Mycal. Res. 97 (2): 141-149 (1993)
Printed in Great Britain
141
Basidiospore viability and germination in ectomycorrhizal and
saprotrophic basidiomycetes
STEVEN L. MILLER
Department of Botany, University of Wyoming, Laramie, Wyoming 82071, U.s.A.
PILAR TORRES
Facultad de Biolog{a, Departamento de Biologza Vegetal, Universidad de Murcia, Murcia, Spain
TERRY M. McCLEAN
Department of Botany, University of Wyoming, Laramie, Wyoming 82071, U.SA.
Spores of the ectomycorrhizal basidiomycetes Rhizopogan rubescens and Suillus tomentosus were induced to germinate and stained with
fluorescent stains FDA and DAPI to assess germinability and viability. Basidiospores of several saprotrophic species including
Pleuratus astreatus, Marasmius areades, Agaricus brunnescens, Coprinus quadrifidus and Conocybe lactea were also assayed for comparison.
Nearly all spores of both the ectomycorrhizal and saprotrophic fungi contained intact nuclei. The percentage of R. rubescens and
S. tamentasus spores that stained with FDA increased throughout the study. Peaks in FDA staining corresponded with germination
events which occurred every 9 to 13 days. The FDA staining in spores of P. astreatus and M. oreades was high and corresponded
with high rates of germinability. Germination and FDA staining of the other saprotrophic species were variable. In ectomycorrhizal
and several saprotrophic species FDA staining was unreliable as an indicator of viability but served as a good predictor of
dormancy. Non-fluorescent vital and nuclear stains were compared with the fluorescent stains and gave similar results. Equations for
estimating viability, dormancy, and activation of spores are presented.
Spores of ectomycorrhizal fungi are difficult to germinate.
Several techniques developed by Fries and others (Fries, 1976,
1977, 1982, 1983a; Fries & Birraux, 1980; Birraux & Fries,
1981; Theodorou & Bowen, 1987) have improved the ability
to germinate these spores, but with some the germination
rates remain less than 1 % after several months' incubation,
and some species of ectomycorrhizal fungi do not respond.
Poor germination has hindered our knowledge of mating
compatibility, somatic incompatibility and population biology
in ectomycorrhizal fungi.
Vital stains have been used to determine viability of fungal
spores and hyphae (Soderstrom, 1977, 1979; Calich, Purchio
& Paula, 1978), seeds (Cottrell, 1948), pollen (Hauser &
Morrison, 1964; Heslop-Harrison & Heslop-Harrison, 1970)
and plant and animal protoplasts (Rotman & Papermaster,
1966; Widholm, 1972; Larkin, 1976). Two such stains are
fluorescein diacetate (FDA) and tetrazolium salts. The
fluorochrome, FDA, is a non-fluorescent compound that can
permeate intact cell membranes. In liVing protoplasts it is
hydrolyzed to the strongly fluorescent carboxyfluorescein
(Bornman, 1989). Succinate dehydrogenase, one of the
tricarboxylic acid cycle enzymes, is restricted in fungi to sites
in active mitochondria (Zalokar, 1965). Colourless tetrazolium
salts, of which 3-[4,5-dimethylthiazol-2-yl]2-5, diphenyl tetrazolium bromide (MTT) is one, form a red diformazan
compound when reduced by actively respiring mitochondria
(Berlyn & Miksche, 1976). Positive MTT staining indicates
functioning respiratory metabolism. Viability as measured by
either stain indicates enzyme activity within the cell although
each functions with a different enzyme system.
Our examination of ectomycorrhizal fungal spores with
several vital stains showed that spore viability of many
species of ectornycorrhizal basidiomycetes is low. However,
the same spores examined with transmission electron
microscopy appear to contain a full complement of organelles
and cytoplasm and an intact nucleus and plasmalemma (Miller,
1988; Miller & Miller, 1988), and spores with viability of less
than 1 % formed abundant ectomycorrhizas when inoculated
onto the roots of appropriate host plants. These facts suggest
that the vital stains being used may not be good predictors of
spore viability in some fungi.
The objectives of this study were to germinate spores of
ectomycorrhizal basidiomycetes and to examine the efficacy
of commonly used vital and nuclear stains. Comparisons were
made with several saprotrophic basidiomycetes with known
germinability.
Basidiospore viability in ectomycorrhizal fungi
142
Figs 1-4. Light micrographs of Suillus tomentosus basidiospores from an agar-spore film on lodgepole pine roots (bar = 3 fJm). Fig. 1.
Spore with high phase-contrast refractivity. Figs 2-3. Spores with low phase-contrast refractivity. Fig. 4. Spore with germination
vesicle at apex. Note the dull appearance of the cytoplasm with phase-contrast microscopy. Figs 5-8. Epifluorescence micrographs of
the Suillus tomentosus basidiospores in Figs 1-4 treated with FDA stain (bar = 3 fJm). Fig. 5. Spore with no cytoplasmic FDA staining.
Arrows indicate faint autofluorescence of outer wall layer. Figs 6-7. Spores with slight FDA positive staining of the cytoplasm. Fig. 8.
Bright positive FDA staining of germinating spore. Figs 9-12. Light micrographs of Rhizopogon rubescens basidiospores from an agarspore film on lodgepole pine roots (bar = 3 fJm). Fig. 9. Spore with high refractivity (top) and spore with low refractivity (bottom)
under phase-contrast microscopy. Figs 10-11. Spores with low phase-contrast refractivity. Fig. 12. Spore with germination vesicle at
apex and dull appearance of the cytoplasm with phase-contrast microscopy. Figs 13-16. Epifluorescence micrographs of the Rhizopogon
rubescens basidiospores in Figs 9-12 treated with FDA stain (bar = 3 fJm). Fig. 13. Spore with no cytoplasmic FDA staining (top. barely
visible) and spore with high positive FDA staining of the cytoplasm (bottom). Figs 14-15. Basidiospores with FDA positive staining of
the cytoplasm. Fig. 16. Positive FDA staining of germinating basidiospore. Arrow indicates faint autofluorescence of outer wall layer.
s. L. Miller,
P. Torres and T. M. McClean
143
Figs 17-19. Germinating basidiospores of Suillus tomentosus from an agar-spore film on lodgepole pine roots. Fig. 17. Cluster of spores
germinating in close proximity (bar = 10 Ilm). Fig. 18. Spores showing germination vesicle at apex (bar = 3 Ilm). Fig. 19. Germinated
spore with hyphal development (bar = 5 Ilm). Figs 20-22. Basidiospores of Rhizopogon rubescens. Fig. 20. Spores from a spore slurry
(bar = 10 Ilm). Note refractive and non-refractive spores under phase-contrast microscopy. Figs 21-22. Basidiospores from an agarspore film on lodgepole pine roots. Fig. 21. Spores showing germination vesicle at apex (bar = 3 Ilm). Fig. 22. Spore following
germination vesicle formation. Arrows indicate lysis (bar = 5 Ilm). Figs 23-25. Basidiospores containing one or more nuclei (indicated
by the arrows) stained with aceto-iron haematoxylin. Fig. 23. Spores of Rhizopogon rubescens (bar = 5 Ilm). Fig. 24. Spores of
Hysterangium separabi/e on basidium (bar = 3 Ilm). Fig. 25. Spore of Gautieria graveo/ens (bar = 3 Ilm). Figs 26-28. Basidiospores stained
with MTI for localization of succinate dehydrogenase (indicated by the arrows). Figs 26-27. Spores of Rhizopogon rubescens
(bar = 5 Ilm). Fig. 26. Note the faint coloration of one spore indicating weak staining. Fig. 27. Showing strong staining of one spore.
Fig. 28. Spores of Ze//eromyces cinnabarinus (bar = 5 Ilm) showing strong, weak and no staining of spores.
144
Basidiospore viability in ectomycorrhizal fungi
100
Rhizopogon rubescens
80
1%
60
~
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40
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20
I
1~ '1
/ X
8%
~
X ...X·X....-
X II %
myc
X 3% X
-X ·X......
oX
100
Suil/us tomentosus
80
60
28%
40
1'IY
20
oX =:::Z==:IX
0
5
10
X
15
1%
IX" / 'X~~%
X;
X
:J
6·9%
X
X'X
myc
.
20
25
Time (d)
30
35
40
Fig. 29. Percentage of ungerminated Rhizopogon rubescens and Suillus
tomentosus basidiospores from agar-spore films on lodgepole pine
roots with positive FDA staining. Numbers above the points indicate
germination events and approximate percentages of spores germinating at that harvest. Arrows below the points indicate initial
appearance of ectomycorrhizas on the pine roots.
MATERIALS AND METHODS
A variety of ectomycorrhizal basidiomycetes with hypogeous
and epigeous fruiting habits including Rhizopogon rubescens
(Tul.) Tulasne, Gautieria graveo/ens Vitt., Rhizopogon subcaeru/escens Smith, Hysterangium separabile Zeller and Zelleromyces
cinnabarinus Smith (hypogeous) and Amanita muscaria (Fr.)
S. F. Gray, Suillus tomentosus (Kauffman), Singer, Snell & Dick,
Lactarius deterrimus Groeger, Chroogomphus rutilus (Fr.) Miller,
and 5uillus brevipes (Peck) Kuntze (epigeous) was used for
various portions of this study, though not all were subjected
to all treatments or procedures. Fresh basidiomes were
collected from a nearby lodgepole pine (Pinus contorta Doug.)
forest except for Z. cinnabarinus which was collected in a
Virginia pine forest. Spore slurries for each species were
prepared by separating the gleba (hypogeous fungi) and the
hymenial tubes or lamellae (epigeous fungi) from the remainder
of the basidiome tissue and blending the fertile tissue in
approximately three times its volume of sterile distilled water
until the slurry was smooth and uniform in consistency.
Slurries were placed in sealed plastic containers for 1 month at
5 °C before use.
Saprotrophic fungi used in this study included basidiomycetes with both light- and dark-coloured spores. Pleurotus
ostreatus (Fr.) Kummer and Marasmius oreades (Fr.) Fries were
considered light-spored, while Conocybe lactea (J. Lange)
Metrod, Coprinus quadrifidus Peck and Agaricus brunnescens
Peck were considered dark-spored. Spore prints were taken
from fresh basidiomes on to clean bond paper. The spore
prints were stored at 5° for approximately 1 month before
use. An isolate of A. brunnescens was also made for use as an
aid to spore germination (Losel. 1964).
Spores of R. rubescens and 5. tomentosus were prepared for
germination by a modification of the Theodorou & Bowen
(1987) technique. Lodgepole pine seeds were surface sterilized
in 30 % hydrogen peroxide and germinated and grown in
sterile sand for 1 month. Initial spore density in each spore
slurry was calculated using a haemocytometer. A sufficient
volume of each spore slurry was added to 20 ml of 1 % water
agar (melted and cooled to 38° in a water bath) to achieve a
spore density of 29 x 10 8 spores ml- I • The root systems of
individual pine seedlings were dipped into the agar-spore
suspension and allowed to cool to envelope the roots in a thin
agar film. Five replicate seedlings were planted in sterile
vermiculite in each of 15 open deep-dish Petri plates. The
seedlings were watered with sufficient sterile distilled water to
moisten the vermiculite and kept under a combination of
fluorescent and incandescent growth lights with photosynthetically active radiation (PAR) of 400 I-lmol m- 2 sec- I
and a photoperiod of 16 h. After 10 days, one set of five
replicate seedlings was harvested three times weekly until the
conclusion of the study. Harvesting consisted of removing the
adherent particles of vermiculite from the root systems of
replicate seedlings and examining the roots under a dissecting
microscope for remnants of the agar-spore film. Pieces of the
agar-spore film from different locations on the root system
were carefully removed with fine dissecting implements and
stained with FDA as described below.
Spores of each of the saprotrophic fungi were taken from
the spore print and placed in sterile distilled water. Aliquots
of each spore suspension were placed in replicate screw-cap
culture tubes containing 5 ml of potato dextrose broth with
30 mg 1-1 streptomycin and 3 mg 1-1 benamyl. In addition,
one 4 mm agar plug of the vigorously growing A. brunnescens
tissue isolate was floated on the surface of the broth in each
spore-inoculated A. brunnescens tube. Beginning immediately
one tube of each species of saprotrophic fungus was harvested
each day by centrifuging the spores at 800 g, decanting the
liquid and staining the spores as outlined below. The growing
A. brunnescens tissue was removed prior to harvesting the
spores.
Staining and microscopy
Two fluorescent stains and two non-fluorescent stains were
used. The fluorescent stains were FDA (Sigma) and DAPI (4'6-diamino-2-phenylindole, Sigma), a DNA stain. The nonfluorescent stains were MTT (Sigma) and haematoxylin (a
stain for chromatin). Stained spores from slurries or from
germination experiments were examined with phase-contrast
microscopy or with epifluorescence microscopy on an
Olympus BHS microscope with a total magnification of 400 or
1000 x . For epifluorescence microscopy, the microscope was
equipped with a blue dichroic filter cube and 455 nm exciter
filter for FDA staining and an ultraviolet dichroic filter cube
for DAPI staining. Light micrographs were taken with
Technical Pan film exposed at ISO 125 and developed in
Kodak D-19 for 4 min at 21° (Kodak) and epifluorescent
S. L. Miller, P. Torres and T. M. McClean
micrographs were taken with TMAX 3200 film (Kodak)
exposed at ISO 3200 and developed in T-MAX RS developer
for 11 min at 21°.
Fluorescent staining
FDA vital stain. The stock solution was prepared by
dissolving 5 mg FDA ml- 1 in acetone and was stored at
- 20° until used. At the time of staining 0'1 ml of the stock
solution was added to 25 ml 0'05 M phosphate buffer at
pH 7'6 giving a final dilution of 20 IJg ml- 1 FDA (Bornman,
1989). Spores from slurries and small thin pieces of agar-spore
film from germination experiments were placed in the stain,
allowed to incubate for approximately 1 min and examined.
Positive staining was indicated when cytoplasm in the spores
had a greenish fluorescent colour distinguishable from
background fluorescence under the appropriate wavelength of
light.
DAPI nuclear stain. Spores were fixed overnight in 8%
glutaraldehyde in a-os M phosphate buffer at pH 6'8 and
washed three times with the same buffer. The DAPI stock
solution was prepared from a modification of Lachapelle &
Boothroyd (1986) by dissolving 10 IJg DAPI ml- 1 of 0'2 M
phosphate-buffered saline (PBS) (Na 2 HP0 4 • 7 H 2 0, 20'7 g 1-1;
NaH 2 P0 4 .H 2 0, 3'2 g 1-1; NaCl, 0'9 g 1-1), pH 7'5. The stock
solution was kept at 5° until used. At the time of staining,
2'5 ml of the DAPI stock solution were added to 50 ml PBS
for a final concentration of 0'5 IJg ml- 1, and placed in a testtube containing the fixed spores. Positive staining was
recorded when a spore contained at least one discrete bluish
nucleus.
Non-fluorescent staining
MTT, tetrazolium vital stain. Spores were incubated for
30 min at 37° in a reaction mixture containing 1 ml 0'1 %
MTT (w lv, aqueous), 1 ml 0'05 M sodium phosphate buffer at
pH 7'0, and 1 ml 0'05 M succinic acid as a substrate. Positive
staining was indicated when reddish compounds were formed
in the mitochondria.
Aceto-iron-haematoxylin nuclear stain. The spores were
fixed as for DAPI staining. The aceto-iron-haematoxylin
staining protocol described by Wittman (1962) with chrome
alum, aluminium alum and iodic acid as mordants was used.
Positive staining was indicated when the spores contained at
least one purplish or purple-black nucleus.
145
staining were generally spores that appeared to be near
germination.
In both ectomycorrhizal fungi two populations of spores
were distinguished with phase-contrast microscopy, those in
which the cytoplasmic contents and wall appeared golden and
were highly refractive and those in which the interior and wall
appeared dull gray and not refractive. Positive staining and
low refractivity were highly correlated in both species.
The percentage of FDA positive spores increased throughout the experiment for both ectomycorrhizal fungi (Fig. 29)
but was variable among pieces of the agar-spore film examined.
The spores exhibited alternating events of high and low FDA
staining. This effect was most pronounced in S. tomentosus.
Spore germination was first observed after 13 days for R.
rubescens and 15 days for S. tomentosus, although the percentage
of total spores germinating was low (Fig. 29). Spore
germination was also variable in different pieces of agar-spore
film and appeared positively correlated with proximity to
developing short roots. Pieces of agar-spore film harvested
from locations near short roots generally had more germinating
spores than those harvested from sections of long roots.
Highest percentages of spore germination for S. tomentosus
occurred at day 38 while for R. rubescens maximum germination
occurred at day 36.
Spore germination occurred intermittently throughout the
study and more spores were found to be germinated at some
harvests than at others (Fig. 29). Peaks in germination occurred
approximately 9-13 days after the preceding peak with an
intervening period of little or no germination. Maximum
germination for both R. rubescens and S. fomentosus corresponded with or lagged approximately 2 days behind peak
FDA staining (Fig. 29).
100
80
60
40
Marasmius areades
20
o
100 t----------------~
26%
80
RESULTS
60
Spores of both S. fomenfosus and R. rubescens exhibited a
positive FDA reaction. Light and epifluorescence micrographs
of S. tomentosus spores are shown in Figs 1-8 and 17-19;
micrographs of R. rubescens are shown in Figs 9-16 and 20-22.
The intensity of staining varied from a pale image barely
detectable against background fluorescence (Figs 6-7) to
bright fluorescence (Figs 8, 13, 15). At each harvest some
spores did not stain and appeared dark (Figs 5,13). Quenching
of the stain was also variable and was weakly correlated with
intensity of staining. Spores that exhibited strongest FDA
40
10
6%
Pleura/us as/rea/us
20
o
o
2
3
Time (d)
Fig. 30. Percentage of ungerminated Marasmius oreades and Pleurotus
ostreatus basidiospores with positive FDA staining. Numbers show
germination events and approximate percentages of spores germinating at that harvest.
MYC 97
146
Basidiospore viability in ectomycorrhizal fungi
Table 2. Percentage of total spores of ectomycorrhizal and saprotrophic
basidiomycetes stored for one month exhibiting positive vital (FDA, MTT)
and nuclear (DAPI, haematoxylin) staining
lllO
Coprinlls '1lwdntidlls
~O
Fluorescent
Non-fluorescent
toO
FDA
MTT haematoxylin
Amanita musearia
Chroogomphus rutilus
Lac/arius deterrimus
Rhizopogon rubeseens 1 2
Rhizopogon rubeseens 2
Rhizopogon subeaeruleseens 1
Rhizopogon subeaeruleseens 2
Rhizopogon subeaeruleseens 3
Suillus brevipes 1
Suillus brevipes 2
Suillus tomen/osus 1
Suillus tomen/osus 2
Suillus tomentosus 3
..0
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0
tOO
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Conon1l e ta,'lea
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8
6
10
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4
81
65
78
2
17
37
0
97
95
99
100
99
97
99
100
99
98
99
97
0
Ii
25
22
32
4
10
92
76
80
12
33
65
a
99 1
99
99
100
99
98
99
100
100
99
98
99
1 Indicates that
the dark spore wall may have prevented accurate
observation of staining.
2 Different numbers following the specific epithet denote basidiomes
collected from different localities.
-::l
3
~
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~O
0
100
.-lgariclIsbrtmn..sc..ns
80
60
~
20
1"00
100%
0
0
2
4
3
5
6
7
8
9
10
II
Time (d)
Fig. 31. Percentage of ungerminated Coprinus quadrifidus, Conocybe
laetea and Agaricus brunnescens basidiospores with positive FDA
staining. Numbers show germination events and approximate
percentages of spores germinating at that harvest.
Table 1. Percentage of total spores of saprotrophic basidiomycetes
exhibiting vital staining with FDA and MTT after storage for one month
FDA
Marasmius oreades
Pleuratus ostreatus
Agaricus bisporus
Conoeybe lac/ea
Coprinus quadrifidus
85
6B
1
10
35
FDA
(boiled)
MIT
0
0
0
0
0
99
99
60 1
65
78 1
MIT
(boiled)
0
a
0
0
0
Replicate spore preparations boiled for 10 min and cooled before staining
served as controls.
1 Indicates that the dark spore wall may have prevented accurate
observation of staining.
Many S. tomentosus spores germinated simultaneously (Fig.
17), whereas single germinating spores of R. rubescens were
most commonly observed. Spores of both fungi began
germination with a small vesicle that developed usually on the
apical end of the spore, however the germination vesicle
developed obliquely or laterally near the apical end in many
R. rubescens spores (Fig. 21). All developmental stages were
observed for S. tomentosus from early vesicle formation to
enlargement of the vesicle and hyphal growth (Figs 17-19).
The later stages of germination were infrequently encountered
in R. rubescens because many spores lysed shortly after vesicle
formation (Fig, 22). Many lysed R. rubescens spores were in
contact with a single hypha lacking clamp connections.
There were differences in FDA staining and germination
between the light- and dark-spored species of saprotrophic
fungi as well as among the three species of dark-spored
saprotrophic fungi. In both M. oreades and P. ostreatus initial
FDA staining was high, 68 to 85 %, and increased within 2 or
3 days to near 100 % (Fig. 30). Likewise, initial spore
germination occurred within the first I or 2 days and had
reached 100% by day 3 (Fig. 30).
Several patterns of FDA staining and germination were
evident in the dark-spored species (Fig. 31). Coprinus quadrifidus
exhibited the stepwise increase in FDA staining with intervals
of peak germination similar to the ectomycorrhizal fungi.
Germination began within 3 days with additional events of
germination occurring every I to 3 days. Intermittent or
periodic spore germination continued in this species throughout the 45 days of the experiment (data not shown) but
germination never reached 100 %.
Spores of A. brunnescens showed little or no FDA staining
but began to germinate at day 5 (Fig. 3 I). By day 6 all spores
had germinated. FDA staining was evident within the spores
after germination vesicle formation. Developing hyphae were
also strongly FDA positive.
Approximately 10% of Conocybe lactea spores were FDA
positive when taken directly from the spore print. The
percentage of C. lactea spores exhibiting positive FDA
staining increased during days I and 2 then gradually
decreased to zero by day 7. By day 45 (not shown) no spores
had germinated and FDA staining remained negligible.
The two vital staining techniques using MTT and FDA
S. L. Miller, P. Torres and T. M. McClean
gave slightly different results for both the saprotrophic and
ectomycorrhizal fungi examined (Tables I and 2). For both
ectomycorrhizal and saprotrophic fungi MTT gave consistently higher percentages of stained spores than did FDA,
although comparable results were achieved on a relative basis
(Tables I and 2). Neither technique effectively stained spores
of saprotrophic fungi that had been subjected to boiling (Table
I). Frequently red diformazan deposits of the MTT positive
reaction were difficult to detect in the thick-walled darkcoloured spores of A. brunnescens, Coprinus quadrifidus and
Chroogomphus rutilus (Tables I and 2). The FDA stain was
restricted to the cytoplasm of the spores (Figs 5-8 and 13-16).
The diformazan deposits from the MTT stain varied from
small red bodies scattered throughout the spore to large red
or reddish purple globules coinciding with the location of the
lipid droplets in the spores (Figs 26-28).
The two techniques for nuclear staining with DAPI and
haematoxylin gave consistently similar results (Table 2). Both
allowed detection of intact nuclei in most or all of the spores
examined (Table 2, Figs 23-25). As with vital staining,
however, the purple-black haematoxylin-stained nuclei were
occasionally difficult to detect in thick-walled, darkly coloured
spores.
Regardless of the vital stain used, the level of FDA and
MTT staining for spores of ectomycorrhizal fungi was
generally lower than for those of saprotrophic fungi, especially
in the species with thin-walled light-coloured spores (Tables I
and 2). Different species of ectomycorrhizal fungi as well as
slurries of the same species from basidiomes collected in
different localities showed considerable variation in staining
(Table 2). Spores of Amanita muscaria exhibited the lowest
overall level of staining, being unresponsive to all stains used.
Two spore slurries of Suillus brevipes and one slurry of R.
subcaerulescens showed the highest FDA and MTT staining
(Table 2). The other two R. subcaerulescens slurries exhibited
low levels of FDA and MTT staining. S. tomentosus showed
variation of nearly 40% in FDA staining and over 50% in
MTT staining among the three spore preparations.
DISCUSSION
The high levels of MTT and FDA staining and complete
germination in Marasmius and Pleurotus indicated that for
these saprotrophic fungi, viability, as determined by staining,
equalled germinability. However, these vital stains were poor
predictors of viability and germinability for ectomycorrhizal
fungi and dark-spored saprotrophic basidiomycetes. Based on
initial FDA staining levels for S. tomentosus and R. rubescens a
greater number of spores germinated after a month than were
initially considered viable. In addition, the percentage of
viable spores as determined by the FDA assay continued to
increase in S. tomentosus, R. rubescens and Coprinus quadrifidus
spores until germination. This suggests that a simplistic
interpretation of vital stain data for fungal spores may be
misleading and that other factors merit consideration.
One important factor that may affect the both the
germinability of spores and their viability as measured by vital
staining methods is dormancy. Sussman (1966a, b, 1968),
Sussman & Halvorson (1966) and Sussman & Douthit (1973)
147
defined several types of dormancy in fungal spores. Constitutive dormancy is a condition wherein germination is
delayed due to an innate property of the dormant stage such
as a barrier to the penetration of nutrients, a metabolic block
or the production of a self-inhibitor. Exogenous dormancy is
a condition wherein development is delayed due to unfavourable chemical or physical conditions in the environment.
In the present case, both types of dormancy may be in
operation.
The FDA and MTT vital staining procedures are both mild
in their effects on spores. In each case, the stain must enter the
spore to become effective. However, constitutive dormancy
mechanisms may have inhibited adequate entrance of stain
into the cytoplasm just as they prevent nutrients from
entering the spore so that only non-dormant or weakly
dormant spores may have responded to FDA or MTT
staining. That MTT staining is slightly more invasive due to
the elevated temperatures required for incubation and that
FDA and MTT work on different enzymes may have
accounted for the consistently higher level of staining with
MTT.
At the same time, most spores were shown to contain at
least one intact nucleus. Fixation of spores before staining in
both fluorescent and non-fluorescent procedures may have
allowed these stains to enter even the dormant spores. From
this perspective, the vital stains appear to have provided a
better measure of dormancy than viability, while the best
prediction of viability was given by nuclear staining data. By
using the attributes of either fluorescent or non-fluorescent
'vital' and nuclear stains in combination the following
equations can be used to estimate not only the viability but
dormancy and activation of spores:
Viable = T-F = N
Active = N-U
Dormant = N - 5
Where: T = number of total spores; F = number of obviously
damaged spores or spores that contain degraded nuclear material;
N = number of spores with an intact nucleus after DAP! or
haematoxylin staining; U = number of spores unstained with FDA
or MTT; 5 = number of spores stained with FDA or MTT.
The pattern of vital staining and germination in most
saprotrophic and ectomycorrhizal fungi examined in this
study can now be better explained. In light-spored saprotrophic fungi the number of FDA or MTT positive spores
equalled the number of spores with an intact nucleus and the
spores exhibited high activation and therefore weak or no
dormancy. Also, the sum of active and dormant spores
equalled the number of spores with an intact nucleus. Boiled
spores had neither intact nuclei nor FDA or MTT positive
reactions. Most spores of the ectomycorrhizal fungi were
initially alive but dormant. Association with pine roots over
time resulted in breaking of dormancy, increasing activation
and finally germination. The increase in FDA staining and
intermittent or periodic germination suggests that different
cohorts of spores were present, some that germinated early
and others that required more time or environmental inputs
before breaking dormancy and germinating. It is unknown
whether the pattern of spore germination is intermittent or
periodic.
to-2
Basidiospore viability in ectomycorrhizal fungi
Different basidiomes and different populations of the same
fungal species may also express various abilities or strategies
for dormancy, activation and germination. Typically no two
basidiomes had the same proportion of dormant or active
spores. This was especially evident in the three R. rubescens
spore slurries made from basidiomes collected at different
localities, and in the slurries used for the germination
experiments. The spores may be under genetic control that
results in some spores being ready to germinate and some
remaining dormant. The proportion of spores in each category
may be determined genetically, by age or developmental
stage or may be dependent on local environmental conditions.
Such diversity enhances survivability and implies that
researchers who utilize single-spore isolates derived from only
a low percentage of germinated spores may bias subsamples
of genetic diversity.
The correlation between the steady increase in FDA
staining and intermittent or periodic germination events,
especially in S. tomentosus, can also be explained by a
stimulatory effect of growing hyphae on spore germination.
Fries (1978, 1982, 1983 a) has demonstrated that exposure of
spores to actively growing hyphae of the same species
induces germination of some ectomycorrhizal fungi. This was
substantiated by observation of mycelium near clusters of
germinating S. tomentosus spores. Although mycorrhizas were
formed by both S. tomentosus and R. rubescens spores and
hyphae were undoubtedly present, fewer hyphae and fewer
clusters of germinating spores were observed in R. rubescens.
The spores may also have been affected by homing reactions
described by Fries (1981, 1983 b) and Fries & Swedjemark
(1985) where inter- or intraspecific hyphae are attracted to and
destroy spores. The homing reactions may have been stronger
in R. rubescens than in S. tomentosus, resulting in fewer
germinating spores and more lysis.
Other ectomycorrhizal fungi examined did not react well to
the combination of stains. Spores of A. muscaria were neither
active nor dormant and even the nuclear stains failed to
provide evidence of viability. The conclusion must be that the
spores were dead. Maintenance of A. muscaria spores other
than in spore slurries should be attempted to determine if this
fungus responds more favourably to different storage methods.
Discrepancies between staining and germination patterns
for some saprotrophic fungi studied required a more complex
explanation. Agaricus brunnescens spores exhibited moderate
levels of succinate dehydrogenase localization, but no esterase
activity. This would be expected if the storage product of A.
brunnescens spores consisted primarily of glycogen or some
other non-lipoidal material lacking ester bonds. O'Sullivan &
Losel (1971), however, found the lipid reserve of A. brunnescens
spores to be rich; only the phospholipid fraction was
unusually small. Another possibility would be that A.
brunnescens spores may possess another type of constitutive
dormancy in which enzymes for lipid catabolism, including
esterases, might be inhibited until after germination. That
would explain the lack of esterase localization with FDA
before germination vesicle formation and the relatively high
succinate dehydrogenase localization.
The pattern of staining observed in Conocybe laetea spores
where activation increased early in the experiment but
148
gradually subsided to a level lower than initial values suggests
involvement of both constitutive and exogenous dormancy
mechanisms. After the spores were exposed to liquid nutrient
solution, dormancy of an early cohort may have broken,
increasing activation. However, because composition of the
nutrient solution was not satisfactory or because the
temperature or some other environmental factor was not
suitable, activation did not proceed to germination and no
other cohorts of spores were activated.
The positive correlation between low refractivity under
phase-contrast microscopy and high FDA staining was most
apparent in spores of saprotrophic and ectomycorrhizal fungi
that were light in colour and had thin or moderately thin
walls. This included Marasmius, Pleurotus, Suillus and Rhizopogon. While not infallible, this correlation was adequate to
allow refractivity to serve as a rough predictor of physiological
activity. The refractivity may be due to lipids present in
spores of all these species. In dormant spores the lipid droplets
were large whereas spores that had become activated and
closer to germination had more but smaller lipid droplets. The
loss of lipid in the spore and softening of the spore wall prior
to germination vesicle formation may account for the loss of
phase refractivity (N. P. Money, Colorado State University,
pers. comm.).
There were a few problems concerning what constituted
positive FDA and MTT staining. Spores of certain fungi,
particularly brown-spored species of agarics such as Conocybe,
Cortinarius, Hebeloma and the gasteromycetes Hymenogaster
and Gautieria autofluoresced, that is they fluoresced with
epifluorescence microscopy even without the addition of
FDA. The autofluorescence was usually brown, orange,
yellow or red and so was different in colour from the green
FDA reaction and often masked low levels of FDA staining.
In addition, spores of R. rubescens and S. tomentosus appeared
to have both cytoplasmic fluorescence and autofluorescence of
an outer wall layer which was most evident as a faint halo in
spores with quenched fluorescence.
Interpretation of the MTT staining reaction also presented
problems in some fungi. Because succinate dehydrogenase in
fungi is localized only in mitochondria (Zalokar, 1965) only
the mitochondria should reduce MTT to diformazan. In
species in which the cytoplasm is mostly dominated by one or
more large lipid droplets such as Laetarius and Russula, and to
a lesser extent Suillus and Rhizopogon, the lipid droplet often
appeared stained by MTT. In the environment of the cell,
factors other than succinate dehydrogenase may reduce MTT
giving a false positive reaction. This is evident in spores
incubated in the reaction mixture for several hours. However,
ultrastructural examination of spores in which the lipids
appeared to be staining showed that the large lipid droplets
had compressed the cytoplasm against the spore wall and that
mitochondria in the cytoplasm were distributed around the
lipid droplets (S. L. Miller, unpublished). Therefore viewing of
MTT positive spores may give the impression that the lipid
is stained when the lipid is actually surrounded by
mitochondria but resolution is not possible with the light
microscope. Further experiments concluding with the germination of these spores are necessary.
As can be seen, spore viability is a relative and complex
149
S. L. Miller, P. Torres and T. M. McClean
concept and there is no single parameter that can be used to
predid vitality or germination. The procedures that we have
outlined provide assays for spore dormancy and activation
that can be used in a variety of fungi including ectomycorrhizal
species. We can now determine the effeds of different
environmental fadors such as temperatures, nutrients, inhibitors on activation of spores without relying on germination
as the sole determinant of viability. The steps required to
move spores from activation to germination merit further
study in both saprotrophic and ectomycorrhizal fungi.
Fries, N. & Birraux, D. (1980). Spore germination in Hebeloma stimulated by
living plant roots. &perien/ia 36, 1056--1057.
Fries, N. & Swedjemark, G. (1985). Sporophagy in Hymenomycetes.
Experimental Mycology 9, 74-79.
Hauser, E. j. P. & Morrison, j. H. (1964). The cytochemical reduction of nitroblue tetrazolium as an index of pollen viability. American Journal of Botany
51, 748-752.
Heslop-Harrison, j. & Heslop-Harrison, Y. (1970). Evaluation of pollen
viability by enzymatically induced fluorescence; intracellular hydrolysis of
fluorescein diacetate. Stain Technology 45, 115-120.
Lachapelle, M. & Boothroyd, E. R. (1986). Nuclear behavior and cell wall
composition in relation to budding in mutants of the yeast Saccharomyces
cerevisiae. Canadian Journal of Botany 64, 193-200.
We would like to thank Dr Rick Kerrigan for his suggestions
on germination of Agaricus brunnescens spores. This project
was funded in part by NSF Grant BSR-8805983, USDA Grant
91-37101-6759, a grant from the University of Wyoming!
National Park Service Research Center and a postdoctoral
fellowship from the Spanish government.
Larkin, P. J. (1976). Purification and viability determinations of plant
protoplasts. Planta 128, 213-216.
Losel. D. M. (1964). The stimulation of spore germination in Agaricus bisporus
by living mycelium. Annals of Botany 28, 541-554.
Miller, S. L. (1988). A systematic overview of spore symmetry and
tegumentation in hypogeous and gasteroid Russulales. Canadian Journal of
Bolany 66, 2561-2573.
Miller, S. L. & MiUer, O. K., jr (1988). Spore release in hypogeous and
gasteroid Russulales. Transactions of the Brilish Mycological Sociely 90,
513-526.
O'Sullivan, j. & Lose!, D. M. (l971). Spore lipids and germination in Agaricus
bisporus. Archiv fii.r Mikrobiologie 80, 27.
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