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Rhizopogon olivaceotinctus increases its inoculum potential in heated soil independent of competitive release from other ectomycorrhizal fungi


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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.
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Rhizopogon olivaceotinctus increases its inoculum
potential in heated soil independent of
competitive release from other ectomycorrhizal
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:
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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
, Maren L. Hale
, and Nhu H. Nguyen
Department of Plant and Microbial Biology, University of California Berkeley, California 94720-3102;
Department of Tropical Plant and Soil
Sciences, University of HawaiʻiatMānoa, Honolulu, Hawaiʻi 96922
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.
Received 22 May 2019
Accepted 15 August 2019
Conservation; seedling
bioassays; soil heating; spore
Rhizopogon olivaceotinctus is a rarely collected false
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
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 speciesapparent 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., Bullards 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
2019, VOL. 111, NO. 6, 936941
© 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
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
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 StubbyCell 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 23seedsofPinus muricata that
were collected locally, surface sterilized with 30% H
imbibed with water overnight, and allowed to germinate
at the University of California (UC) Berkeley Oxford
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
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,
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.
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
= 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.
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
= 7.9008, df =1,
P= 0.005).
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.
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
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 Sapsiss(1991)mantra
pyrodiversity promotes biodiversityis likely to apply
to ectomycorrhizal fungi as well as plants.
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.
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Maren L. Hale
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... 3B) can survive wildfires (Baynes et al. 2011;Greene et al. 2010), and some fungi likely have persistent, heat-resistant spores, for example, Rhizopogon olivaceotinctus (FIG. 3K;Baar et al. 1999;Glassman et al. 2016;Bruns et al. 2019), Ascobolus carbonarius, and Trichophaea abundans (conidia and ascospores; Msh and Webster 1968b). Others may survive as mycelia in mycorrhizae or soil, as exemplified by ...
... Rhizopogon olivaceotinctus-dominated root colonization after the 1995 Vision Fire burned down a Pinus muricata forest (Baar et al. 1999) and after the 2013 Rim Fire burned down a Pinus ponderosa forest (Glassman et al. 2016), both in California. Its great abundance in the roots of juvenile trees is likely due to its ability to produce and maintain prolific thermotolerant spore banks in soil (Bruns et al. 2019). Many more endemic pyrophilous fungi may exist that are thus far at best known only as sequences in community surveys or, more likely, wholly undescribed. ...
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Fires occur in most terrestrial ecosystems where they drive changes in the traits, composition, and diversity of fungal communities. Fires range from rare, stand-replacing wildfires to frequent, prescribed fires used to mimic natural fire regimes. Fire regime factors, including burn severity, fire intensity, and timing, vary widely and likely determine how fungi respond to fires. Despite the importance of fungi to post-fire plant communities and ecosystem functioning, attempts to identify common fungal responses and their major drivers are lacking. This synthesis addresses this knowledge gap and ranges from fire adaptations of specific fungi to succession and assembly fungal communities as they respond to spatially heterogenous burning within the landscape. Fires impact fungi directly and indirectly through their effects on fungal survival, substrate and habitat modifications , changes in environmental conditions, and/or physiological responses of the hosts with which fungi interact. Some specific pyrophilous, or "fire-loving," fungi often appear after fire. Our synthesis explores whether such taxa can be considered cosmopolitan, and whether they are truly fire-adapted or simply opportunists adapted to rapidly occupy substrates and habitats made available by fires. We also discuss the possible inoculum sources of post-fire fungi and explore existing conceptual models and ecological frameworks that may be useful in generalizing fungal fire responses. We conclude with identifying research gaps and areas that may best transform the current knowledge and understanding of fungal responses to fire.
... Thus, these spores can be dispersed independent of host plant presence, allowing the spore bank to spread beyond the host tree stand. Since Rhizopogon species are often dominant in EcM fungal spore banks (Buscardo et al. 2011;Bruns et al. 2019;Shemesh et al. 2020), they play an integral role as a major source of fungal inoculum for seedlings after disturbances such as wildfire and clear cutting (Horton et al. 1998;Kipfer et al. 2010;Glassman et al. 2016). ...
... Although these methods do not necessarily retrieve all EcM fungi available, the EcM fungal communities detected would represent the first fungi to colonize host roots. Thus, they are suitable for investigating EcM fungi related to seedling establishment or reforestation after severe disturbances (Baar et al. 1999;Buscardo et al. 2011;Glassman et al. 2016;Miyamoto and Nara 2016;Bruns et al. 2019). The species of seedlings used in bioassays can affect the composition of the EcM fungi detected (Miyamoto and Nara 2016;Murata et al. 2017), and the host species should be carefully selected. ...
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Ectomycorrhizal (EcM) fungal spores play an important role in seedling establishment and forest regeneration, especially in areas where compatible host tree species are absent. However, compared to other Pinaceae trees with a wide distribution, limited information is available for the interaction between the endangered Pseudotsuga trees and EcM fungi, especially the spore bank. The aim of this study was to investigate EcM fungal spore bank communities in soil in remnant patches of Japanese Douglas-fir (Pseudotsuga japonica) forest. We conducted a bioassay of 178 soil samples collected from three P. japonica forests and their neighboring arbuscular mycorrhizal artificial plantations, using the more readily available North American Douglas-fir (Pseudotsuga menziesii) as bait seedlings. EcM fungal species were identified by a combination of morphotyping and DNA sequencing of the ITS region. We found that EcM fungal spore banks were present not only in P. japonica forests but also in neighboring plantations. Among the 13 EcM fungal species detected, Rhizopogon togasawarius had the second highest frequency and was found in all plots, regardless of forest type. Species richness estimators differed significantly among forest types. The community structure of EcM fungal spore banks differed significantly between study sites but not between forest types. These results indicate that EcM fungal spore banks are not restricted to EcM forests and extend to surrounding forest dominated by arbuscular mycorrhizal trees, likely owing to the durability of EcM fungal spores in soils.
... Species of Rhizopogon produce spores with a thick wall that allows them to persist over many years in the soil, sometimes becoming more reactive (i.e., germinating easily and colonizing compatible hosts) as they remain longer in the soil Nguyen et al., 2012). Some species such as R. olivaceotinctus have spores that can survive and germinate after heat treatments of up to 65 °C (Izzo et al., 2006;Peay et al., 2009Peay et al., , 2010Bruns et al., 2019). This trait allows R. olivaceotinctus to outcompete other, less heat-tolerant fungi. ...
This study investigated broad patterns in communities of ectomycorrhizal fungi from three Florida habitats (sandhills, scrub, and pine rocklands) and the ability of spore bank fungi to associate with Pinus elliottii (slash pine) and Pinus densa (south Florida slash pine). Efforts to replant pines in the endangered pine rocklands are vital to the persistence of this habitat, yet little is known about the ectomycorrhizal fungi communities or how they may differ from those in other pine-dominated habitats in Florida. We used high-throughput amplicon sequencing (HTS) to assess baseline fungal communities and greenhouse bioassays to bait ectomycorrhizal fungi using seedlings. HTS soil data recovered 188 ectomycorrhizal species but only a few subsequently colonized the bioassay seedlings. We recovered 21 ectomycorrhizal species on pine seedlings including common spore bank fungi such as Cenococcum, Suillus, and Tuber, but Rhizopogon species were dominant across all sites and habitats. Habitat type and site were significant variables influencing the community composition of the total soil fungal community, soil ectomycorrhizal community, and the fungi found on seedling root tips. However, we found no significant differences between the ectomycorrhizal communities on seedling roots from the two Pinus species.
... Some suilloid species produce desiccation-resistant spores that can remain dormant for several years in soils ) and germinate in response to chemical cues produced by compatible hosts (Theodorou & Bowen, 1987;Kikuchi, Matsushita, Suzuki, & Hogetsu, 2007). Moreover, several studies have reported that spores are heat resistant and that germination may be stimulated by heat, suggesting an adaptation to fire disturbance (Izzo, Canright, & Bruns, 2006;Peay, Garbelotto, & Bruns, 2009;Murata, Nagata, & Nara, 2017b;Bruns, Hale, & Nguyen, 2019). These unique spore features are thought to promote quick establishment of EM associations with Pinaceae hosts at invasion fronts and in disturbed habitats (Baar, Horton, Kretzer, & Bruns, 1999;Ashkannejhad & Horton, 2006;Policelli et al., 2019). ...
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Microbial symbionts are essential for plant niche expansion into novel habitats. Dormant propagules of ectomycorrhizal (EM) fungi are thought to play an important role in seedling establishment in invasion fronts; however, propagule bank communities above the treeline are poorly understood in the Eurasian Arctic, where treelines are expected to advance under rapid climate change. To investigate the availability of EM fungal propagules, we collected 100 soil samples from Arctic tundra sites and applied bioassay experiments using Larix cajanderi as bait seedlings. We detected 11 EM fungal operational taxonomic units (OTUs) by obtaining entire ITS regions. Suillus clintonianus was the most frequently observed OTU, followed by Cenococcum geophilum and Sebacinales OTU1. Three Suillus and one Rhizopogon species were detected in the bioassay seedlings, indicating the availability of Larix-specific suilloid spores at least 30 km from the contemporary treeline. Spores of S. clintonianus and S. spectabilis remained infective after preservation for 14 mo and heat treatment at 60 °C, implying the durability of the spores. Long-distance dispersal capability and spore resistance to adverse conditions may represent ecological strategies employed by suilloid fungi to quickly associate with emerging seedlings of compatible hosts in treeless habitats.
... Heat treatment had a positive effect on the occurrence of R. togasawarius, and it clearly increased the frequency of its colonization ( Figure 3, Table 2). Bruns et al. (2019) showed that the rate of EcM formation by R. olivaceotinctus spores was significantly higher in heated soils than in non-heated ones. Thus, the increased frequency of heat-treated spore colonization may be due to R. togasawarius heat tolerance, or to reduced competition with other EcM fungi susceptible to heat. ...
Ectomycorrhizal (EcM) fungal spore banks can facilitate seedling establishment where compatible host tree species are absent. Although Rhizopogon togasawarius spore banks play an important role in the early establishment stage of endangered Japanese Douglas-fir (Pseudotsuga japonica) seedlings, the dispersal ecology of this EcM fungus is poorly understood. The objective of this study was to clarify the spatial distribution and dispersal distance of R. togasawarius spore banks that extend inside and outside extant P. japonica forests. We evaluated R. togasawarius spore banks in five remnant forests and neighboring arbuscular mycorrhizal artificial plantations using Douglas-fir (P. menziesii) seedling bioassays. Forty-five to ninety-five surface soils were collected along lines of increasing distance from P. japonica forests to neighboring plantations. In each forest, 60%–84% of the soils collected inside or within 50 m of forest boundaries of P. japonica forests harbored R. togasawarius spore banks. The occurrence of R. togasawarius decreased significantly with increasing distance from the forest boundaries. Moreover, R. togasawarius was detected in samples several hundred meters away from forest boundaries. These results suggest that R. togasawarius has a dispersal capacity to extend the range of the spore banks outside the forest of host trees.
Mycorrhizae are mutualisms between plants and fungi that evolved over 400 million years ago. This symbiotic relationship commenced with land invasion, and as new groups evolved, new organisms developed with varying adaptations to changing conditions. Based on the author's 50 years of knowledge and research, this book characterizes mycorrhizae through the most rapid global environmental changes in human history. It applies that knowledge in many different scenarios, from restoring strip mines in Wyoming and shifting agriculture in the Yucatán, to integrating mutualisms into science policy in California and Washington, D.C. Toggling between ecological theory and natural history of a widespread and long-lived symbiotic relationship, this interdisciplinary volume scales from structure-function and biochemistry to ecosystem dynamics and global change. This remarkable study is of interest to a wide range of students, researchers, and land-use managers.
Root‐associated fungi play a critical role in plant ecophysiology, growth, and subsequent responses to disturbances, so they are thought to be particularly instrumental in shaping vegetation dynamics after fire in the boreal forest. Despite increasing data on the distribution of fungal taxonomic diversity through space and time in boreal ecosystems, there are knowledge gaps with respect to linking these patterns to ecosystem function and process. Here we explore what is currently known about post‐fire root‐associated fungi in the boreal forest. We focus on wildfire impacts on mycorrhizal fungi and the relationships between plant‐fungal interactions and forest recovery in an effort to explore whether post‐fire mycorrhizal dynamics underlie plant‐soil feedbacks that may influence fire‐facilitated vegetation shifts. We characterize the mechanisms by which wildfire influences root‐associated fungal community assembly. We identify scenarios of post‐fire plant‐fungal interactions that represent putative positive and negative plant‐soil feedbacks that may impact successional trajectories. We highlight the need for empirical field observations and experiments to inform our ability to translate patterns of post‐fire root‐associated fungal diversity to ecological function and application in models. We suggest that understanding post‐fire interactions between root‐associated fungi and plants is critical to predict fire effects on vegetation patterns, ecosystem function, future landscape flammability, and feedbacks to climate.
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Mega‐fires of unprecedented size, intensity, and socio‐economic impacts have surged globally due to climate change, fire suppression, and development. Soil microbiomes are critical for post‐fire plant regeneration and nutrient cycling, yet how mega‐fires impact the soil microbiome remains unclear. We had a serendipitous opportunity to obtain pre‐ and post‐fire soils from the same sampling locations after the 2016 Soberanes mega‐fire burned with high severity throughout several of our established redwood‐tanoak plots. This makes our study the first to examine microbial fire response in redwood‐tanoak forests. We re‐sampled soils immediately post‐fire from two burned plots and one unburned plot to elucidate the effect of mega‐fire on soil microbiomes. We used Illumina MiSeq sequencing of 16S and ITS1 to determine that bacterial and fungal richness were reduced by 38‐70% in burned plots, with richness unchanged in the unburned plot. Fire altered composition by 27% for bacteria and 24% for fungi, whereas the unburned plots experienced no change in fungal and negligible change in bacterial composition. We observed pyrophilous taxa that positively responded to fire were phylogenetically conserved, suggesting shared evolutionary traits. For bacteria, fire selected for increased Firmicutes and Actinobacteria. For fungi, fire selected for the Ascomycota classes Pezizomycetes and Eurotiomycetes and for a Basidiomycota class of heat‐resistant Geminibasidiomycete yeasts. We build from Grime’s Competitor‐Stress tolerator‐Ruderal (C‐S‐R) framework and its recent microbial applications to show how our results might fit into a trait‐based conceptual model to help predict generalizable microbial responses to fire.
Fire can impact terrestrial ecosystems by changing abiotic and biotic conditions. Short fire intervals maintain grasslands and communities adapted to frequent, low-severity fires. Shrub encroachment that follows longer fire intervals accumulates fuel and can increase fire severity. This patchily distributed biomass creates mosaics of burn severities in the landscape—pyrodiversity. Afforded by a scheduled burn of a watershed protected from fires for 27 years, we investigated effects of woody encroachment and burn severity on soil chemistry and soil-inhabiting bacteria and fungi. We compared soils before and after fire within the fire-protected, shrub-encroached watershed and soils in an adjacent, annually burned, non-encroached watershed. Organic matter and nutrients accumulated in the fire-protected watershed but responded less to woody encroachment within the encroached watershed. Bioavailable nitrogen and phosphorus and fungal and bacterial communities responded to high severity burn regardless of encroachment. Low severity fire effects on soil nutrients differed, increased bacterial but decreased fungal diversity, and effects of woody encroachment within the encroached watershed were minimal. High severity burns in the fire-protected watershed led to a novel soil system state distinct from non-encroached and encroached soil systems. We conclude that severe fires may open grassland restoration opportunities to manipulate soil chemistry and microbial communities in shrub-encroached habitats.
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Microorganisms commonly inhabit all environments in which they can survive. The number of bacteria in soil depends on its structure, moisture and nutrient content, and ranges from a few hundred to several thousand per gram of soil. Qualitative and quantitative composition of bacteria mainly depends on physico-chemical agents, soil and vegetation cover, the content of biogenic elements, but also on the salinity and pollution. In the case of forest soils number of bacteria amounts to about 4.8×10⁹ per 1 cm3 of soil. In the rhizosphere, the soil directly surrounding plant roots, there are organisms that affect the biochemical activity of plants. The main representatives of bacteria, which are present in the rhizosphere layer, are species of the genera: Pseudomonas and Bacillus, Acidobacteria that protect plants against attack of pathogens. Soil microorganisms form a symbiosis with vascular plants. Because of their properties, they are effective antagonists against fungi that cause plant diseases (leaf spots, roots and shoot apices disease, as well as rot). This group includes such species as: Sclerotinia sclerotiorum, Rotrytis cinerea and Colletotrichum gloeosporioides or the species belonging to Oomycetes, for example Phytophthora and Pythium. Bacteria also protect plants against harmful insects and inhibit the growth of fungal diseases. The beneficial role of bacteria is observed in the development of truffles as well. They are responsible for providing nitrogen to the mycelium forming fruiting bodies. Bacteria improve plant growth and protect their host against drought. Understanding the diversity of bacteria that have important role in the functioning of ecosystems, including forest ecosystems, remains a challenge for microbiologists.
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There are 234 fungal species listed in the record of decision (ROD) for amendments to Forest Service and Bureau of Land Management planning documents within the range of the northern spotted owl. There are four strategies to established guidelines for the survey and management of various organisms, including amphibians, mammals, bryophytes, mollusks, vascular plants, lichens, arthropods, and fungi. Strategy 1 (S1) entailed compiling all known distribution and ecological information on 147 fungus species. Other strategies convey protection or encourage the collecting of additional geographic and habitat information. Upon further taxonomic examination of the S1 fungal species, it was determined that only 135 separate species existed, with the others reduced to synomomy. Most of these S1 fungal species are poorly known and uncommon to rare. A few S1 fungal species were revealed to be much more common than previously thought. This handbook was designed to facilitate understanding of the life history of all S1 and protection buffer species and to aid in their discovery and identification. Each species is represented by a condensed description, a set of distinguishing features, and information on substrate, habitat, and seasonality. We also present a list of known sites within the range of the northern spotted owl, a distribution map and additional references to introduce the available literature on a particular species. A set of artificial taxonomic keys is presented to aid the worker in identification. A partially illustrated glossary helps introduce the novice to mycological terms.
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After severe wildfires, pine recovery depends on ectomycorrhizal (ECM) fungal spores surviving and serving as partners for regenerating forest trees. We took advantage of a large, severe natural forest fire that burned our long-term study plots to test the response of ECM fungi to fire. We sampled the ECM spore bank using pine seedling bioassays and high-throughput sequencing before and after the California Rim Fire. We found that ECM spore bank fungi survived the fire and dominated the colonization of in situ and bioassay seedlings, but there were specific fire adapted fungi such as Rhizopogon olivaceotinctus that increased in abundance after the fire. The frequency of ECM fungal species colonizing pre-fire bioassay seedlings, post-fire bioassay seedlings and in situ seedlings were strongly positively correlated. However, fire reduced the ECM spore bank richness by eliminating some of the rare species, and the density of the spore bank was reduced as evidenced by a larger number of soil samples that yielded uncolonized seedlings. Our results show that although there is a reduction in ECM inoculum, the ECM spore bank community largely remains intact, even after a high-intensity fire. We used advanced techniques for data quality control with Illumina and found consistent results among varying methods. Furthermore, simple greenhouse bioassays can be used to determine which fungi will colonize after fires. Similar to plant seed banks, a specific suite of ruderal, spore bank fungi take advantage of open niche space after fires.The ISME Journal advance online publication, 16 October 2015; doi:10.1038/ismej.2015.182.
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Germination behavior of 45 tree, shrub, subshrub, and liana taxa from fire-prone coastal sage scrub and chaparral was investigated. Nearly 1/3 of the species had seeds that germinated readily upon wetting, and germination was not further stimulated by any fire-related cue. Most coastal sage subshrubs germinate readily in the absence of fire-related stimuli and can thus colonize other forms of disturbance. For many of these species, germination was inhibited in the dark. This may result in a portion of the seed pool remaining dormant until fire since, in the case of several species, dark inhibition is overcome by charate. Chaparral shrubs and trees that germinate readily upon wetting seldom establish seedlings after fire. Seedling establishment and population expansion for such species is dependent upon extended fire-free periods. In contrast, woody species that fail to germinate without some fire-related cue have seedling establishment and potential population expansion restricted to postfire conditions. -from Author
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Ecologists have long acknowledged the importance of seed banks; yet, despite the fact that many plants rely on mycorrhizal fungi for survival and growth, the structure of ectomycorrhizal ( ECM ) fungal spore banks remains poorly understood. The primary goal of this study was to assess the geographic structure in pine‐associated ECM fungal spore banks across the North American continent. Soils were collected from 19 plots in forests across North America. Fresh soils were pyrosequenced for fungal internal transcribed spacer ( ITS ) amplicons. Adjacent soil cores were dried and bioassayed with pine seedlings, and colonized roots were pyrosequenced to detect resistant propagules of ECM fungi. The results showed that ECM spore banks correlated strongly with biogeographic location, but not with the identity of congeneric plant hosts. Minimal community overlap was found between resident ECM fungi vs those in spore banks, and spore bank assemblages were relatively simple and dominated by R hizopogon , W ilcoxina , C enococcum , T helephora , T uber , L accaria and S uillus . Similar to plant seed banks, ECM fungal spore banks are, in general, depauperate, and represent a small and rare subset of the mature forest soil fungal community. Yet, they may be extremely important in fungal colonization after large‐scale disturbances such as clear cuts and forest fires.
In this study we analyzed the spatial structure of ectomycorrhizal fungi present in the soils as resistant propagules (e.g. spores or sclerotia) in a mixed-conifer forest in the Sierra Nevada, California. Soils were collected under old-growth Abies spp. stands across approximately 1 km and bioassayed with seedlings of hosts that establish best in stronger light (Pinus jeffreyi) or that are shade-tolerant (Abies concolor). Ectomycorrhizal fungi colonizing the roots were characterized with molecular techniques (ITS-RFLP and DNA sequence analysis). Wilcoxina, five Rhizopogon species and Cenococcum were the most frequent of 17 detected species. No spatial structure was detected in the resistant propagule community as a whole, but P. jeffreyi seedlings had higher species richness and associated with seven Rhizopogon species that were not detected on A. concolor seedlings. We drew two conclusions from comparisons between this study and a prior study of the ectomycorrhizal community on mature trees in the same forest: (i) the resistant propagule community was considerably simpler and more homogeneous than the active resident community across the forest and (ii) Cenococcum and Wilcoxina species are abundant in both communities.
In this study we examine the distribution of Rhizopogon species in spore banks from five California pine forests. Four of the forest sites were discontinuous populations of Pinus muricata and a fifth was a Pinus ponderosa stand in Sierra National Forest. Rhizopogon species were retrieved by bioassaying the soils with pine seedlings followed by isolation of axenic cultures from individual root tips with typical Rhizopogon ectomycorrhizal morphology. The cultures were screened by ITS-RFLP and all unique patterns were sequenced. These sequences then were compared with those derived from identified sporocarp material. Bioassaying proved to be an efficient way to bring Rhizopogon species into culture. Approximately 50% of the pots contained ectomycorrhizal tips with Rhizopogon-like morphology, and axenic Rhizopogon cultures were obtained from half these pots. Our results showed that Rhizopogon spores usually are well distributed within local forest areas, while there is significant structuring of species at the regional scale. Spore longevity and homogenization by soil and water movement might explain their distribution within local forest areas, while the regional pattern might be explained by limited long distance dispersal or climatic and edaphic differences.
Activation of Byssochlamys fulva ascospores was influenced by temperature and the suspending medium. At 60 C maximal activation occurred in hydrochloric and nitric acid solutions. Both the concentration of hydrogen ions and the type of anion were critical; little activation was achieved above pH 1.6, and pH 1 solutions of sulfuric and phosphoric acid were not stimulatory. Storage of activated spores as aqueous suspensions at 32 C resulted in about a 50% reversion to a dormant state.
Soil protists are commonly suggested being solely bacterivorous, serving together with bacterivorous nematodes as the main controllers of the bacterial energy channel in soil food webs. In contrast, the fungal energy channel is assumed to be controlled by arthropods and mycophagous nematodes. This perspective accepted by most soil biologists is, however, challenged by functional studies conducted by taxonomists that revealed a range of mycophagous protists. In order to increase the knowledge on the functional importance of mycophagous protists we isolated and initiated cultures of protist taxa and tested eight for facultative feeding on diverse fungi in microcosm experiments. Two different flagellate species of the genus Cercomonas, the testate amoeba Cryptodifflugia operculata and four genera of naked amoebae (Acanthamoeba sp., Leptomyxa sp., two Mayorella spp. and Thecamoeba spp.) fed and grew on yeasts with four taxa (Cercomonas sp., Leptomyxa sp., Mayorella sp., and Thecamoeba sp.) also thriving on spores of the plant pathogenic hyphal-forming fungus Fusarium culmorum.