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Mycologia
ISSN: 0027-5514 (Print) 1557-2536 (Online) Journal homepage: https://www.tandfonline.com/loi/umyc20
Rhizopogon olivaceotinctus increases its inoculum
potential in heated soil independent of
competitive release from other ectomycorrhizal
fungi
Thomas D. Bruns, Maren L. Hale & Nhu H. Nguyen
To cite this article: Thomas D. Bruns, Maren L. Hale & Nhu H. Nguyen (2019)
Rhizopogon�olivaceotinctus increases its inoculum potential in heated soil independent
of competitive release from other ectomycorrhizal fungi, Mycologia, 111:6, 936-941, DOI:
10.1080/00275514.2019.1657354
To link to this article: https://doi.org/10.1080/00275514.2019.1657354
Published online: 11 Oct 2019.
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Rhizopogon olivaceotinctus increases its inoculum potential in heated soil
independent of competitive release from other ectomycorrhizal fungi
Thomas D. Bruns
a
, Maren L. Hale
a
, and Nhu H. Nguyen
b
a
Department of Plant and Microbial Biology, University of California Berkeley, California 94720-3102;
b
Department of Tropical Plant and Soil
Sciences, University of HawaiʻiatMānoa, Honolulu, Hawaiʻi 96922
ABSTRACT
Rhizopogon olivaceotinctus is a rarely collected ectomycorrhizal fungus that has been found
primarily in California and southern Oregon. Prior work has shown that it (i) is common in soil
spore banks associated with pine forests from these areas; (ii) is rare or absent on trees in
undisturbed forests in these same areas; (iii) exhibits an increased abundance on pine seedlings
following fire or experimental soil heating; and (iv) has spores that are more resistant to heat than
those of several other ectomycorrhizal species tested to date. Here, we reject the hypothesis that
the increased abundance of the species following soil heating is caused only by reduced
competition with other ectomycorrnizal fungi and show instead that heating alone significantly
increases the inoculum potential of its spores. We argue that this is likely caused by heat
stimulation of the spores, a process that has precedent in saprotrophic fungi and plant seeds.
This result, in combination with those of previous studies, shows that Rhizopogon olivaceotinctus is
well adapted to fire.
ARTICLE HISTORY
Received 22 May 2019
Accepted 15 August 2019
KEYWORDS
Conservation; seedling
bioassays; soil heating; spore
longevity
INTRODUCTION
Rhizopogon olivaceotinctus is a rarely collected false
trufflethatformsectomycorrhizaewithpine(Taylor
and Bruns 1999) and perhaps other members of the
Pinaceae (Smith and Zeller 1966). A search in
MyCoPortal, under this name and under its previous
name (Alpova olivaceotinctus), yields only 23 collec-
tions across nine herbaria. Some of these collections
were made at the same date and location, suggesting
that they may be parts of the same collection that were
distributed to different herbaria. All but two originate
from California, and most seem to be derived from two
California sites where R. olivaceotinctus has been
recollected multiple times. In addition, there is one
collectionfromsouthernOregonandonefromthe
mountains outside Mexico City. Because of its apparent
rarity, it was listed as one of 135 potentially rare species
in the Northwest Forest Plan (Castellano et al. 1999).
Part of the species’apparent rarity could be due to its
nearly black basidiomes and its fruiting below ground:
these features make it hard to find and could lead to it
being undercollected. Nevertheless, it is certainly not
well known from its fruiting.
In contrast to the rarity of its basidiome collections,
R. olivaceotinctus has been found throughout California
hard pine (i.e., Pinus subg. Pinus) forests in the soil
spore banks (Taylor and Bruns 1999; Kjøller and Bruns
2003; Izzo et al. 2006b; Rusca et al. 2006; Glassman
et al. 2015). These observations were based on bioassays
in which pine seedlings were planted into samples of
forest soil that had been dried to kill active mycelium
and select for colonization by resistant propagules (i.e.,
basidiospores). When these assays were based on
diluted forest soils, the frequency of colonization by
R. olivaceotinctus was not very high. This contrasts
with species such as R. salebrosus that increase in colo-
nization frequency with soil dilution (Rusca et al. 2006).
Thus, spores of R. olivaceotinctus are widespread but
not necessarily abundant in California pine forests.
Rhizopogon olivaceotinctus appears to be rare or
absent as an actively growing fungus in undisturbed
forests. In two sites where its spores were detected by
bioassay, mycorrhizae on mature trees also were
sampled (Taylor and Bruns 1999; Izzo et al. 2005a),
but R. olivaceotinctus was not found. To our knowl-
edge, it has never been reported from the roots of
mature trees in undisturbed forests. Some of the rare
collections of its fruiting bodies do come from mature
forests (e.g., Bullard’s Bar, Yuba County, California),
but picnic areas and campgrounds are listed in the
location descriptions. Izzo et al. (2005b) analyzed
sequence data from rodent scat, showing that three of
CONTACT Thomas D. Bruns pogon@berkeley.edu
MYCOLOGIA
2019, VOL. 111, NO. 6, 936–941
https://doi.org/10.1080/00275514.2019.1657354
© 2019 The Mycological Society of America
Published online 11 Oct 2019
the five Rhizopogon species that were common in the
spore bank were also being found and eaten by the local
rodents. Two of these species were not found on roots
of mature trees in the same forests, and the third
represented less that 1% of the colonized root tip sam-
ples; thus, all three species were presumably rare on
roots but were found successfully as basidiomes by
rodents. However, Rhizopogon olivaceotinctus was not
found either on mature trees or in the rodent scat even
though it was present in the spore bank at that location.
The only collection site where we have found it fruiting
is a post-fire site in Point Reyes National Seashore, in
association with approximately 11-y-old trees.
In contrast to the situation in undisturbed forests,
ectomycorrhizal roots colonized by R. olivaceotinctus
can be abundant on pine seedlings in nature following
stand-replacing fires (Baar et al. 1999; Glassman et al.
2016), and its frequency increased on seedlings in post-
fire relative to pre-fire bioassays (Baar et al. 1999;
Glassman et al. 2016). Similarly, its frequency increased
on seedlings when soils were artificially heated in the
laboratory to mimic the effects of fire (Izzo et al. 2006a;
Peay et al. 2010). In addition, Peay et al. (2009) used
vital stains and heat treatments of water suspensions of
spores to show that R. olivaceotinctus spores are more
heat resistant than those of several other mycorrhizal
fungi, including Rhizopogon salebrosus.
Taken together, the results from these studies lead to
the view that R. olivaceotinctus is a post-fire ruderal
species that fruits in the early years following fire,
establishes a spore bank, and remains dormant until
the next disturbance or stand-replacing fire. Bioassay
results from unheated soils show that it does not need
fire for its spores to germinate and colonize seedlings,
but heat tolerance of its spores may give it an advantage
in surviving fire. In fact, heat-tolerant spores could be
sufficient to explain its increased frequency following
fire where less heat-tolerant competitors are eliminated.
Alternatively, if it is indeed adapted to fire, one might
expect its spores to be positively stimulated by the
direct or indirect effects of fire, similar to the behavior
of spores of some saprotrophic fungi (Shear and Dodge
1927; Hardison 1976; Splittstoesser et al. 1972) and
seeds of some fire-adapted plants (Keeley 1987;
Kauffman and Martin 1991; Keeley et al. 2011).
In this study, we tested the hypothesis that the
increased abundance of R. olivaceotinctus following
heating is caused by direct heat stimulation and not
only by reduced competition with other ectomycorrhi-
zal fungi. To accomplish this, we used soil that con-
tained known quantities of R. olivaceotinctus spores but
lacked other ectomycorrhizal inoculum and then com-
pared the inoculum potential of this soil in heated
versus unheated treatments. Our results show that heat-
ing increases the inoculum potential of the spores, even
when competitive release is eliminated as a possible
mechanism.
MATERIALS AND METHODS
Collection and inoculation of soil.—Spores were
extracted from a single fresh collection of multiple
basidiomes that was collected in Point Reyes National
Seashore in a young, Pinus muricata forest that had
established following a 1995 wild fire. A spore slurry
containing 2.5 × 10
6
spores was mixed into 28 L of
nonsterile soil/sand mix that lacked ectomycorrhizal
(EM) inoculum, and 1.6 L portions of this inoculated
soil were placed in 16 6.6-inch terracotta pots and
buried in a grassland in 2006 (Bruns et al. 2009). One
of these pots was retrieved after 12 y, and the contents
were used as inoculum for this experiment.
Mixing and heat treatment.—Inoculated soil was
divided into two treatments: heated and unheated. Soil
to be heat-treated was placed in a 39.5 × 23 cm stainless
steel pan and spread evenly to a depth of approximately
0.9 cm. Four K-type thermocouples were placed in the
center (by depth) of the soil and arrayed approximately
equidistant along the long axis of the pan (FIG. 1). The pan
was then placed in a drying oven, and the temperature was
monitored and recorded. After 40 min, the soil reached
a peak temperature of 56.7 ± 1.44 C. The pan was removed
from the oven and allowed to cool slowly to room
temperature. This temperature was selected because our
preliminary experiment had shown that R. olivaceotinctus
spores remained viable after 3 h at 65 C.
Heated and unheated soil treatments were diluted
4-fold with sterile soil and then with sterile coarse sand,
to obtain a 1:1 soil/sand mixture and a final spore
concentration of 22 spores/mL. This concentration
was selected based on a pilot study conducted several
years earlier that showed that half the seedlings were
colonized in unheated soil at this concentration.
Bioassays.—Approximately 50 mL of the heated or
unheated soil mixtures was added to 98 (49/treatment)
Cone-tainers (RLC-4 Super “Stubby”Cell Cone-tainers;
Stuewe & Sons, Corvallis, Oregon), with a small amount
of polyester filling (obtained at a local fabric store)
stuffed in the bottom to prevent leakage. Each Cone-
tainer was planted with 2–3seedsofPinus muricata that
were collected locally, surface sterilized with 30% H
2
O
2
,
imbibed with water overnight, and allowed to germinate
at the University of California (UC) Berkeley Oxford
MYCOLOGIA 937
Tract Insectary greenhouse. Seedlings were thinned to
one per Cone-tainer if more than one was successfully
established, and they were grown for 7 mo (Sep 2017 to
Apr 2018).
Scoring of mycorrhizal condition.—Seedlings were
harvested from the Cone-tainers, and root systems were
washed until free of most soil. Roots were examined
under a dissecting scope and scored as mycorrhizal or
nonmycorrhizal. Seedlings that died in the greenhouse
were tallied as dead (42 total). If death occurred near the
end of the experiment and the root system was intact, they
were scored for mycorrhizae (30 total), but their living/
dead status was also recorded. Twelve seedlings that died
earlier in the experiment were not scored for mycorrhizal
status.
Sequencing of ectomycorrhizae.—Twenty-four root
tips were harvested from 24 seedlings, including six
representatives from each category (heated mycorrhizal,
heated nonmycorrhizal, unheated mycorrhizal, and
unheated nonmycorrhizal) to verify colonization by
R. olivaceotinctus. Root tips were removed, placed in
sterile water, and refrigerated at 4 C for a few days until
DNA was extracted. For extraction, individual root tips
were placed into strip tubes containing 10 µL of Extract-
N-Amp extraction solution (Sigma-Aldrich, St. Louis,
Missouri).Tubeswerespundownusingaminicentrifuge
and heated in a thermal cycler for 10 min at 65 C, 10 min at
95 C, and cooled to room temperature. A 20-µL aliquot of
Extract-N-Amp Dilution Solution (Sigma-Aldrich) was
added to each tube, and the strip tubes were stored in the
refrigerator overnight (4 C).
Polymerase chain reaction (PCR) amplification of the
internal transcribed spacer (ITS) region for each sample
was conducted using primers ITS1F (forward) and ITS4
(reverse) and standard conditions (White et al. 1990;
Gardes and Bruns 1993). The concentration of DNA
was determined with the QuBit DNA HS kit (Thermo
Fisher Scientific, Waltham, Massachusetts), and those
with concentrations greater than 10 ng/µL were diluted
with sterile water to reach 10 ng/µL. Samples were sent
to the UC Berkeley DNA Sequencing Facility for PCR
cleanup and Sanger sequencing. All sequences were
identified with the National Center for Biotechnology
Information (NCBI) Basic Local Alignment Search
Tool (BLAST) to confirm that the ectomycorrhizal
roots were R. olivaceotinctus. One exemplar was depos-
ited in Genbank: MN235715.
Statistical analysis of heat and colonization.—The
number of colonized versus uncolonized seedlings was
compared between heat-treated and nontreated soils
with a chi-square (goodness of fit) test as implemented
with R (R Core Team 2014). Initially this test used all
seedlings (i.e., both living and dead), and the test was
rerun with only the living seedlings.
RESULTS
The heating treatment produced a fairly uniform tem-
perature across the soil (FIG. 1). A total of 97 seedlings
established and grew, but almost half (42) died prior to
the end of the experiment from unknown causes. All
but 12 died in the last weeks of the experiment, and the
mycorrhizal status was still easily determined for the 30
other dead seedlings. BLAST results indicated that the
EM root samples all contained R. olivaceotinctus, and
no other EM fungi were detected.
Soil heating increased the number of seedlings colo-
nized by R. olivaceotinctus (FIG. 2). Of the 45 seedlings
recovered from the heated soil, 41 were mycorrhizal
(84%) and 4 were nonmycorrhizal (8%). Of the 40
recovered from the unheated soil, 21 were mycorrhizal
(44%) and 19 were nonmycorrhizal (43%) (χ
2
= 14.099,
Figure 1. Heat treatment and temperature profile. A. Pan of soil
with four thermocouples implanted. B. Temperatures over time
for each of the thermocouples during heating in drying oven
and cooling after removal.
938 BRUNSETAL.:HEATSTIMULATIONOFRHIZOPOGON OLIVACEOTINCTUS
df =1,P= 0.0002). When all dead seedlings are
dropped from the analysis, the sample size decreased
substantially: heated mycorrhizal (31); heated nonmy-
corrhizal (0); unheated mycorrhizal (17); unheated
nonmycorrhizal (7). Nevertheless, the difference in
treatments remained significant (χ
2
= 7.9008, df =1,
P= 0.005).
DISCUSSION
The inoculum potential of R. olivaceotinctus spores
increased approximately 2-fold in heated soil relative to
the unheated control (FIG. 2). In addition, the percen-
tage of seedlings colonized in the heat-treated soil (84%)
is very similar to the percentage colonized by undiluted
soil in year 2 of the longevity experiment (82%; Bruns
et al. 2009), even though the spore concentration was
4-fold more dilute in the heated soil. Previous results
had shown that colonization by R. olivaceotinus
increased with heating of native forest soil (Izzo et al.
2006a;Peayetal.2010), but those increases could have
been caused by heat tolerance of its spores (Peay et al.
2009) coupled with reduced competition from other
ectomycorrhizal fungi that lacked such tolerance. In the
current study, we have eliminated the confounding fac-
tor of competition with other ectomycorrhizal species.
Increased colonization under these conditions demon-
strates that the spores become more effective at coloni-
zation after being heated in the soil.
The increased inoculum potential observed here has
two possible causes: direct stimulation or activation of
the basidiospores, or indirect changes in the colonization
efficiency of basidiospores once they germinate. Indirect
effects would include any process that increases the dis-
tance mycelium of a germinating spore can grow through
the soil, the probability of that mycelium finding a root,
or the probability of colonizing a root once found.
Reduction of antagonistic soil organisms such as bacteria,
spore-feeding fungi, and mycophagous soil fauna (Fries
and Swedjemark 1985;LeveauandPreston2008;Geisen
et al. 2016; Siebyla and Hilszczanska 2017)bytheheat
treatment would be such a possible mechanism, and it is
also conceivable that microbes that positively stimulate
colonization (Garbaye 1994) are increased with heating.
These factors were not tested for or controlled in our
experiment, but one might expect them to have general
effects on post-fire ectomycorrhizal fungi rather than
a specific effect on this particular species.
Direct stimulation or activation of basidiospores is
the simplest explanation. It has precedence with heat
activation of some ascospores (Shear and Dodge 1927;
Splittstoesser et al. 1972; Hardison 1976) and also has
obvious parallels with seeds of fire-adapted plants that
are often heat-scarified (Keeley 1987; Kauffman and
Martin 1991; Keeley et al. 2011). Here, we envision
a similar process in which either the dormancy of
basidiospores is broken by heat or heat stimulates ger-
mination directly. From previous work, we know that
several species of Rhizopogon have a percentage of
spores that are not initially receptive to colonization
but become receptive over time (Bruns et al. 2009).
Viewed within this context, a heat-mediated breaking
of dormancy of R. olivaceotinctus is a very plausible
mechanism that fits well with the observed increase in
R. olivaceotinctus following wildfires (Baar et al. 1999;
Glassman et al. 2016).
To our knowledge, this is the first ectomycorrhizal
fungus for which heat treatments have increased inocu-
lum potential. This behavior, in combination with the
heat tolerance of the spores (Peay et al. 2009), its
increased frequency of occurrence following heating
of forest soil (Izzo et al. 2006a; Peay et al. 2010), and
its increased prevalence after forest fires (Baar et al.
1999; Glassman et al. 2016), makes a strong case for
classifying this as a fire-adapted species.
Peay et al. (2010)showedthatR. olivaceotinctus spores
are not stimulated by ash alone in natural soils with other
competing species present. In fact, R. olivaceotinctus was
completely absent on the bioassay seedlings growing in
unheated soil with or without ash additions in that study.
However, the results may have shown a synergistic effect
between ash and heat, as the number of root tips colo-
nized by R. olivaceotinctus almost doubled when ash was
added to heated soil compared with heated soil without
ash additions.
The longevity of spores of R. olivaceotinctus in the
soil is a necessary precondition for heat tolerance and
heat stimulation to be advantageous, because the fire
Figure 2. Mycorrhizal and nonmycorrhizal seedlings in heated
and unheated soil.
MYCOLOGIA 939
return interval in California pine forests ranges from 10
y in dry mid-elevation pine to over 100 y in Sierra
lodgepole pine (Martin and Sapsis 1991). Here, we
show that the spores remain viable for at least 12 y, as
this sample was collected in 2006 and was buried in the
soil since that time (Bruns et al. 2009). Greater periods
of longevity can be inferred from two fires in which the
age of forest was known. The Rim Fire burned in
aPinus ponderosa forest that was about 50 y old, and
the Mount Vision Fire burned in Pinus muricata forest
that was about 60 y old. Both sites had abundant post-
fire colonization of seedlings by R. olivaceotinctus, and
neither site had detectable colonization by this species
prior to the fire (Baar et al. 1999; Grogan et al. 2000;
Glassman et al. 2016). Thus, we conclude that the
spores are likely to last at least several decades. Spore
longevity seems to be widespread within at least the
pine-associated Rhizopogon species (Bruns et al. 2009),
and multiple Rhizopogon species are common coloni-
zers of seedlings in these same post-fire settings (Baar
et al. 1999; Grogan et al. 2000; Smith et al. 2005;
Glassman et al. 2016).
Although Rhizopogon species are diverse and abun-
dant in California pine forests and are common on post-
fire seedlings, we have not found other Rhizopogon spe-
cies that increase in frequency of colonization after soil
heating experiments or that show a relative increase in
colonization in post-fire forest soil (Baar et al. 1999;Izzo
et al. 2006a; Glassman et al. 2016). Post-fire success of
these other Rhizopogon species may be due to spore
abundance, longevity, and responsiveness alone if soil
heating does not reach killing temperatures. In Europe,
however, Kipfer et al. (2010) reported that R. roseolus
increases its abundance with heat treatment of forest
soil. Their results may be due to heat tolerance and
competitive release, or they may show that other species
in the genus also increase their inoculum potential with
heating. One additional candidate for this behavior is
R. olivaceoniger (A.H. Smith). This is an eastern North
American species that is morphologically very similar to
R. olivaceotinctus. Both species were initially placed in
a separate subgenus because of their gel-filled glebas, and
both have similar dark colors and relatively long narrow
spores. Rhizopogon olivaceoniger also shares an addi-
tional feature with R. olivaceotinctus; it appears to be
a very rare fruiter. A MyCoPortal search revealed only
two collections of it, and both were from the holotype
location.
These results have significance for conservation in two
ways. First, having species such as R. olivaceotinctus in
a forest increases the resilience of the system because it
broadens the conditions under which mycorrhizal inocu-
lum survives catastrophic wildfire. In regions such as
California where large, intense forest fires are becoming
more common, survival of inoculum is likely to affect the
speed and trajectory of forest regeneration. Second, the
fact that there are fire-adapted mycorrhizal species means
that different fire regimes are likely to select for different
species of ectomycorrhizal fungi, just as they do for plants
and animals. Thus, Martin and Sapsis’s(1991)mantra
“pyrodiversity promotes biodiversity”is likely to apply
to ectomycorrhizal fungi as well as plants.
FUNDING
Funding was provided by National Science Foundation
(NSF) grant DEB 236096 to T.D.B. for the early collections
and long-term storage of R. olivaeotinctus spores, and by
Department of Energy grant DE-SC0016365 for current
funding on post-fire fungi.
ORCID
Thomas D. Bruns http://orcid.org/0000-0002-2943-8669
Maren L. Hale http://orcid.org/0000-0003-4980-9823
Nhu H. Nguyen http://orcid.org/0000-0001-8276-7042
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