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Mycologia
ISSN: 0027-5514 (Print) 1557-2536 (Online) Journal homepage: https://www.tandfonline.com/loi/umyc20
Cultural characterization and chlamydospore
function of the Ganodermataceae present in the
eastern United States
Andrew L. Loyd, Eric R. Linder, Matthew E. Smith, Robert A. Blanchette &
Jason A. Smith
To cite this article: Andrew L. Loyd, Eric R. Linder, Matthew E. Smith, Robert A. Blanchette
& Jason A. Smith (2019): Cultural characterization and chlamydospore function of the
Ganodermataceae present in the eastern United States, Mycologia
To link to this article: https://doi.org/10.1080/00275514.2018.1543509
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Published online: 24 Jan 2019.
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Cultural characterization and chlamydospore function of the Ganodermataceae
present in the eastern United States
Andrew L. Loyd
a
, Eric R. Linder
a
, Matthew E. Smith
b
, Robert A. Blanchette
c
, and Jason A. Smith
a
a
School of Forest Resources and Conservation, University of Florida, Gainesville, Florida 32611;
b
Department of Plant Pathology, University of
Florida, Gainesville, Florida 32611;
c
Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota 55108
ABSTRACT
The cultural characteristics of fungi can provide useful information for studying the biology and
ecology of a group of closely related species, but these features are often overlooked in the order
Polyporales. Optimal temperature and growth rate data can also be of utility for strain selection of
cultivated fungi such as reishi (i.e., laccate Ganoderma species) and potential novel management
tactics (e.g., solarization) for butt rot diseases caused by Ganoderma species. Historically, the
taxonomy of the laccate (shiny) Ganoderma species has been unresolved and many species have
been treated together as G. lucidum. The cultural characteristics of Ganoderma species from the
United States are needed to understand the biology of these unique species that have all been
lumped under this name. Culture morphology, average growth rate, optimal temperatures, and
resiliency to elevated temperature exposure were characterized for isolates of Ganodermataceae
taxa from the eastern United States, including Ganoderma curtisii, G. martinicense, G. meredithiae,
G. ravenelii, G. sessile, G. tsugae, G. tuberculosum, G. cf. weberianum, G. zonatum, and Tomophagus
colossus. We documented differences in linear growth rates and optimal temperatures between
taxa. Isolates of G. sessile and T. colossus grew the fastest, and isolates of G. meredithiae,
G. ravenelii, and G. tsugae grew the slowest. Isolates of G. sessile, G. martinicense, G. cf. weberianum,
and T. colossus constitutively produced chlamydospores on malt extract agar, and these species
were the only species to survive long-term exposure (30 or 40 d) to 40 C. We hypothesize that
chlamydospores function as survival structures that serve as propagules resilient to adverse
temperature conditions, especially heat. Cultural characteristics of G. martinicense, G. ravenelii,
G. tuberculosum, and G. cf. weberianum collected from the United States are described for the first
time.
ARTICLE HISTORY
Received 7 Feburary 2018
Accepted 30 October 2018
KEYWORDS
Chlamydospores;
Ganodermataceae;
polypores; survival
INTRODUCTION
Ganoderma Karst. is a large and diverse genus, and
these fungi cause white rot of the roots and lower
bole of many tree species (Murrill 1902; Elliott and
Broschat 2001; Schwarze and Ferner 2003). Species of
Ganoderma are found worldwide in both urban and
natural settings and are generally associated with
declining and dead trees. The taxonomy of North
American Ganoderma species is confusing (Moncalvo
et al. 1995; Zhou et al. 2015) but has recently been
resolved for the laccate Ganoderma species in the
United States (Loyd et al. 2018a). In the past century,
the name G. lucidum sensu lato has been used for any
laccate (varnished or polished) Ganoderma species
growing on hardwood trees (Gilbertson and Ryvarden
1986; Adaskaveg and Gilbertson 1988,1989;
Hapuarachchi et al. 2015). It is now recognized that
G. lucidum sensu stricto (Curtis) Karst only occurs in
Europe and possibly some parts of China (Moncalvo
et al. 1995; Postnova and Skolotneva 2010; Zhou et al.
2015; Hennicke et al. 2016). Since there are many
species of laccate Ganoderma in North America (Loyd
et al. 2018a), it is likely that these species differ in their
physiology, anatomy, and ecological niches.
The laccate Ganoderma species have nuanced differ-
ences in basidiomata morphology, host preference, and
geographic limitations, and these features have been
studied extensively in many parts of the world
(Murrill 1902,1908; Steyaert 1980; Gilbertson and
Ryvarden 1986; Welti and Courtecuisse 2010; Loyd
et al. 2018a). The in vitro cultural growth habits such
as growth rate and optimal temperature ranges have
been rarely reported for species of Ganoderma (Nobles
1965; Bazzalo and Wright 1982; Adaskaveg and
Gilbertson 1989). However, the in vitro growth rates
of the laccate Ganoderma species can be used as
CONTACT Andrew L. Loyd aloyd@bartlett.com
Supplemental data for this article can be accessed on the publisher’s Web site.
MYCOLOGIA
https://doi.org/10.1080/00275514.2018.1543509
© 2019 The Mycological Society of America
Published online 24 Jan 2019
a diagnostic feature to distinguish some species of
Ganoderma, such as G. colossum, G. zonatum, and
G. tsugae (Adaskaveg and Gilbertson 1988,1989).
Similarly, previous research found that decay rates are
generally proportional to linear growth rates at an
optimal temperature for Ganoderma taxa (Adaskaveg
and Gilbertson 1986a). This suggests that rapidly grow-
ing isolates would likely decay woody substrates
quicker than slower growing isolates. This proxy can
be used in rapid cultural experiments that estimate
decay rates of common wood decay fungi.
In addition to growth rates, optimal temperature
ranges can distinguish some Ganoderma species, such as
G. tsugae (20–25 C) and G. colossum (35–40 C)
(Adaskaveg and Gilbertson 1989). Furthermore, investi-
gating the optimal temperature ranges of wood-degrading
fungi can be useful for understanding geographic distri-
butions. This approach has been used to study the limita-
tions of Fomes fomentarius and F. fasciatus in the eastern
United States (McCormick et al. 2013). Experiments that
investigate optimal temperature ranges can also elucidate
the etiology of pathogenic Ganoderma species, such as
G. zonatum, and potentially lead to novel management
strategies for landscape professionals.
Lastly, optimal temperatures and growth rates can be
important in strain selection for the reishi cultivation
industry to help determine the best strains, as observed
with other medicinal fungi (Dresch et al. 2015).
Ganoderma species have been used medicinally for
thousands of years in Asia where they are commonly
referred to as “reishi”or “lingzhi”(Stamets 2000).
Reishi have been prescribed as a supplement in tradi-
tional Eastern medicine for use as an anti-
inflammatory, immune-enhancing, and cancer therapy
drug (Wang et al. 2012; Hennicke et al. 2016). Recently,
Loyd et al. (2018d) showed that cultivated “Grow-your-
own”reishi kits sold in the United States are labeled as
G. lucidum but in fact almost always contain another
species such as the Asian species G. lingzhi. Optimal
temperature and growth rate data could be helpful for
industrial cultivators or hobby growers in choosing
a tenacious strain of reishi and growing it at the correct
growth temperature, assuming it is labeled with the
correct species.
In addition to mycelial growth, chlamydospores are
produced by some species of laccate Ganoderma (Nobles
1965; Adaskaveg and Gilbertson 1989). Chlamydospores
are asexually produced, thick-walled spores that are found
in numerous groups of fungi and fungal-like organisms
(e.g., Fusarium spp., Phytophthora spp., and some wood
decay fungi) (Adaskaveg and Gilbertson 1986b,1989;
Erwin and Ribeiro 1996;Chang2003;Bennett2012).
Chlamydospores are generally considered survival
structures that can withstand adverse environmental con-
ditions (e.g., dry conditions, heat, cold, flooding, etc.)
(Erwin and Ribeiro 1996;Chang2003;Bennett2012).
Chlamydospores could play an important role in the life
cycle of this group of wood decay fungi, and few studies
have characterized the function of chlamydospores in the
Ganodermataceae (Chang 2003).
Due to the ambiguous use of the name G. lucidum in
North America (where the name has been historically
used for many laccate Ganoderma species), the cultural
growth habits published in the literature for this taxon
in North America are questionable and should be reas-
sessed (Atkinson 1908; Haddow 1931; Nobles 1948;
Overholts 1953; Gilbertson and Ryvarden 1986;
Adaskaveg and Gilbertson 1989). The objectives of
this research are to (i) characterize the cultural mor-
phology, (ii) determine the optimal temperature ranges
and average growth rates, and (iii) determine the resi-
liency following elevated temperature (40 C) exposure
of Ganodermataceae taxa collected in the eastern
United States. It is likely that the physiology and gen-
eral biology of these species are different. Determining
differences in their physiology is needed to better
understand the role they play in tree decline and death.
MATERIALS AND METHODS
Isolate collection and colony morphology.—Thirty-
two dikaryotic isolates of laccate Ganoderma species
from the eastern United States (TABLE 1) were made
from basidiomata by excising small pieces (<1 cm
3
)of
context tissue with a sterile scalpel and placing them
onto medium made with a base of malt extract agar
(MEA) (Difco Laboratories, Franklin Lakes, New
Jersey) according to the manufacturer’s instructions
with the addition of streptomycin sulfate (100 mg/L)
(Fisher Scientific, Waltham, Massachusetts), benomyl
95% (4 mg/L) (Benlate, Sigma-Aldrich, St. Louis,
Missouri), and lactic acid (1 mL/L) (Fisher Scientific).
Isolates were subcultured to obtain pure cultures and
then maintained on MEA without antibiotics. All
isolates were maintained for long-term storage on
colonized pieces of MEA medium submerged in
sterile deionized water (diH
2
O). Culture morphology
was assessed visually on MEA after 8 d of growth at
each temperature for each taxon. Colony morphology
was described using the guidelines of Stalpers (1978),
with colors based on Ridgway (1912). Each isolate was
examined microscopically for the presence/absence of
chlamydospores. Chlamydospores were examined by
making slide mounts of fresh mycelium from the
center of an 8-d-old culture in a drop of 5% KOH
and then visualizing using a Nikon Eclipse 55i light
2LOYD ET AL.: CULTURAL CHARACTERIZATION OF THE GANODERMATACEAE IN THE USA
microscope (Melville, New York). Measurements were
made for at least 20 chlamydospores per isolate using
micrographs and the photo analysis software ImageJ
(www.imagej.net). The measurements from each
isolate were averaged for each taxon. Cultures are
archived and managed at the Center for Forest
Mycology Research (CFMR) Culture Collection and
Herbarium, United States Department of Agriculture
(USDA) Forest Service, Madison, Wisconsin,
maintained by the Northern Research Station and
housed in the Forest Products Laboratory. In
addition, representative basidiomata are archived at
the CFMR herbarium as well as duplicates at the
University of Florida Mycological Herbarium (FLAS)
in Gainesville, Florida.
DNA extraction, PCR, and sequencing.—Samples
were identified using the macro- and
micromorphological features of basidiomata based
from published descriptions (Loyd et al. 2018a), and
these identifications were confirmed through
Table 1. Isolates of the laccate Ganoderma species used in these experiments, with metadata including isolate names, locations, ITS
accession numbers, and host substrates.
Isolate Taxon Location Associated substrate
a
Culture collection
b
Accession no.
WD2085 G. boninense Japan Unknown Unknown KJ143906
WD2028 G. boninense Japan Unknown Unknown KJ143905
102NC G. curtisii North Carolina, USA Hardwood CFMR MG654073
UMNGA1 G. curtisii South Carolina, USA Hardwood CFMR MG654117
UMNFL28 G. curtisii Florida, USA Hardwood CFMR MG654097
UMNFL60 G. curtisii Florida, USA Hardwood CFMR MG654105
CBS100131 G. curtisii North Carolina, USA Unknown Unknown JQ781848
CBS100132 G. curtisii North Carolina, USA Unknown Unknown JQ781849
Cui9166 G. lingzhi Shandong, China Unknown Unknown KJ143907
Dai12479 G. lingzhi Anhui, China Unknown Unknown JQ781864
MT26/10 G. lucidum Czech Republic Unknown Unknown KJ143912
Rivoire4195 G. lucidum France Unknown Unknown KJ143909
231NC G. martinicense North Carolina, USA Hardwood CFMR MG654182
246TX G. martinicense Texas, USA Hardwood CFMR MG654185
UMNSC7 G. martinicense South Carolina, USA Hardwood CFMR MG654177
UMNTN1 G. martinicense Tennessee, USA Cactus CFMR MG654178
LIPSW-Mart08-55 G. martinicense French West Indies Unknown Unknown KF963256
LIPSW-Mart08-44 G. martinicense French West Indies Unknown Unknown KF963257
124FL G. meredithiae Florida, USA Conifer CFMR MG654188
UMNFL50 G. meredithiae Florida, USA Conifer CFMR MG654103
UMNFL64 G. meredithiae Florida, USA Conifer CFMR MG654106
CWN04670 G. multipileum Taiwan, China Unknown Unknown KJ143913
Dai9447 G. multipileum Hainan, China Unknown Unknown KJ143914
151FL G. ravenelii Florida, USA Hardwood CFMR MG654208
MS187FL G. ravenelii Florida, USA Hardwood CFMR MG654211
CBS194.76 G. resinaceum Netherlands Unknown Unknown KJ143916
Rivoire4150 G. resinaceum France Unknown Unknown KJ143915
113FL G. sessile Florida, USA Hardwood CFMR MG654307
114FL G. sessile Florida, USA Hardwood CFMR MG654308
117TX G. sessile Texas, USA Hardwood CFMR MG654309
UMNFL22 G. sessile Florida, USA Hardwood CFMR MG654232
UMNFL10 G. sessile Florida, USA Hardwood CFMR MG654227
JV1209/27 G. sessile Arizona, USA Unknown Unknown KF605630
NY00985711 G. sessile New York, USA Unknown Unknown KJ143918
UMNMI30 G. tsugae Michigan, USA Conifer CFMR MG654326
UMNNC4 G. tsugae North Carolina, USA Conifer CFMR MG654329
UMNWI1 G. tsugae Wisconsin, USA Conifer CFMR MG654333
Dai12760 G. tsugae Connecticut, USA Unknown Unknown KJ143920
UMNFL82 G. tuberculosum Florida, USA Hardwood CFMR KY646215
PLM540 G. tuberculosum Florida, USA Hardwood CFMR MG654368
PLM684 G. tuberculosum Florida, USA Hardwood CFMR MG654369
LIPSW-Mart08-45 G. tuberculosum French West Indies Unknown Unknown KF96325
261FL G. cf. weberianum Florida, USA Hardwood Unknown MG654370
UMNFL100 G. cf. weberianum Florida, USA Hardwood CFMR MG654373
123FL G. zonatum Florida, USA Palm CFMR MG654416
UMNFL85 G. zonatum Florida, USA Palm CFMR MG654402
UMNFL59 G. zonatum Florida, USA Palm CFMR MG654395
UMNFL54 G. zonatum Florida, USA Palm CFMR MG654393
PLM639 G. zonatum Florida, USA Palm CFMR -
FL-02 G. zonatum Florida, USA Palm Unknown KJ143921
TC-02 T. cattienensis Vietnam Unknown Unknown KJ143923
255FL T. colossus Florida, USA Cycad CFMR MG654427
UMNFL110 T. colossus Florida, USA Unknown CFMR MG654429
Note. Isolates in boldface were produced in this study, and the rest were used as reference sequences for each species.
a
Associated substrates were based on collection information if available. If it was not available, then it is labeled as “unknown.”
b
Culture collections are noted for where cultures are stored if known. “CFMR”is the fungal culture collection at the US Forest Service Wood Products
Laboratory in Madison, Wisconsin. If collection is not known, isolates are labeled as “unknown.”
MYCOLOGIA 3
sequencing and analysis of the internal transcribed
spacer (ITS) region of the ribosomal DNA (rDNA)
(Murrill 1902,1908; Overholts 1953; Steyaert 1972;
Steyaert 1980; Gilbertson and Ryvarden 1986; Welti
and Courtecuisse 2010; Loyd et al. 2018a). DNA was
extracted from fresh mycelium of each isolate with the
Extract-N-Amp rapid DNA kit (Sigma-Aldrich) per the
manufacturer’s recommendations. Amplification of the
ITS region was performed with the primers ITS1f and
ITS4 on a MJ Mini thermocycler (Bio-Rad, Hercules,
California) with thermocycling conditions of an initial
cycle of 94 C for 4 min and followed with 37 cycles of
94 C for 50 s, 55 C for 50 s, and 72 C for 1 min that
produced an amplicon of ~700–800 nucleotides (White
et al. 1990; Gardes and Bruns 1993). Amplicons were
cleaned with Exo-SAP-IT (ThermoFisher, Waltham,
Massachusetts) according to the manufacturer’s
instructions. Sanger sequencing was performed using
both forward and reverse primers at the
Interdisciplinary Center for Biotechnology Research
(ICBR) at the University of Florida. Forward and
reverse sequences were aligned and edited using
Geneious 10 (Aukland, New Zealand). ITS sequences
have been deposited to the National Center for
Biotechnology Information (NCBI) GenBank database
(TABLE 1).
Phylogenetic analysis.—The ITS sequences were
queried against reliable, reference sequences from recent
phylogenetic studies using the Basic Local Alignment
Search Tool (BLAST) (Altschul et al. 1997;Zhouetal.
2015;Loydetal.2018a). In order to verify the
phylogenetic placement and relatedness of our isolates,
51 sequences (including the sequences generated in this
study and reliable reference sequences) were used in
a phylogenetic analysis. Sequences were aligned using
the MAFFT (Katoh et al. 2002) plugin in Geneious 10.
The alignment was visually edited to remove ambiguities
and minimize differences that could have resulted from
sequencing error. The alignment contained 460
nucleotide characters and was used for phylogenetic
analysis based on maximum likelihood with the RAxML
(Stamatakis 2014) plugin in Geneious 10. The RAxML
analysis used a general time reversible (GTR)
evolutionary model with rapid bootstrapping and 1000
bootstrap replications. The analysis was rooted with
Tomophagus species that belong to Ganodermataceae
but are placed outside the genus Ganoderma.The
alignment has been submitted to TreeBASE under the
submission number 23202 (http://purl.org/phylo/tree
base/phylows/study/TB2:S23202).
Optimal temperature and growth rate.—To
determine the optimal temperature and linear
growth rates, one to five isolates of each taxon
(TABLE 1) were cultured onto 20 mL of MEA in
Petri dishes and placed in incubators set at 15, 25,
30, and 40 C or kept at ambient temperature (~22
C). Additional temperatures (45, 50, and 55 C) were
included to test the temperature range of T. colossus.
Colony diameter (cm) was measured in two
directions daily for 8 d for each isolate. An
average of the two diameters (mm) was used to
calculate the average linear growth rate (mm/d) for
each taxon by averaging the linear growth rates of
each isolate of a given taxon for each day. This was
repeated three times, and the incubators were
randomly assigned temperatures independently in
each repetition.
Survival at elevated temperatures over time.—To
test the resiliency to elevated temperature exposure over
time, one to five isolates of Ganodermataceae taxa were
cultured onto MEA for 7 d at 28 C and then used to
colonize sterilized rye grain in half-filled 237-mL glass jars
incubated at 28 C until fully colonized (approximately 3
wk). Commercially available birch dowels (Fungi Perfecti,
Olympia, Washington) (0.8 × 2.54 cm) were soaked in
diH
2
O for 24 h and then sterilized by autoclaving for 45
min in 237-mL glass jars at 121 C at 103 kPa. Hydrated,
sterile wooden dowels were infested with grain inoculum
of each respective isolate and were incubated at 28 C for 6
wk. After the dowels were fully colonized, they were
placed into glass test tubes and put into an incubator set
at 40 C. At each time point and temperature, there were
three replicates per isolate of each taxon, except G.cf.
weberianum, which had six replicates because there was
only one isolate of this taxon. At 5, 12, 20, and 30 or 40 d,
individual dowels of each replicate of each
representative isolate were plated onto MEA, where
they were incubated at 28 C for 5 d. After incubation,
all replicates of each isolate were scored as alive or dead,
and percent survival was calculated for each taxon by
averaging the mean survival of each replicate per isolate
of each taxon. In addition, colony growth area (cm
2
)
was measured for each colony by subtracting the area of
the colonized wooden dowel from the total area of the
fungal colony. Colony growth area was measured by
analyzing photographs calibrated to size with Assess 2.0
software (APS, Minneapolis, Minnesota). Colony
growth areas of all replicates of each isolate for
a given taxon were averaged. The experiment was
repeated twice.
4LOYD ET AL.: CULTURAL CHARACTERIZATION OF THE GANODERMATACEAE IN THE USA
RESULTS
Isolate collection and identification.—Based on
a survey of the laccate Ganoderma species in the eastern
United States conducted between 2013 and 2017 (Loyd
et al. 2018a), 10 described taxa have been collected and
identified: G. curtisii (Berk.) Murrill, G. martinicense
Welti & Court., G. meredithiae Adask. & Gilb.,
G. ravenelii Steyaert, G. sessile Murrill, G. tsugae Murrill,
G. tuberculosum Murrill, G.c.f.weberianum (Bres. & Henn.
Ex Sacc.) Steyaert, G. zonatum Murrill, and Tomophagus
colossus (Fr.) Murrill (syn. G. colossus). For this study, 31
isolates were selected as representatives of G. curtisii (n = 4),
G. martinicense (n = 4), G. meredithiae (n = 3), G. ravenelii
(n = 2), G. sessile (n = 5), G. tsugae (n = 3), G. tuberculosum
(n = 3), G.cf.weberianum (n = 1), G. zonatum (n = 4), and
T. colossus (n = 2) (TABLE 1). This represents all of the
known species of laccate Ganoderma in the eastern United
States (Loyd et al. 2018a). Based on phylogenetic analysis of
ITS sequences (TABLE 1), the 10 morphologically
identified Ganodermataceae taxa clustered well with
reference sequences, with the exception of G. meredithiae
(FIG. 1). This result is consistent with the findings of Loyd
et al. (2018a), who proposed that G. meredithiae was
conspecific with G. curtisii basedonamultilocus
phylogenetic analysis and morphology. However, Loyd
et al. (2018a)alsorecognizedthatG. meredithiae is
physiologically different in that it grows on pines in the
southeastern United States and has a slow in vitro growth
rate on MEA. Accordingly, Loyd et al. (2018a)proposedthe
informal classification of G. curtisii f. sp. meredithiae to
differentiate isolates of G. curtisii from pine that have slow
in vitro growth on MEA.
Colony morphology.—Ganoderma isolates generally
produced white colonies with yellow, orange, or beige
pigmentation toward the center of the colony after 8
d of growth at each temperature optimum
(SUPPLEMENTARY FIG. 1). Colony pigments were
more pronounced when isolates were grown at
temperatures above the temperature optimum for each
taxon. Isolates of Tomophagus colossus were woolly and
had brown pigments after 8 d of growth on MEA. Colony
morphologies were similar for all isolates of a given taxon.
Descriptions are summarized in TABLE 2.
Chlamydospore production.—In 8-d-old cultures
grown on MEA at 30 C, chlamydospores were
produced by G. martinicense, G. sessile, G. cf.
weberianum, and Tomophagus colossus (FIG. 2).
Chlamydospores of these four taxa were readily
produced in 8-d-old MEA cultures; adverse conditions
were not required for their production.
Chlamydospores of G. martinicense and T. colossus
were pigmented and ornamented, whereas those of
G. sessile and G. cf. weberianum were hyaline and
smooth. Chlamydospore morphology and
measurements are described in TABLE 2.
Optimal temperature and growth rate.—All isolates
of each Ganoderma taxon grew at 15, 22, 25, 30, and 35 C,
whereas no growth was observed at 40 C, with the
exception of some negligible growth (<1 mm/d) in a few
isolates of G. sessile and G. martinicense (FIG. 3). Isolates
of T. colossus grew at 22, 25, 30, 35, 40, and 45 C, but no
growthwasobservedat50or55C,andnegligiblegrowth
at 15 C (<1 mm/d) (FIG. 3). Isolates of G. tsugae grew
optimally at the coolest temperature range with no
difference in growth between 22 and 25 C. Most
Ganoderma species grew in the optimal temperature
range of 25–30 C, including isolates of G. curtisii,
G. meredithiae, G. ravenelii, G. sessile, G. tuberculosum,
G.cf.weberianum,andG. zonatum. Ganoderma
martinicense grew optimally at a slightly higher
temperature range of 30–35 C, and T. colossus grew
optimally between 35 and 40 C (TABLE 2).
At the respective optimal temperature range for each
taxon, there were differences in linear growth rates
between some of the taxa. Isolates of T. colossus grew
the fastest at its optimal temperature of 40 C, growing on
average at 15.3 ± 0.8 mm/d, whereas isolates of
G. ravenelii had the slowest average linear growth rate
of 1.6 ± 0.2 mm/d at its optimal temperature of 25
C(FIG. 3). At each respective optimal temperature, iso-
lates of each taxon were grouped as growing slow, mod-
erate, or fast. Isolates of G. meredithiae, G. ravenelii,and
G. tsugae grew slowly (1.4–3.4 mm/d). Isolates of
G. curtisii, G. martinicense, G. tuberculosum, G.cf.weber-
ianum,andG. zonatum grew moderately fast
(4.6–8.5 mm/d). Lastly, isolates of G. sessile and
T. colossus grew fast (9.3–16.1 mm/d) (TABLE 2).
Survival at elevated temperatures over time.—
There were differences in survival of Ganoderma species
when exposed to 40 C over different periods of time. After
12 d of exposure, only the chlamydospore-producing taxa
survived (G. martinicense, G. sessile, G.cf.weberianum,
and T. colossus)(TABLE 3). However, there were
differences in the vigor and resilience of the
chlamydospore-producing taxa. Isolates of T. colossus
were the most vigorous; colony growth area after 30 d of
exposure to 40 C was 92% of the colony growth area after
5 d of exposure to 40 C. However,its optimal temperature
range was between 35 and 40 C. Gandoderma sessile was
MYCOLOGIA 5
the most vigorous of the other three chlamydospore-
producing Ganoderma species. The colony growth area
of isolates after 40 d exposure to 40 C was on average 72%
of the colony growth area after 5 d of exposure to 40
C. Isolates of G. martinicense and G.cf.weberianum
survived 30 d of exposure to 40 C, but the vigor of
colony growth was strongly reduced after this exposure
(FIG. 4).
DISCUSSION
Cultural characteristics such as colony morphology,
chlamydospore production, and average growth rate
are often overlooked during of studies of the
Polyporales. These characteristics can be useful diag-
nostic features, distinguish physiological differences,
and explain geographic distribution limits among simi-
lar taxa (Nobles 1965; Adaskaveg and Gilbertson 1986b,
1989; McCormick et al. 2013). There were differences
in colony morphology, chlamydospore production, lin-
ear growth rates, and optimal temperatures for isolates
of the 10 Ganodermataceae taxa investigated here
(TABLE 2).
Optimal temperature data supported the geographic
distribution of Ganoderma tsugae, which had an opti-
mal temperature between 22 and 25 C. This species is
typically found in northern latitudes following the geo-
graphic distribution of Tsuga canadensis (Gilbertson
Figure 1. Tree topology derived from a RAxML phylogenetic analysis of an alignment of 460 characters derived from ITS sequences
of Ganoderma species from this study or reference sequences. Statistical values shown are ML bootstrap values above 70%. Samples
in boldface were generated in this study. Tomophagus species were used to root the tree.
6LOYD ET AL.: CULTURAL CHARACTERIZATION OF THE GANODERMATACEAE IN THE USA
Table 2. Summary of cultural characterization for 10 taxa in the Ganodermataceae in the eastern United States.
Chlamydospores Cultural characteristics
Taxon Authority
Presence/
absence
a
Shape Size
b
Optimal
temperature
range (°C)
Average
linear growth
rate (mm/d)
c
Colony morphology
Ganoderma
curtisii
(Berk.) Murrill
1902
Absent ——25–30 5.6 ± 1.0 Plumose, felty colony margin and appressed to the medium; densely white, and
with age developed yellow to orange pigments
Ganoderma
martinicense
Welti & Court.
2010
Present Hyaline to pigmented, ovate to
spherical or irregularly shaped with
protruding appendages
17.1
(13.5–21.1) ×
12.2
(9.2–17.3)
30–35 7.9 ± 0.6 Fringed or even margin appressed to the medium; felty in texture, and at maturity
lacked aerial mycelia; densely white, and with age developed yellowish pigments
Ganoderma
meredithiae
Adask. & Gilb.
1988
Absent ——25–30 2.6 ± 0.6 Plumose, felty colony margin and appressed to the medium; densely white, and
with age developed yellow to orange pigments
Ganoderma
ravenelii
Steyaert 1980 Absent ——25–30 1.6 ± 0.2 Plumose, felty colony margin and appressed to the medium; densely white, and
with age developed yellow to orange pigments
Ganoderma sessile Murrill 1902 Present Hyaline, elliptical to obpyriform to
ovate, and smooth
16.0
(12.0–26.0) ×
11.0
(9.5–12.0)
25–30 11.0 ± 1.7 Fringed or even margin appressed to the medium; felty in texture, and at maturity
lacked aerial mycelia; densely white, and farinaceous (mealy) in texture
Ganoderma
tsugae
Murrill 1902 Absent ––20–25 3.0 ± 0.4 Slightly fringed or even margin appressed to the medium, and were floccose to
felty in texture; densely white, developing yellowish pigments toward the center of
the colony
Ganoderma
tuberculosum
Murrill 1908 Absent ——25–30 6.5 ± 1.2 Slightly fringed or even margin appressed to the medium, and were cottony but
becoming felty over time; densely white, and with age produced bright yellow
pigments
Ganoderma cf.
weberianum
(Bres. & Henn.
Ex. Sacc.)
Steyaert 1972
Present Hyaline, elliptical to obpyriform to
ovate, and smooth
17.1
(14.1–20.1) ×
12.0
(9.6–14.1)
25–30 6.7 ± 1.2 Fringed or even margin appressed to the medium; felty in texture, and at maturity
lacked aerial mycelia; densely white, and farinaceous (mealy) in texture
Ganoderma
zonatum
Murrill 1902 Absent ——25–30 7.3 ± 0.9 Fringed to even margin that were floccose becoming felty over time, with aerial
mycelium common in young cultures but rare in mature cultures; yellowish to
ochraceous to cream colored pigments in the center of the colony that were
lacunose (sunken or depressed) and crustose
Tomophagus
colossus
(Fr.) Murrill
1905
Present Pigmented, globose, with
protruding appendages
16.1
(15.1–17.6)
35–40 15.3 ± 0.8 Even margin that was cottony to woolly with aerial hphae; white aging to tan or
brown throughout the entire colony
a
Presence/absence of chlamydospores was noted on malt extract agar (MEA) following 8 d of growth in the dark at each taxon’s optimal temperature.
b
At least 20 chlamydospores were measured for G. martinicense (n = 4), G. sessile (n = 5), G. cf. weberianum (n = 1), and T. colossus (n = 2).
cAverage linear growth rate was based on growth on MEA at each respective optimal peak temperature for each taxon. The standard deviation is also given.
MYCOLOGIA 7
and Ryvarden 1986; Loyd et al. 2018a). Similarly,
T. colossus has a limited geographic distribution in
tropical locales and has only been reported within the
United States in Florida. Tomophagus colossus has an
optimal temperature range between 35 and 40 C, which
is consistent with its distribution in areas with constant
warm temperatures and rare freezing (Gilbertson and
Ryvarden 1986; Adaskaveg and Gilbertson 1989).
Lastly, G. sessile has the largest geographic distribution
in the eastern United States and is found in most states
east of the Rocky Mountains (Gilbertson and Ryvarden
1986; Loyd et al. 2018a). Relative to other Ganoderma
species, isolates of G. sessile had fast linear growth at
the optimal temperature of 30 C but also had fast linear
growth from 15 to 30 C (FIG. 3). The fast linear growth
across the wide temperature range is consistent with
the large geographic distribution of this species from
tropical, subtropical, and temperate regions of the east-
ern United States. We hypothesize that the fast growth
rate and chlamydospore production of G. sessile has
aided in its survival across this wide geographic range.
Linear growth rates have been hypothesized to be
proportional to decay rates (Adaskaveg and Gilbertson
1986a). These data suggest that dikaryotic isolates of
G. sessile and T. colossus would have a faster decay rate
relative to the other taxa based on their fast linear
growth rates. On the contrary, isolates of G. ravenelii,
G. meredithiae, and G. tsugae had the slowest linear
growth rates at each temperature optimum. Based on
this previous hypothesis, these slow-growing taxa
would decay wood slower relative to the faster growing
taxa. Of the isolates with slow linear growth rates,
G. meredithiae and G. tsugae predominately decay con-
iferous wood. Coniferous sapwood is generally more
decay-resistant than the sapwood of hardwoods due to
the production of resins and terpenes (Baietto and
Wilson 2010). The slow in vitro growth could be due
to a lack of nutrition in the artificial medium, or the
Figure 2. Chlamydospores produced by four taxa of the Ganodermataceae. A. Ornamented and pigmented double-walled intercalary
ovate chlamydospore produced by G. martinicense (231NC). B. Dextrinoid, terminal chlaymospore produced by G. sessile (113FL)
stained in Melzer’s reagent. C. Ovate to obpyriform chlamydospores produced by G. c.f. weberianum (UMNFL100). D. Mature glogose,
double-walled pigmented and ornamented chlamydospores of T. colossus (UMNFL110). Bars = 20 μm.
8LOYD ET AL.: CULTURAL CHARACTERIZATION OF THE GANODERMATACEAE IN THE USA
result of decay specialization on conifers because linear
growth can also be affected by water-soluble sapwood
extracts from pine (Loyd et al. 2018b). More studies are
required to elucidate these differences in physiology.
These results also confirm the slow growth rate of
G. meredithiae as compared with G. curtissi, as pre-
viously reported (Adaskaveg and Gilbertson 1988;
Loyd et al. 2018a). This finding of no phylogenetic
differences between G. meredithiae and G. curtissi but
with G. meredithiae apparently specialized for growth
on conifers supports the renaming of G. meredithiae to
G. curtissi f. sp. meredithiae. Recent studies of ecologi-
cal transitions in wood decay fungi suggest that most
white rot fungi, including species of Ganodermataceae
and other Polyporales, are specialized for growth on
angiosperms with relatively few taxa specialized on
gymnosperms (Krah et al. 2018). Although Krah et al.
(2018) suggest that there are likely many ecological
transitions between angiosperm specialization, gymnos-
perm specialization, and generalism, our findings sug-
gest that in the case of Ganodermataceae, it is likely
that G. tsugae and G. curtissi f. sp. meredithiae may
represent evolutionarily recent switches to decay on
conifers.
In addition to understanding differences in physiol-
ogy, these optimal temperature and growth data could
Figure 3. Average optimal temperatures and growth rates of 31 isolates representing 10 taxa within the Ganodermataceae in the
eastern United States. Each isolate is labeled with a different color in each taxon box. The averages and standard error bars are based
on three replications.
Table 3. Percent survival of Ganodermataceae taxa exposed to
40 C over time.
Percentage of isolate survival at elevated
temperature
a
Taxon n
b
5
c
12
c
21
c
30
c
40
c
G. curtisii 414 0 0 nt 0
G. martinicense 4 100 100 75 58 nt
G. meredithiae 350 0 0 nt 0
G. ravenelii 20 0 0 0 nt
G. sessile 4 100 100 100 nt 100
G. tsugae 20 0 0 0 nt
G. tuberculosum 3 80 33 0 nt 0
G. cf. weberianum 1 100 100 33 83 nt
G. zonatum 5 0 0 0 nt 0
T. colossus 2 100 100 100 100 nt
Note. nt = not tested.
a
Scored isolate survival after 5 d of incubation at 28 C on malt extract agar
with 3 replicates per isolate
b
Number of unique isolates for each species.
c
Days exposed at 40 C.
MYCOLOGIA 9
also be utilitarian and benefit reishi cultivators who
may choose to select fast-growing species and specific
optimal growth temperatures for producing spawn of
a given taxon. In addition, chlamydospore-producing
species would likely have a longer shelf life across
adverse temperature conditions. During strain selec-
tion, this morphological feature could help reishi
spawn producers decide on strains they wish to culti-
vate and sell.
Another utilitarian use of this data is designing novel
management tactics for Ganoderma butt rot of palms
caused by Ganoderma zonatum. Ganoderma butt rot is
the most important disease of palms in Florida (Elliott
and Broschat 2001). Isolates of G. zonatum had the
greatest decrease (−6.7 mm/d) in average growth rate
when the temperature was increased from 30 to 35
C. Isolates of G. zonatum also failed to grow when
exposed to 40 C for only 5 d. Isolates of G. zonatum
are not tolerant to high temperatures, which could
explain why pathogenicity tests conducted previously
in Florida were unsuccessful (Elliott and Broschat 2001;
Loyd et al. 2018c). Based on these data, we hypothesize
that the spread of inoculum, presumably by basidios-
pores, is likely most successful in the cooler months.
Furthermore, due to the phasing out of fumigants such
as dazomet, soil removal and replacement is recom-
mended for replanting palms where palms have been
removed due to Ganoderma butt rot (Elliott and
Broschat 2001). Based on the low tolerance of high
temperatures, future research should focus on the
Figure 4. Effects of exposure to 40 C (elevated temperature) over time on colony growth area of Ganodermataceae taxa, where no
growth indicates mortality or no growth. Number of isolates used in this experiment are indicated in each taxon box as “n=x.”
10 LOYD ET AL.: CULTURAL CHARACTERIZATION OF THE GANODERMATACEAE IN THE USA
potential use of soil solarization and steam sterilization
to eliminate G. zonatum inoculum. Our results suggest
that these alternative practices may be useful for mana-
ging this important palm pathogen.
Chlamydospore-producing taxa were the only ones able
to survive long-term exposures to 40 C. The double-walled
chlamydospores produced by G. martinicense, G. sessile, G.
c.f. weberianum,andT. colossus function as survival struc-
tures and can persist in wood despite the fact that the fungi
are not actively growing. In a previous study, Loyd (2018)
found that chlamydospores produced by G. sessile are
constitutively produced and begin maturing after only 3
d of growth at the optimal temperature on MEA. Similarly,
Loyd (2018) found that young 2-d-old cultures of G. sessile
that lacked chlamydospores were not able to survive 8 d at
40 C. In contrast, mature 7-d-old cultures with abundant
chlamydospores survived well under the same conditions.
Based on these survival data, we suspect that the chla-
mydospores of the other Ganodermataceae taxa are also
regularly produced during regular growth. With the
exception of T. colossus, the other three chlamydospore-
producing Ganoderma species do not actively grow at 40
C. This suggests that the chlamydospores, not the hyphae,
allow these fungi to revive following exposure to elevated
temperatures. In addition to the function of chlamydos-
pores, there were morphological differences distinguish-
ing chlamydospores produced by G. sessile and G.cf.
weberianum as compared with those produced by
G. martinicense and T. colossus. The evolutionary history
of chlamydospore production in the Ganodermataceae is
still unresolved and requires further investigation of addi-
tional taxa. However, species in the G. resinaceum clade
all produce morphologically similar chlamydospores
(Hong and Jung 2004;Loydetal.2018a)thatlikelyhave
similar survival functions.
Overall, there were differences in colony morpholo-
gies, chlamydospore production, average linear growth
rates, optimal temperature ranges, and resiliency to ele-
vated temperatures of the Ganodermataceae taxa investi-
gated. This work demonstrates that cultural
characteristics can be useful for understanding the basic
biology of Ganodermataceae taxa and can be used to
infer information about the general ecology, physiology,
and utility for the laccate Ganoderma species. Continued
research focusing on the laccate Ganoderma taxa in the
United States is needed to better understand this cosmo-
politan genus of wood decay and medicinal fungi.
ACKNOWLEDGMENTS
The authors would like to thank the generous collectors for
collecting several of the basidiomata that were cultured for this
study.
FUNDING
This project was partially funded by the F.A. Bartlett Tree
Experts company and a grant from the International Society
of Arboriculture Florida Chapter, and the authors are greatly
appreciative. Matthew Smith’s participation in this work was
supported by the USDA NIFA McIntire-Stennis project
1011527.
ORCID
Andrew L. Loyd http://orcid.org/0000-0002-6463-2635
Matthew E. Smith http://orcid.org/0000-0002-0878-0932
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