
reaction with
R.
flavoflbrillosus
was found
on
both mycorrhizas
and
sporocarps.
This consistency in chemical reaction between fungal
mantle and sporocarp
is a
helpful diagnostic feature
for identifying field mycorrhizas.
Newman {1988) discussed
the
shortcomings
of in
vitro studies showing interplant linkages
and the
need
for
caution
in
interpreting their ecological
significance.
Our
dual culture trial, however,
provides important data
as to
when fungi
can
establish
on
both 'preferred' and secondary host,
an
aspect missed
in
conventional
one
fungus-one host
syntheses. Importantly,
our
dual culture results
expose
the
pitfalls
of
using only one growth system
when assigning specificity groups, especially
if a
fungus
can
modify
its
host range according
to the
conditions under which
it is
tested. Dual-culture
studies should advance
our
understanding
of the
dynamics
of
specificity
in
complex forest ecosystems
where influences
by
companion plants, ectomycor-
rhizal
or not,
must certainly affect
the
biological
processes.
ACKNOWLliDGEMENTS
We thank the Natural Sciences and Engineering Research
Council
of
Canada
for a
postdoctoral fellowship
to the
senior author. Our sincere thanks also to Linda Tackaberry
for invaluable help with root processing and editing, Doni
McKay for greenhouse assistance, and Michael Castellano
and James Trappe
for
sporocarp identification
as
well
as
valuable advice throughout
the
study.
We
thank Elaine
Ingham
and
Steven Miller
for
help
and
advice with
the
EDA stain.
In
addition,
we are
grateful
to
Anders
Dahlberg
and
James Trappe
for a
critical review
of the
manuscript.
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