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Growing evidence for facultative biotrophy in saprotrophic fungi:
data from microcosm tests with 201 species of wood-decay
basidiomycetes
Gabriel R. Smith
1
, Roger D. Finlay
2
, Jan Stenlid
2
, Rimvydas Vasaitis
2
and Audrius Menkis
2
1
Department of Biology, Stanford University, 371 Serra Mall, Stanford, CA 94305, USA;
2
Department of Forest Mycology and Plant Pathology, Uppsala BioCenter, Swedish University of
Agricultural Sciences, PO Box 7026, Uppsala SE-75007, Sweden
Author for correspondence:
Gabriel R. Smith
Tel: +1 650 723 0552
Email: grsmith@stanford.edu
Received: 2 January 2017
Accepted: 24 February 2017
New Phytologist (2017)
doi: 10.1111/nph.14551
Key words: biotrophy–saprotrophy
continuum, ectomycorrhizal (ECM)
evolution, facultative biotrophy, Picea abies,
Pinus sylvestris, plant–fungus interaction,
saprotrophic fungi, symbiosis.
Summary
Ectomycorrhizal (ECM) symbioses have evolved a minimum of 78 times independently from
saprotrophic lineages, indicating the potential for functional overlap between ECM and sapro-
trophic fungi. ECM fungi have the capacity to decompose organic matter, and although there
is increasing evidence that some saprotrophic fungi exhibit the capacity to enter into faculta-
tive biotrophic relationships with plant roots without causing disease symptoms, this subject is
still not well studied.
In order to determine the extent of biotrophic capacity in saprotrophic wood-decay fungi
and which systems may be useful models, we investigated the colonization of conifer seedling
roots in vitro using an array of 201 basidiomycete wood-decay fungi. Microtome sectioning,
differential staining and fluorescence microscopy were used to visualize patterns of root colo-
nization in microcosm systems containing Picea abies or Pinus sylvestris seedlings and each
saprotrophic fungus.
Thirty-four (16.9%) of the tested fungal species colonized the roots of at least one tree
species. Two fungal species showed formation of a mantle and one showed Hartig net-like
structures. These features suggest the possibility of an active functional symbiosis between
fungus and plant.
The data indicate that the capacity for facultative biotrophic relationships in free-living
saprotrophic basidiomycetes may be greater than previously supposed.
Introduction
The ectomycorrhizal (ECM) symbiosis, involving a polyphyletic
group of fungi spanning Basidiomycota, Ascomycota and
Mucoromycotina (formerly Zygomycota) (Hibbett et al., 2007;
Tedersoo & Smith, 2013), is widespread and has important
effects on ecosystem composition and function (van der Heijden
et al., 2015). In this association, fungi encase the root tips of host
plants in a sheath of fungal material called a mantle, and prolifer-
ate in the apoplastic space between root cortical cells (Smith &
Read, 2008). This proliferation between root cortical cells is
called a Hartig net, and is the interface across which nutrients
obtained by the fungus from the soil are traded from fungus to
plant in return for photosynthetically fixed carbon (C) (Smith &
Read, 2008). Although only c. 2% of terrestrial plant species
form this type of association (Tedersoo et al., 2010), the boreal
and temperate forest biome that they dominate occupies a dispro-
portionately large global area; ECM fungi and their associated
plant species thus have a significant influence on global biogeo-
chemical cycling (Averill et al., 2014). In particular, many species
in the obligately ECM (Briscoe, 1959; Tedersoo et al., 2010)
plant order Pinaceae are highly economically and ecologically
important, as they are dominant members of forests in boreal
and temperate zones (Liston et al., 2003).
In contrast to ECM fungi, free-living saprotrophic wood-decay
fungi derive C from dead organic material. Using extracellular
enzymes and the nonenzymatic Fenton reaction (Eastwood et al.,
2011), they are able to access nutrients in recalcitrant forms such
as those bound within the lignocellulosic matrix of wood. As pri-
mary decomposers of forest lignocellulose (Baldrian &
Valaskova, 2008), the most abundant organic substance in the
world, as well as one of the most difficult to degrade (Tanesaka
et al., 1993), some of these fungi strongly influence the recycling
of nutrients within forested ecosystems, as well as soil respiration
(Osono, 2007). Saprotrophic fungi can also influence commu-
nity composition of co-occurring bacteria (Folman et al., 2008)
and affect soil C storage through their interactions with ECM
fungi (Averill et al., 2014; Fernandez & Kennedy, 2016).
Despite the established ecological differences between ECM
and saprotrophic fungi, evidence for functional overlap between
the two groups continues to grow (Koide et al., 2008). The
capacity of ECM fungi to contribute to decomposition was first
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theorized by Frank in 1894 (reviewed by Lindahl & Tunlid,
2015); their ability to oxidize organic matter is now supported by
both laboratory (Rineau et al., 2012; Vaario et al., 2012) and field
studies (Talbot et al., 2013; B€odeker et al., 2014; Phillips et al.,
2014). This is a hallmark of their evolutionary history (Shah
et al., 2016); the ECM life habit has evolved from saprotrophic
lineages and persisted a minimum of 78 times, independently
(Tedersoo & Smith, 2013).
This repeated convergent evolution of ECM symbiosis in fungi
has resulted in an array of functionally similar but genetically
diverse associations, illustrated by a diversity of retained genes
coding for carbohydrate-active enzymes (Kohler et al., 2015) and
thus a diversity of enzymatic and nonenzymatic decomposition
ability (Rineau et al., 2012, 2013). Despite their differences,
however, ECM fungi do share broad genetic commonalities,
including increases in genes coding for nitrogen (N) and phos-
phorus (P) transporters (Martin et al., 2010) and reductions in
numbers of genes coding for plant cell wall degrading enzymes
(PCWDEs) compared with saprotrophic relatives (Martin et al.,
2008, 2010; Nagendran et al., 2009; Kohler et al., 2015).
On the one hand, it may be expected that PCWDE loss helps
to facilitate the adoption of symbiosis, because fungi expressing
PCWDEs could trigger plant immune responses, precluding
mutualism (Plett & Martin, 2011). Indeed, loss of a decomposi-
tion pathway has been implicated as the determining event in
adoption of the ECM condition in Amanita (Wolfe et al., 2012),
and Eastwood et al. (2011) have also suggested that ECM transi-
tions in Boletales may be correlated with diminished ligninolytic
capabilities in brown-rot ancestors. On the other hand, the ECM
life habit has been adopted many times independently (Hibbett
et al., 2000; Tedersoo & Smith, 2013), which suggests that to
move from saprotrophy to ECM symbiosis is no great leap and
that the genetic adaptations required in order to make the switch
are relatively small. Some fungi that live both as necrotrophs and
as saprotrophs have been shown to regulate gene expression,
including that of genes coding for carbohydrate-active enzymes,
according to the substrate on which they are growing (Olson
et al., 2012); thus, PCWDEs could simply be downregulated in
symbiotic tissue, rather than lost. A further possibility is that neo-
functionalization has occurred in some cases, resulting in moder-
ate use of PCWDEs for plant cell wall remodeling in
establishment of symbiotic structures. This hypothesis is sup-
ported by transcriptomic evidence from the model ECM fungus,
Laccaria bicolor (Veneault-Fourrey et al., 2014). Because ancestral
saprotrophic abilities underlie the crucial capacity of ECM fungi
to access organic nitrogen pools (B€odeker et al., 2014; Shah et al.,
2016), complete loss of genes coding for associated enzymes
could even be maladaptive. Hence, although establishment of
symbiosis may require that PCWDEs not be highly expressed in
symbiotic tissue, complete loss of PCWDEs is unlikely to be a
necessity in early stages of adaption to an ECM lifestyle, and
could even be detrimental.
In summary, many different lineages of saprotrophic fungi
have independently adopted an ECM life habit throughout evo-
lutionary history and the major known genetic differences
between extant saprotrophic and ECM fungi do not appear to
preclude development of biotrophic relationships between sapro-
trophic fungi and living plants. We therefore expect that extant
saprotrophic fungi exhibit a range of capacity for facultative
biotrophic relationships with plant roots, just as extant ECM
fungi exhibit a range of capacity for facultative saprotrophy or
decomposition (Colpaert & Laere, 1996; Vaario et al., 2012;
B€odeker et al., 2014).
In support, some saprotrophic fungi can form functional
orchid mycorrhizas in nature (Martos et al., 2009; Lee et al.,
2015), and molecular studies using environmental samples some-
times recover saprotrophic DNA from mycorrhizal root tips
(Tedersoo & Smith, 2013), even though these ‘molecular scraps’
are often discounted (Selosse et al., 2010). Established wood-
decay fungi have been found in the roots of apparently healthy
trees both in forest nurseries (Menkis et al., 2005) and in forest
ecosystems (Menkis et al., 2012), and laboratory experiments
have confirmed colonization of living fine roots by several of
these fungi (Vasiliauskas et al., 2007). Because nearly a third of
saprotrophic lineages sister to ECM lineages are wood-decay
fungi (Tedersoo et al., 2010), further research on this subject is
likely to offer insight into the evolution of ECM symbioses as
they now exist. Nevertheless, little work has been done to identify
how common the ability of saprotrophic wood-decay fungi to
enter into biotrophic relationships with plants is, nor which sys-
tems are likely to be most similar morphologically and anatomi-
cally to ECM fungi, and thus most relevant to the study of the
evolution of ECM symbiosis. In order to identify the extent of
biotrophic capacity in saprotrophic wood-decay fungi, and to
identify patterns of root colonization, we undertook a survey of
201 species of saprotrophic wood-decay basidiomycetes. The
fungi were grown in axenic culture with seedlings of two ECM
host species, Pinus sylvestris and Picea abies, which are widespread
and economically important tree species in north temperate and
boreal zones. The colonized plant roots were examined using
microtome sectioning, differential staining and fluorescence
microscopy.
Materials and Methods
Establishment of microcosm systems
Stock cultures of 201 species of wood-decay basidiomycete fungi
were obtained from the culture collection of the Department of
Forest Mycology and Plant Pathology, Swedish University of
Agricultural Sciences (Table 1, Supporting Information
Table S1), and were grown out on modified Melin–Norkrans
(MMN) agar medium (Marx, 1969) in Petri dishes. Experimen-
tal 9-cm diameter microcosm systems were constructed based
upon mycorrhizal synthesis methods first used by Duddridge
(1986), with adjustments by Vasiliauskas et al. (2007). Briefly,
sterile, 2-wk-old seedlings of Pinus sylvestris L. and Picea abies (L.)
H. Karst were aseptically inoculated with 5 95 mm agar plugs
from actively growing fungal cultures, and microcosms were
established in Petri dishes filled with a solid growth substrate con-
sisting of sterilized fine sphagnum peat : vermiculite : 1/10
strength liquid MMN mixture in the ratio 1 : 4 : 2 (v/v/v)
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(Rosling et al., 2004). The pH of this medium was 5.5. Three
replicate microcosms were constructed for each tree species–fun-
gus combination. Control systems were also established using
sterile agar plugs. In order to keep the microcosms axenic after
inoculation, holes for seedling shoots were sealed around the stem
with sterile anhydrous lanolin and the system itself was wrapped
with parafilm as in other microcosm studies (Duddridge, 1986;
Finlay, 1989; Rosling et al., 2004; Vasiliauskas et al., 2007),
ensuring inaccessibility to airborne spores and contaminants.
Monitoring of seedling growth and fungal colonization of
roots
Inoculated microcosms were incubated under controlled condi-
tions in a climate chamber at 16°C with a photoperiod of
16 h : 8 h, light : dark. The photosynthetic photon flux density
within the climate chamber was c. 300 lmol m
2
s
1
; to ensure
that the light affected only the shoot of the plant, microcosms
were wrapped with aluminum foil. Microcosms were regularly
monitored for changes in seedling vitality and for external fea-
tures of fungal colonization of fine living roots.
The axenic microcosm systems were incubated for
6 months, and then subjected to a preliminary assessment
under a Leica M165 FC dissection microscope (Wetzlar,
Germany) for fungal affinity for roots. We define an affinity
for roots here as hyphal growth on fine root tips as opposed
to growth in the surrounding medium, as demonstrated by
at least one microcosm replicate and at least three root tips.
Systems showing fungal colonization of roots were frozen
and stored at 20°C until further analysis.
Table 1 List of wood-decay basidiomycetes that colonized roots in microcosm systems with Pinus sylvestris and/or Picea abies seedlings, and observed pat-
terns of root colonization
Fungal species
Pinus sylvestris Picea abies
Pattern of root colonization Pattern of root colonization
Surface Epidermis Cortex Vascular Surface Epidermis Cortex Vascular
Amylostereum ferreum ++ ++
Amylostereum laevigatum ++ ++
Armillaria cepistipes*+na
†
na na
Armillaria mellea*++ ++
Bjerkandera adusta*++
Ceratobasidium sp. 257*++
Chondrostereum purpureum*++ ++ +
Collybia butyracea*++
Coniophora cerebella ++
Creolophus cirrhatus ++
Fomes fomentarius*++ +
Grifola frondosa ++ ++
Gymnopus sp. 406*++ ++ ++ +
Heterobasidion parviporum ++ ++
Hymenochaete tabacina
a
++ +
Hypholoma capnoides*++ +
Hypholoma fasciculare ++
Lenzites betulina
b
++ +
Marasmius androsaceus*++ +
Marasmius scorodonius*++ ++
Mycena abramsii*++ +
Mycena epipterygia*++ +
Mycena galopus*++ +
Mycena sp. 480
c
++ +
Phellinus chrysoloma*++
Phellinus igniarius*++ ++ ++ +
Phellinus nigricans*++ +
Phellinus tremulae +
Pholiota gummosa ++ +
Pholiota squarrosa ++ +
Pleurotus ostreatus ++ ++
Schizophyllum commune*++
Stereum ostrea +
Stereum sanguinolentum* ++ +
Fungi in microcosms in which seedling mortality was observed are marked in bold; incidence of seedling mortality is indicated in the footnote as a
proportion (%) of three replicates. Asterisks denote fungal species whose identity in microcosm systems was confirmed by internal transcribed spacer (ITS)
rDNA sequencing from root tips. A list of tested fungi that did not colonize roots can be found in Supporting Information Table S1.
a
Picea abies 66%;
b
P. abies 66%;
c
P. abies 33%;
†
data not available.
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Determination of root colonization patterns
From each selected system, colonized root tips (Fig. 1) were sam-
pled under the Leica dissection microscope. A minimum of three
such root tips, representing each selected fungus–plant system,
were carefully excised and 5–10-lm cross-sections taken using a
Leica CM1850 cryomicrotome. Staining was then carried out
using Biotium CF
TM
488A wheat germ agglutinin (Hayward, CA,
USA), staining fungal tissue green, and propidium iodide, stain-
ing plant tissue red, according to the procedure of Doehlemann
et al. (2009). Following staining, slides were examined using a
Leica DM5500 B light microscope and photographed using fluo-
rescence filter cube A4. Photographs were then assessed for pat-
terns of hyphal colonization in specific, established root regions
for each colonized fungus–tree combination: surface, epidermis,
cortex and vascular tissues. Surface colonization included both
aggregated and/or individual fungal hyphae on the outer surface
of fine roots, visible in stained cross-sections. Epidermal or cortex
colonization included the presence of fungal hyphae in each of
these established root regions (Russell, 1977), and colonization
within the endodermis was classified as that of vascular tissue. In
order to minimize the possibility of erroneous classification,
assessments were based upon presence or absence of clearly
stained hyphae in these regions in multiple photographs per
species. In addition, features present in roots that were stained
green but deviated from known fungal morphology (e.g. crys-
talline or blotchy) were considered artifacts of the staining pro-
cess rather than fungal tissue, and were not included in
classification determinations. Finally, fungal tissue not integrated
into cross-sections was also not included in classifications due to
the possibility of the tissue having been displaced in the section-
ing process. Fungi exhibiting morphology similar to that of ecto-
mycorrhizal species were noted.
Molecular identification of fungal species
In order to confirm the identity of fungi that showed affinity for
roots in microcosm systems, sequencing of the internal tran-
scribed spacer of fungal rDNA (ITS rDNA) was carried out from
root tips. Three individual root tips were collected in different
parts of the microcosm systems. Extraction of fungal DNA from
the roots, amplification and sequencing were performed as in
Vasiliauskas et al. (2007).
Results
After 6 months of cultivation in the climate chamber, the major-
ity of seedlings inoculated with wood-decay fungi and all non-
inoculated control seedlings were healthy-looking: shoots and the
majority of needles were green and without signs of decline,
whereas root systems remained intact and free of visible decay.
Examination under the dissection microscope confirmed that all
control systems remained free of fungal colonization. Five (2.5%)
fungi were associated with variable degrees of seedling mortality
in P. sylvestris and six (3%) in P. abies (Tables 1, S1). In particu-
lar, the fungi Kuehneromyces mutabilis and Phanerochaete rimosa
were associated with mortality in all three replicate seedlings of
P. sylvestris (Table S1).
Of the 201 fungal species tested, 34 (16.9%) showed an affin-
ity for roots (Table 1; Fig. 1). Among these, 29 (85.3%) colo-
nized roots exclusively of P. sylvestris, two (5.9%) exclusively of
P. abies, and three (8.8%) colonized roots of both tree species.
Among the fungi that colonized roots of P. sylvestris, 32 (100%)
showed colonization of the root surface, 30 (93.8%) colonization
of the epidermis, 21 (65.6%) colonization of cortical tissue and 9
(28.1%) colonization of vascular tissue (Table 1; Fig. 2). Among
the fungi that colonized roots of P. abies, five (100%) showed col-
onization of the surface, four (80.0%) colonization of epidermis
and four (80.0%) colonization of the cortex; none showed colo-
nization of vascular tissue (Table 1; Fig. 2). Although not scored,
development of intracellular hyphae was sometimes observed
(e.g. Fig. 2c,d; see also Figs S3–S6). Further photographic exam-
ples of root colonization by tested saprotrophic wood-decay fungi
can be found in the Supporting Information (Figs S1–S6).
Two species, Coniophora cerebella and Hypholoma capnoides,
formed mantle-like structures on P. sylvestris (Figs S2, 2b), which
we define as thick proliferations of hyphae visible on the epider-
mal surface of a root cross-section. Phellinus igniarius developed
intercellular hyphae resembling a Hartig net on P. abies (Fig. 2a).
Dichotomously branching root tips, which are characteristic fea-
tures of pines colonized by certain ECM fungi (Persson, 2002),
were sometimes observed, but were not scored (e.g. Fig. S1).
The fungi that colonized roots of both tree species showed dif-
ferent patterns of root colonization for each tree species. For
example, in P. abies,Chondrostereum purpureum,Gymnopus sp.
406 and P. igniarius colonized surface, epidermis and cortex. By
contrast, in P. sylvestris,C. purpureum colonized only surface and
epidermis, whereas Gymnopus sp. 406 and P. igniarius colonized
surface, epidermis, cortex and vascular tissue.
Eighteen microcosm systems of P. sylvestris and four of
P. abies, which were inoculated with different fungal species
(Table 1), were used for sequencing of fungal ITS rDNA from
the root tips. The remaining microcosm systems were not avail-
able as all root tips were used for cross-sectioning. Amplification
was successful for between one and three root tips per microcosm
system. Sequencing confirmed the identity of each inoculated
fungus and no other fungi were detected, indicating that there
was no fungal contamination. The sequences are available from
GenBank under accession numbers KY352513–KY352531.
Discussion
Our data demonstrate that colonization of living fine tree roots
by wood-decay fungi and formation of structures similar to those
found in the ectomycorrhizal (ECM) symbiosis is a relatively rare
phenomenon; colonization occurred in only 16.9% of our tested
fungal species. Nevertheless, these results also reveal the potential
for several wood-decay fungi to be used as model systems in
studying the evolution of root symbioses and their functioning.
In particular, the wood-decay fungus Phellinus igniarius showed
development of intercellular hyphae around the cortical cells of
Picea abies (Fig. 2a), similar in appearance to a Hartig net, the
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organ of nutrient exchange in the ECM symbiosis. Formation of
such features in association with tree seedlings has, to the best of
our knowledge, so far only been observed in vitro in one other
saprotrophic wood-decay fungus, Phlebiopsis gigantea (Vasili-
auskas et al., 2007).
Even though the pure culture techniques used in our study are
well-established methods of determining the mycorrhizal status
of fungi (Duddridge, 1986; Finlay, 1989; Rosling et al., 2004;
many more reviewed by Vasiliauskas et al., 2007), differences in
important variables, such as pH and temperature, between natu-
ral and laboratory conditions can affect the ability of a fungus to
form mycorrhizal associations (Riffle, 1973). Intraspecific genetic
variation is a further factor that may influence the ability of
saprotrophic fungi to colonize living fine roots; for example,
although Eastwood et al. (2011) previously observed colonization
of Pinus sylvestris roots by Serpula lacrymans, this species showed
no affinity for roots in the present study (Table S1). Demonstra-
tion of mycorrhizal formation in vitro therefore does not
necessarily prove that such interactions occur frequently in vivo,
in the field, nor does an absence of interaction in laboratory
microcosms preclude the possibility that they could occur under
other conditions. Because multiple species of saprotrophic fungi
previously shown to colonize living fine roots in vitro (Vasili-
auskas et al., 2007) have also been observed on tree roots and in
rhizosphere soil both in tree nurseries and in forest ecosystems
(Menkis et al., 2005, 2012), the present relationships are unlikely
to be restricted to laboratory conditions. Indeed, saprotrophic
fungi from several of the genera that we have observed colonizing
root tips in this study, such as Gymnopus,Marasmius and
Mycena, also associate symbiotically with heterotrophic orchids
in nature (Martos et al., 2009; Lee et al., 2015).
The effects of ecological interactions in axenic culture can also
differ from those found in nature, as demonstrated in the present
study by necrotrophs such as Armillaria mellea (Fig. 1a),
Chondrostereum purpureum and Fomes fomentarius, which did not
cause visible disease symptoms nor increased mortality in
(a) (b)
(c) (d)
Fig. 1 Patterns of fine root colonization of
conifer seedlings by wood-decay fungi. (a)
Armillaria mellea on Pinus sylvestris; (b)
Pholiota gummosa on P. sylvestris; (c)
Lenzites betulina on P. sylvestris; (d)
Heterobasidion parviporum on P. sylvestris.
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replicate host seedlings despite colonization of roots (Table 1).
The possible mutualistic status of the observed symbioses can
therefore be conclusively determined only by studies specifically
quantifying nutrient and C transfer between fungus and plant.
Differences in colonization patterns show that a degree of
host-specificity is present in the demonstrated relationships, and
that the same saprotrophic fungus may develop qualitatively dif-
ferent symbioses on different hosts, just as the same mycorrhizal
fungus may form different kinds of mycorrhizas on different
hosts (Grelet et al., 2010). Specific varieties of root colonization
are therefore not inherent fungal traits, but rather emergent prop-
erties developed in interaction with plants. For example, although
P. igniarius showed intercellular cortical colonization similar to
an ECM Hartig net in association with P. abies (Fig. 2a), colo-
nization of vascular tissue in association with P. sylvestris
(Table 1) suggests a relationship different from those developed
by mycorrhizal fungi, none of which are known to grow within
living plant vascular tissue. Variation between plant species is also
clear in the overall colonization patterns observed in P. abies and
P. sylvestris: fewer fungal species associated with the former, colo-
nization never extended into vascular tissue, and mortality of all
three replicate seedlings was never observed. Although this would
seem to suggest lower receptivity to fungal colonization in
P. abies as compared with P. sylvestris, formation of Hartig net-
like structures by wood-decay fungi has now been documented
twice on the former (Fig. 2a; Vasiliauskas et al., 2007), but never
on the latter.
Only 34 of the 201 fungi tested showed any affinity for
roots after 6 months, suggesting that despite the potential
source of labile C represented by plant roots, sustained fungal
responsiveness may not be universal. A further possibility is
that the majority of fungal species were repelled by plant
(a) (b)
(c) (d)
Fig. 2 Differentially stained fine root cross-
sections of conifer seedlings grown for
6 months together with wood-decay fungi:
fungal material is stained green and plant
material red. (a) Intercellular colonization of
cortical cells of Picea abies by Phellinus
igniarius; (b) formation of mantle-like
structures on fine root of Pinus sylvestris by
Hypholoma capnoides; (c) colonization of
fine root epidermis and outermost cortical
cells of P. abies by Stereum sanguinolentum,
(d) colonization of surface, epidermis, cortex,
and vascular tissue of P. sylvestris by
Marasmius scorodonius.
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immunity. Indeed, preventing colonization of living roots by
pathogenic fungi is a primary role of plant immune responses,
which can be triggered by chitin found in fungal cell walls
(Zipfel, 2014). Saprotrophic enzymes, which normally do not
facilitate entry into living cells, can also provoke an immune
reaction (Plett & Martin, 2011) due to detection by the plant
of endogenous byproducts of plant cell wall damage
(Zamioudis & Pieterse, 2011). Our observance of intracellular
hyphal development in colonized root tips of outwardly
healthy plants is consistent with downregulated expression of
such enzymes in symbiotic fungal tissue, a phenomenon simi-
lar to that observed in fungi such as Heterobasidion spp.,
which can live both necrotrophically and saprotrophically, reg-
ulating enzyme expression according to substrate (Olson et al.,
2012).
The sensitivity of plant immune systems to microbe-associated
molecular patterns such as chitin (Zipfel, 2014), many of which
are present both in pathogens and in mutualists, results in a
requirement that symbiotic fungi, whether mutualistic or
pathogenic, make use of effector proteins to attenuate plant
immune response and facilitate establishment of symbiosis
(Zamioudis & Pieterse, 2011). Thus, as plant health depends on
simultaneously repelling microbial pathogens while accepting
microbial mutualists, the formation of ECM root tips involves a
complex co-evolved molecular exchange between fungus and host
(Garcia et al., 2015).
In particular, the development of the Hartig net by the ECM
fungus Laccaria bicolor has been shown to require expression of
an effector protein, mycorrhizal induced small secreted protein 7
(MiSSP7), in symbiotic fungal tissue (Plett et al., 2011). Further-
more, mutants not expressing MiSSP7 are unable to engage in
functional symbiosis (Plett et al., 2011), underscoring the impor-
tance of the Hartig net for bidirectional nutrient transfer and
demonstrating that its formation does not occur spontaneously
but, rather, as part of a larger pattern of plant–fungus communi-
cation. The development of Hartig net-like structures by
P. igniarius thus suggests the use of effectors such as MiSSP7 and
potentially constitutes an independent ECM symbiotic origin
event. Although secretomic analysis indicates that wood-decay
fungi produce a reduced complement of effector proteins com-
pared with obligate biotrophs (Kim et al., 2016), P. igniarius in
particular has not been examined, and to our knowledge no tran-
scriptomic or secretomic analysis has been carried out using
saprotrophic fungi grown with seedlings as in the present study.
Our results indicate that although facultative biotrophy is far
from ubiquitous among saprotrophic basidiomycetes, it may be
more common than previously supposed; this can be confirmed
by future functional analysis. These findings thus support current
hypotheses of multiple, independent ECM origin events
throughout evolutionary history (Tedersoo & Smith, 2013) and
extend scientific knowledge concerning the functional diversity
of saprotrophic fungi. We also demonstrate here the potential for
use of multitrophic nonmycorrhizal basidiomycetes as model sys-
tems in the study of ECM evolution and signaling. Coupled with
modern genomic and transcriptomic tools, systems such as these
can offer new insights into the evolution of this crucial and
fundamental symbiosis. Our forthcoming studies examining the
potential for bidirectional nutrient transfer in the most promising
of these systems, such as those of P. igniarius and P. gigantea in
association with P. abies, will build upon our present findings
and further elucidate these phenomena.
Acknowledgements
Financial support from the Carl Tryggers Foundation, Anna and
Gunnar Vidfelts fund Foundation, and a Fulbright US Student
Grant to G.R.S. are gratefully acknowledged. We also thank
three anonymous reviewers for comments leading to the substan-
tial improvement of this paper.
Author contributions
R.F., J.S., R.V. and A.M. designed the study; R.F., J.S. and R.V.
contributed materials; G.R.S. and A.M. performed the research;
G.R.S. and A.M. collected, analyzed and interpreted the data;
and G.R.S., R.F., J.S., R.V. and A.M. wrote the manuscript.
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Supporting Information
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Fig. S1 Dichotomously branching root tip of Pinus sylvestris colo-
nized by Marasmius androsaceus.
Fig. S2 Cross-section of Pinus sylvestris root tip colonized by
Coniophora cerebella.
Fig. S3 Cross-section of Picea abies root tip colonized by
Chondrostereum purpureum.
Fig. S4 Cross-section of Pinus sylvestris root tip colonized by
Amylostereum laevigatum.
Fig. S5 Cross-section of Pinus sylvestris root tip colonized by
Gymnopus sp. 406.
Fig. S6 Cross-section of Pinus sylvestris root tip colonized by
Mycena abramsii.
Table S1 List of tested fungal species that did not colonize roots
of Pinus sylvestris nor of Picea abies in microcosm systems
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