Ceratobasidioid mycobionts in Russian populations of Goodyera repens and mycorrhizal specificity in Goodyerinae subtribe (Orchidaceae)

Abstract

Members of Ceratobasidiaceae family (more commonly known by its anamorphic name “rhizoctonias”) possess a variety of nutritional modes: plant pathogens, saprotrophs, endophytes and symbionts of orchid mycorrhiza. Links between nutritional modes and taxonomy of these fungi as well as their specificity towards plant host is still ambiguous. The scope of the present study was to explore biodiversity of ceratobasidioid mycobionts of sciophytic terrestrial orchid Goodyera repens , search for evolutionary stable clades within mycobionts of Goodyerinae subtribe uniform by plant host or geographic region and to establish possible connection between ceratobasidioid nutritional modes and morphological characteristics. We consider G. repens a generalist associated with a wide range of distantly related mycobionts. Two unidentified Ceratobasidium species and Thanatephorus ochraceus are reported from G. repens roots for the first time. Ceratobasidiaceae tend to form stable clades specific to either temperate or tropical region. Morphological characteristics of pathogenic and mycorrhizal rhizoctonia isolates tend to form a variety of transitional forms to correlate with nutritional mode.

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Ceratobasidioid mycobionts in Russian populations of Goodyera repens and
mycorrhizal specicity in Goodyerinae subtribe (Orchidaceae)
Nikita Bibikov (  bibik0808@mail.ru )
Lomonosov Moscow State University
Elena Voronina
Lomonosov Moscow State University
Olga Kamzolkina
Lomonosov Moscow State University
Maria Yarmeeva
Lomonosov Moscow State University
Alexander Kurakov
Lomonosov Moscow State University
Research Article
Keywords: orchid mycorrhiza, Ceratobasidium, Goodyera repens, metagenome, mycorrhizal specicity, rhizoctonias
Posted Date: August 18th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-3252508/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full License

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Abstract
Members of Ceratobasidiaceae family (more commonly known by its anamorphic name “rhizoctonias”) possess a variety of nutritional modes: plant
pathogens, saprotrophs, endophytes and symbionts of orchid mycorrhiza. Links between nutritional modes and taxonomy of these fungi as well as their
specicity towards plant host is still ambiguous. The scope of the present study was to explore biodiversity of ceratobasidioid mycobionts of sciophytic
terrestrial orchid
Goodyera repens
, search for evolutionary stable clades within mycobionts of Goodyerinae subtribe uniform by plant host or geographic
region and to establish possible connection between ceratobasidioid nutritional modes and morphological characteristics. We consider
G. repens
a generalist
associated with a wide range of distantly related mycobionts. Two unidentied
Ceratobasidium
species and
Thanatephorus ochraceus
are reported from
G.
repens
roots for the rst time. Ceratobasidiaceae tend to form stable clades specic to either temperate or tropical region. Morphological characteristics of
pathogenic and mycorrhizal rhizoctonia isolates tend to form a variety of transitional forms to correlate with nutritional mode.
Introduction
Orchid mycorrhiza (OM) is the recently emerged type of symbiosis: molecular clock approach estimates its repeated origins as the middle Cretaceous (Strullu-
Derrien et al. 2018). Due to comparatively brief history of coevolution with orchids, symbiotic fungi may not be taxonomically detached from non-mycorrhizal
ones. Even though OM is formed only by members of Orchidaceae, the presence of evolutionary stable clades of specically OM fungi is still ambiguous.
There are numerous examples of OM fungal symbionts with more than one nutritional modes at a time. Thus, OM fungal assemblage can embrace free-living
wood-decayers and soil saprobes along with host-dependent ectomycorrhizal or non-orchid plant pathogenic species (Smith and Read 2008).
Anamorphic rhizoctonias (former genus
Rhizoctonia
species, initially not ascribed to any basidiomycete teleomorphs) were the rst OM symbionts revealed
by means of plant root tissue microscopy and regular isolations in pure culture with subsequent reinoculation (Bernard 1904; Fuller 1909). For a long time
they were regarded as the only symbionts in OM, and even after the other groups of symbiotic fungi were detected in orchid roots, the green orchids
considered to be associated largely with rhizoctonias contrary to achlorophyllous species recruiting diverse saprotrophic and ectomycorrhizal macrofungi (for
more details see Smith and Read 2008). The data obtained during the last two decades allow to consider the more complicated orchid-rhizoctonia
interactions, for the latter turn out to be rather common mycobionts for not only green but non-photosynthetic plant species too (e.g. Bougoure et al. 2009;
Pecoraro et al. 2020).
Despite of the fact that OM research pioneered by Noël Bernard (Selosse et al. 2011) was originated with observation of symbiotic rhizoctonias, its taxonomic
status remained obscure for a quite long period. The rst evidence of the genus polyphyly was provided by Warcup and Talbot (1967) who successfully
obtained teleomorphic stages under cultural condition. The more recent studies applying molecular techniques resulted in consistent conclusions, and current
view of rhizoctonias implies a form complex placed within basidiomycete orders Cantharellales (Ceratobasidiaceae, Tulasnellaceae) and Sebacinales
(Sebacinaceae) (Smith and Read 2008 and references therein; Pilshchikova and Gannibal 2016; Weiß et al. 2016; Oberwinkler et al. 2017). Both taxa
mentioned are known for broad mycorrhizal activity.
Rhizoctonial mycobionts of Ceratobasidiaceae anity require particular attention due to variety of nutritional strategies presented (economically important
plant pathogens, saprotrophs, endophytes and OM symbionts), a number of yet unresolved questions concerning its evolution and specicity, and
controversy of published data on correlation between nutritional mode and morphological and genetic traits (Veldre et al. 2013).
Due to poor cultural morphology of Ceratobasidiaceae various characteristics were tested as diagnostic to build phylogeny and describe new taxa.
Morphology of basidia was used to describe genera (Tu and Kimbrough 1978), while indirect traits were used on species level such as plant host (Constantin
and Dufour 1920), anastomosis groups and nuclear status (Pilshchikova and Gannibal 2016). The two latter characteristics are shown to correlate with
ecological status and ITS phylogeny: multinucleate isolates were described as plant pathogens, whereas binucleate isolates may appear as both pathogens
and OM mycobionts (Kataria, Hoffman, 1988; Soelistijono et al. 2020). However, these traits are not useful to reconsider existing phylogeny of this family.
Therefore, due to uncertain phylogeny, poor morphology and lack of alternative phylogenetic markers in majority of studies Ceratobasidiaceae fungi are not
identied to species level.
Currently near all previously described ceratobasidioid species are placed within either
Ceratobasidium
or
Thanatephorus
(MycoBank 2023). These genera
represent a wide range of fungal life styles, both saprotrophic and biotrophic. Extensive attempts to build a family-scale phylogeny show that ITS phylogeny
of Ceratobasidiaceae partly correlate with nutritional mode, anastomosis group and nuclear status of isolates. However, to build highly supported phylogeny
ITS region should be supplemented with additional phylogenetic and morphological markers (Oberwinkler et al. 2013; Veldre et al. 2013).
Therefore, current knowledge on Ceratobasidiaceae taxonomy allows us to investigate specicity of OM by searching of highly supported taxonomic clades
that are specic for certain plant host or geographic region which may be further described as new species. This approach may reveal evolutionary traits of
mycorrhizal Ceratobasidiaceae. Most studies show that each orchid individual possesses only one mycobiont, but at plant species level there are evidences
of a wide range of mycobionts existence (Yagame et al. 2008; Shefferson et al. 2015; Rammitsu et al. 2019). Therefore, the current study aims on the
biodiversity of ceratobasidioid orchid symbionts study with the example of globally distributed Goodyerinae subtribe with emphasis on model plant species
Goodyera repens
(L.) R.Br. in W.T.Aiton; on the search for Ceratobasidiaceae clades specic for certain plant host or geographic region; and on providing
characteristics of fungal isolates that could shed light on nutrition modes evolution in Ceratobasidiaceae.
Materials and methods
Studied plants description

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Subtribe Goodyerinae (Cranichidae, Orchidoideae) has worldwide distribution and includes approximately 27 genera of terrestrial sciophytic rhizome-forming
orchids (Chen et al. 2019). The most species-rich genera are
Goodyera
inhabiting mostly temperate Europe, Asia and Northern America (Kallunki 1976) and
Anoectochilus
predominantly inhabiting tropical Asia and America (Zettler et al. 2012; Bon et al. 2020).
Goodyera repens
is a rhizome-reproducing clonal orchid that inhabits conifer forests in temperate regions of the northern hemisphere (Łazarski 2021). This
species may be regarded as a model object in OM studies as it was used in the experiments of Cameron et al. (2006) to prove mutualistic nature of OM.
Isotopic evidences assume autotrophy of adult
G. repens
individuals, but further research is required (Hynson et al. 2009; Voronina et al. 2018).
Dominant mycobionts associated with
G. repens
are members of Ceratobasidiaceae, namely
Ceratobasidium cornigerum
(Bourdot) D.P. Rogers, originally
described as
Rhizoctonia goodyerae-repentis
Costantin & L.M. Dufour (Constantin and Dufour 1920; Cameron et al. 2006). This species is binucleate and
regarded as hemi-biotroph possessing the ability to exist as both plant pathogen and OM symbiont (Newton et al. 2010). Moreover, presence of
ectomycorrhizal fungi in
G. repens
roots may indicate the ability of this species to form mycorrhizal networks with conifer trees (Voronina et al. 2018).
Sampling sites and sample collection
The material used for the study were 11 samples of
G. repens
roots and 11 matching soil samples at the root depth within 10 cm range from corresponding
plants. The sampling was conducted in 2021 and 2022 on 11 sites located in 3 regions of European part of Russia: Leningrad region (2 sites in surroundings
of Bolshoe Lesnoe lake, Vyborg district), Moscow region (3 sites on the territory of Moscow State University Zvenigorod biological station), and Karachay-
Cherkessia republic (6 sites in Teberdinskiy natural reserve). All sampling sites were located in conifer forests predominated by
Pinus sylvestris
and
Picea
abies
in Leningrad and Moscow regions and by
P. sylvestris, P. abies
, and
Abies nordmanniana
in Karachay-Cherkessia. Sites L1 and L2 were distant from
conifer trees, T4 was located on a granite stone with scarce soil covering with no access for conifer roots. Altitude of Karachay-Cherkessia sites varied from
1675 to 1987 m above sea level. The short description of sampling sites is summarized in Table1.
Samples were stored under 4oC in absolute ethanol for metagenome analysis and in paper bags for culture isolation.
Table 1
Description of sampling sites
Region Site Coordinates Nearby conifers Altitude, m
Leningrad region L1 60.800425, 28.941970 None ND*
L2 60.801061, 28.950757 ND
Moscow region M1 55.691680; 36.715776
Pinus sylvestris, Picea abies
ND
M2 55.691485; 36.714948
P. abies
ND
M3 55.694775; 36.739656
P. sylvestris, P. abies
ND
Karachay-Cherkessia T1 43.446608, 41.704962
P. sylvestris
1987
T2 43.441927, 41.704291 1982
T3 43.438027, 41.703754 1960
T4 43.437247, 41.706977 None 1763
T5 43.437539, 41.709663
Abies nordmanniana
1685
T6 43.438027, 41.713557
P. abies
1675
*ND – not dened
Isolation and identication of fungal cultures
Fungal cultures were isolated from root fragments of approximately 1 cm long cleared from debris and soil residues, sterilized by placing in 70% ethanol,
amoxicillin solution, surfactant and rinsed in sterile distilled water. Sterilized root fragments were placed on Petri dishes with malt extract agar and cultivated
under 26°C for 7 days.
Fungal isolates were identied by sequencing of ITS region. Genomic DNA was isolated using DNA extraction kit, amplied using ITS1 and ITS4 primers
(chemicals and primers are provided by Evrogen Co, Russia) and sequenced by Evrogen Co.
Metagenome analysis
The samples were processed the same way as for culture isolation followed by disintegrating in mortar. Genomic DNA was isolated using FastDNA SPIN Kit
(MP Biomedicals, USA), fungal ITS2 regions were amplied using NR_5.8SR and NR_ITS4R primers (Evrogen Co, Russia). Next generation sequencing was
performed by BioSpark Co (Russia) on Illumina MiSeq sequencer (Illumina, USA) with generation of 5000 reads per sample. Sequences were processed with
QIIME 1.9.1 algorithm. Analysis was made by BioSpark Co (Russia).
Fluorescence microscopy
Fluorescence microscopy was used for visualization of nuclei in cells of Ceratobasidiaceae cultures and pelotons in
G. repens
roots. Transverse sections of
roots were hand-made with razor blades. Fungal cultures were grown on Petri dishes with malt extract agar covered with sterile cellophane lm. Mycelium

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taken from lm was placed on the glass and dyed with 0.01 µg/ml DAPI solution for 1 minute. Slides were examined under Axioscop 40 FL uorescence
microscope (Carl Zeiss, Germany) using DAPI narrow Zeiss lter with 365 nm excitation.
Pathogenicity test
Pathogenicity of Ceratobasidiaceae isolates was studied on potato tuber slices. Healthy potato tubers were washed, surface-sterilized in 0.5% sodium
hypochlorite solution for 15 min, rinsed in distilled water, peeled and sliced with a sterile blade. Slices were put into sterile wet chambers. Actively growing
mycelium was placed in the center of the slice and incubated at 12oC for 7 days and then at 24oC for other 7 days. Pathogenicity was measured as radiuses
of mycelial growth after 7 and 14 days. Four potato pathogenic strains of
Rhizoctonia solani
J.G. Kühn: R156, P1, P2 and K3- 3 isolated from infected potato
in Russia were used as positive control. In addition to the strain revealed in the current study, two strains of orchid mycobionts were used in the experiment:
Zs5-1 (from
Zeuxine strateumatica
(L.) Schltr.) and Ss1-1 (from
Spiranthes hongkongensis
S.Y. Hu & Barretto) isolated from plant roots in Shenzhen, China.
The experiment was conducted with three replicates for each strain.
Electron microscopy
Ultrastructure of Ceratobasidiaceae isolates (same as in “Pathogenicity test”) was studied by scanning electron microscopy (SEM) on equipment of the
Center for collective use “Electron microscopy laboratory of Moscow State University Biology Faculty”.
Mycelium was grown on malt agar medium for 7 days and xed in 2.5% glutaraldehyde. Samples were dehydrated in ethanol, dried in Hitachi HCP-2 critical
point dryer (Hitachi, Japan), coated by Eico IB-3 ion coater (Eico, Japan) and examined under JSM-6380 scanning electron microscope (JEOL Inc, USA).
Data analysis
Fungal taxa were identied by ITS sequences using GenBank and UNITE databases, aligned by MAFFT algorithm, and analyzed in MEGA-X. Maximum-
likelihood phylogenetic tree was calculated in IQTree online service and visualized in FigTree v. 1.4.4. Scientic names of fungal taxa are given according to
MycoBank database (MycoBank 2023).
Results
Biodiversity and occurrence of G. repens mycobionts on studied territories
Fungal sequences (OTUs — operational taxonomic units) revealed by metagenomic approach by themselves could not indicate the certain fungal clone as a
mycobiont of
G. repens.
However, introduction of additional requirements regarding known ecological role and occurrence of certain clone in the sample may
narrow down the list of putative mycobionts. Therefore, to assume a certain clone as a putative OM mycobiont, it was obliged to fulll the following
requirements: (1) to appear among dominant 25% of OTUs in
G. repens
root sample; (2) do not appear in dominant 25% of OTUs in matching soil sample; (3)
to appear among the taxa known as OM symbionts. These requirements were applied to basidiomycete taxa with
Rhizoctonia
anamorphs containing OM
mycobionts: Sebacinaceae, Tulasnellaceae and Ceratobasidiaceae.
Sebacinaceae OTUs appeared more frequently in soil samples and lacked specicity towards the roots of
G. repens
, Tulasnellaceae OTUs were not revealed
in studied samples. Thus, Ceratobasidiaceae is the only group of orchid mycobionts, members of which could be assumed as putative mycobionts (see
supplementary materials, Figure S1). High share of Ceratobasidiaceae in
G. repens
roots and presence of pelotons (see supplementary materials, Figure S2)
proves that revealed OTUs are related to mycorrhizal fungi.
Allover, metagenomic analysis revealed 5 Ceratobasidiaceae OTUs: 4 belonging to
Ceratobasidium
and one – to
Thanatephorus.
Due to uncertain phylogeny
of Ceratobasidiaceae and lack of highly similar reference sequences, revealed
Ceratobasidium
OTUs were identied to genus level. Species were delimited
based on less than 95% match (see Table2).
Table 2
Revealed orchid mycorrhizal ceratobasidioid taxa. L – Leningrad region, M – Moscow region, T –
Karachay-Cherkessia
Taxon GenBank number Origin Putative mycobiont Isolation method
Ceratobasidium
sp1 OP782630.1 M M, Culture
OQ244428.1 L, M, T M, T Metagenome
Ceratobasidium
sp5 OP800122.1 L, M L
Ceratobasidium
sp6 OP800123.1 L L
Ceratobasidium
sp7 OP782636.1 T T
Thanatephorus ochraceus
OP782644.1 M M
Among revealed OTUs only
Ceratobasidium
sp1 was detected in all regions studied and
Ceratobasidium
sp5 was revealed in Leningrad and Moscow regions.
Ceratobasidium
sp6, 7 and
Thanatephorus ochraceus
(Massee) P. Roberts were specic for Leningrad region, Karachay-Cherkessia, and Moscow region
correspondingly.

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Isolate OP782630.1 from M3 site was obtained using cultural method. ITS2 region of this isolate matched
Ceratobasidium
sp1 and its mycelium was
binucleate.
Shares of Ceratobasidiaceae OTUs in roots of
G. repens
ranged from 12–85% of all fungal OTUs per sample (see Fig.1). The highest shares (> 70%) were
revealed on sites L1, L2, and T4 with no conifer trees nearby.
Allover, 5 Ceratobasidiaceae OTUs were assumed as
G. repens
mycobionts in the studied regions:
Ceratobasidium
sp5 and 6 in Leningrad region,
T.
ochraceus
and
Ceratobasidium
sp1 in Moscow region and
Ceratobasidium
sp1 and 7 in Karachay-Cherkessia.
Global distribution of revealed ceratobasidioid mycobionts
Deeper analysis of revealed Ceratobasidiaceae OTUs distribution and ecology was made by investigation of reference sequences from GenBank database
that were highly similar (> 97%) to revealed OTUs (see Table3).
Table 3
References for revealed Ceratobasidiaceae with 97% similarity threshold
Reference ID Similarity, % Host Region
Ceratobasidium
sp1
MH855688.1 99,03
Quercus pedunculata
Italy
MH248045.1 99,03
Goodyera repens
Moscow region, Russia
AJ419929.1 99,03
Pinus sylvestris
Finland
KP056301.1 98,46
Goodyera repens
Norway
EU668908.1 98,46
Pyrola rotundifolia
Estonia
KF646110.1 97,49
Rosa rugosa
Lithuania
JQ972064.1, JQ972069.1, JQ972066–67.1, JQ972072–73.1 97,3
Platanthera yadonii
California, USA
GQ268595.1 97,3 Dipterocarpaceae Malaysia
Ceratobasidium sp5
KU516417.1 100
Abies alba
Poland
OL437012.1 98,88
Pinus taeda
Idaho, USA
MK397197.1 97,49
Pinus greggii
Mexico
Ceratobasidium sp6
KP056302.1 99,45
Goodyera repens
Norway
MZ078478.1 99,17
Quercus robur
Poland
DQ309181.1 98,62
Calluna vulgaris
Australia
JQ972107.1, JQ972109.1, JQ972111.1, JQ972113–17.1 98,06
Platanthera yadonii
California, USA
MW927755–56.1, MW927758.1, MW927767.1, MW927770–71.1 97,79
Platanthera cooperi
USA
Ceratobasidium sp7
MN006062.1 98,9
Gymnadenia conopsea
ND
HM141046.1 98,34
Goodyera velutina
Japan
HM141010.1 97,79
Goodyera tesselata
Massachusetts, USA
T. ochraceus
MN684576.1 99,23
Taeniophyllum glandulosum
China
AB831841.1 97,69
Neottia
sp. Japan
EU218892.1 97,44 Orchidaceae gen sp. ND
FJ788721–22.1 97,18
Pterygodium alatum
South Africa
Ceratobasidium
sp1 references were revealed in roots of
G. repens
and related habitats in conifer forests: roots of
P. sylvestris
and
Pyrola rotundifolia.
Also
they were detected in association with
Platanthera yadonii
orchid and in unrelated hosts:
Rosa rugosa, Quercus pedunculata
and Dipterocarpaceae.
Ceratobasidium
sp5 references were revealed in association with conifer trees in Poland, USA, and Mexico.
Ceratobasidium
sp6 conspecics were reported
from
G. repens
roots in Norway and conifer forests (
Calluna vulgaris
) in Australia. Its match was also found in association with orchids
P. yadonii
and
P.
cooperi
and in deciduous forests (
Quercus robur
).
Ceratobasidium
sp7 conspecic was found associated with two
Goodyera
species:
G. velutina
and
G.
tesselata
and orchid
Gymnadenia conopsea. T. ochraceus
references were also found in association with different orchids.

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All revealed OTUs except
Ceratobasidium
sp5 were previously reported in orchid roots. Highly similar references of
Ceratobasidium
sp1 and
Ceratobasidium
sp6 were revealed in
G. repens
roots from Moscow region and Norway.
Ceratobasidium
sp5, 7 and
T. ochraceus
are reported in roots of this species for the
rst time.
Phylogeny of Goodyerinae mycobionts
Host and region specicity of ceratobasidioid mycobionts of Goodyerinae orchids was investigated via ITS2 phylogeny. ITS sequences of Ceratobasidiaceae
isolated from Goodyerinae hosts of genera
Anoectochilus, Chamaegastrodia, Cheirostylis, Erythrodes, Goodyera, Hataeria
, and
Zeuxine
were obtained from
GenBank database. Clades with > 80% statistic support were regarded as a single highly conservative unit to assume specicity to certain plant host or
geographic region. The latter were highlighted on a global scale: Pacic region includes Japan and western USA, Atlantic region includes Norway, western
Russia and eastern USA, and tropical region includes Taiwan, Southern China, Thailand, India, Hawaii, and Puerto-Rico.
Outer group was formed by three representatives of other Cantharellales families: Botryobasidiaceae (
Botryobasidium robustius
Pouzar & Hol.-Jech.
MH859491.1), Tulasnellaceae (
Tulasnella cumulopuntioides
S. Fujimori, J.P. Abe, I. Okane & Y. Yamaoka NR_160570.1), and Cantharellaceae (
Cantharellus
paucifurcatus
Buyck & V. Hofst. NR_137854.1). Also Ceratobasidiaceae representatives of known species isolated from non-orchid hosts were included:
Ceratobasidium angustisporum
Warcup & P.H.B. Talbot NR_154601.1,
C. pseudocornigerum
M.P. Christ. MH861653.1,
C. anceps
(Bres. Syd. & P. Syd.) H.S.
Jacks. MH855251.1,
C. papillatum
Warcup & P.H.B. Talbot NR_154600.1,
C. cereale
D.I. Murray & Burpee AJ302008.1,
C. chavesanum
M.P. Melo, J.A. Ventura,
H. Costa & P.C. Ceresini NR_164016.1,
C. cornigerum
AJ301900.1,
C. ramicola
C.C. Tu, Roberts & Kimbr. NR_138368.1,
Ceratorhiza oryzae-sativa
(Sawada)
R.T. Moore MH861282.1,
Ceratorhiza rhizodes
(Auersw.) Z.H. Xu, T.C. Harr. M.L. Gleason & BatzerMH859145.1, and
Thanatephorus cucumeris
(A.B. Frank)
Donk MH855798.1 (see Fig.2).
A total of 14 highly supported groups were revealed by phylogenetic analysis. Two groups restricted to Europe were also united by plant host
G. repens
. Two
groups were specic for Pacic region and include fungi isolated from
Goodyera
spp. and
Hataeria
spp. Five groups specic for tropical region were revealed
in
Anoectochilus
spp.,
Zeuxine
spp. and
Erythrodes plantaginea.
Five groups were not restricted to the certain region.
Clones
Ceratobasidium
sp1 and 6 were clustered with sequences isolated from
G. repens
(clades II, III), with
Ceratobasidium
sp5 on a sister clade with low
support (48%).
Ceratobasidium
sp7 was clustered on a mixed clade IV with fungi isolated from
G. repens, G. tesselata
and
G. schlechtendaliana
from Europe,
USA and Japan.
T. ochraceus
was clustered with a mycobiont of
G. procera
from Japan on a mixed clade VIII.
Orchid mycobionts do not cluster with sequences of known Ceratobasidiaceae species so the identication remains on the genus level.
Cultural characteristics and pathogenicity of mycorrhizal and pathogenic isolates
Anatomy of isolated strain
Ceratobasidium
sp1 was studied in comparison with isolates of 4 plant pathogens and 2 orchid mycobionts. Strains R156 and K3-
3 with teleomorph
Thanatephorus cucumeris
AG 3 were isolated from potato tubers. Strains P1 and P2 isolated from potato stem had teleomorph
Ceratobasidium
sp. AG K. Two orchid mycobionts Zs5-1 and Ss1-1 were isolated from
Zeuxine strateumatica
and
Spiranthes hongkongensis
and had
teleomorph
Ceratobasidium
sp. AG F and L correspondingly.
Nuclear status was revealed using uorescence microscopy. SEM method was used to observe polysaccharide sheath (see Table4, Figure S3).
Table 4
Cultural characteristics and pathogenicity of pathogenic and mycorrhizal Ceratobasidiaceae isolates
Pathogenicity,
14 days,
24oC, mm
Pathogenicity,
7 days, 12oC,
mm
Polysaccharide
sheath Nuclei Anastomosis
group Plant host Origin Nutrition
mode Taxon Isolate
6.6 ± 2.0 4.2 ± 1.9 Weak > 2 AG 3** Potato, tuber Moscow
region Plant
pathogen
Thanatephorus
cucumeris
R156
30.7 ± 8.5 6.0 ± 1.1 AG 3 Smolensk
region K3-3
8.0 ± 1.7 5.0 ± 0.0 Massive 2 AG K Potato, stem Astrakhan
region
Ceratobasidium
sp. P1
12.3 ± 2.5 NP* AG K P2
7.0 ± 0.8 4.3 ± 0.5 ND***
G. repens
root Moscow
region Mycorrhizal C sp1
9.25 ± 2.2 NP AG F
Zeuxine
strateumatica
root
Shenzhen,
China Zs5-1
9.25 ± 1.0 NP AG L
Spiranthes
hongkongensis
root
Ss1-1
*NP – pathogenicity not observed
**Anastomosis groups were identied by closest reference ITS sequence
**ND – no data

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Polysaccharide sheath surrounding hyphae varied in structure and was measured by “weak” and “massive” categories. Weak sheath of thickness
commensurable to cell wall thickness surrounds single hyphae, eventually being observed in branching points and between hyphae for pathogenic
multinucleate
Thanathephorus
isolates on SEM (Figure S3). Massive sheath is thicker than cell wall, surrounds multiple hyphae and clearly seen on SEM on a
surface of hyphae. This type of sheath is intrinsic for pathogenic and mycorrhizal binucleate
Ceratobasidium
isolates.
Four out of seven studied isolates show pathogenicity on potato tubers under 12oC. Two OM isolates: Zs5-1 and Ss1-1 and pathogenic strain P1 did not
show any growth under these conditions. Still, no signicant difference between pathogenicity areas of OM and pathogenic isolates was shown (p > 0.05).
After incubation under 24oC all isolates have shown pathogenicity with no signicant difference between OM and pathogenic strains (p > 0.05).
Discussion
Biodiversity of ceratobasidioid mycobionts of G. repens
Ceratobasidiaceae are known to be the most abundant of
Goodyera
mycobionts (Shefferson et al. 2015). In the current research we proved the preference of
this association and elucidate internal taxonomical and regional specicity of Goodyerinae species and its mycobionts.
Allover, 5 putative mycobionts of Ceratobasidiaceae family were revealed in roots of
G. repens
, 3 of which were revealed in roots of this species for the rst
time and 2 OTUs were not previously reported in roots of
Goodyera
spp.
Ceratobasidium
sp1 was previously revealed in roots of
G. repens
in northern Europe: Norway (Liebel et al. 2015) and Moscow region of Russia (Voronina et
al. 2018). This isolate also had high similarity (99%) with
Rhizoctonia quercus
E. Castell. isolated from
Quercus pedunculata
roots in Italy. However, this
reference isolate is uninucleate anamorph (Castellani, 1934) and connection between these two stages is doubtful.
Ceratobasidium
sp1 was the only isolate
revealed from all three studied regions that allows us to broaden the distribution area of this taxon in association with
G. repens
from Norway and Leningrad
region on the north to Caucasus Mountains on the south. This isolate was clustered with mycobionts of
G. repens
which allows us to presume that these
isolates belong to one species that is specic for
G. repens
in Europe and USA.
Ceratobasidium
sp5 was revealed in association with orchid roots for the rst time. However, the presence of highly similar isolates in conifer forests may
presume its occurrence in OM in Europe and USA. This OTU was revealed in Leningrad and Moscow region and assumed as a mycobiont in Leningrad region.
This isolate did not belong to a branch with high support but was close to clade II represented by mycobionts of
G. repens
.
Ceratobasidium
sp6 is a putative mycobiont in Leningrad region and is highly similar to
Ceratobasidium
sp. previously revealed from
G. repens
in Norway.
These sequences formed a conservative clade III distant from clade II also specic for
G. repens
. Although these clades were uniform by region and plant
host, phylogenetic analysis assumes them as evolutionary diverse.
Ceratobasidium
sp7 was specic for Karachay-Cherkessia in 5 of 6 studied sites which assumes its domination in this region with occasional substitution by
Ceratobasidium
sp1. Similar sequences were found in roots of
Goodyera
spp. and
Gymnadenia conopsea
orchids in Japan and USA which assumes wide
host and geographic range of this taxon. Domination of this mycobiont in Karachay-Cherkessia and its absence in Moscow and Leningrad regions may be
conditioned by subalpine ecosystem with domination of
Abies nordmanniana
and altitudinal zoning:
G. repens
clones were not found below 1675 m. These
characteristics discriminate this region and affect mycobiome of
G. repens.
T. ochraceus
was the only clone identied to species level by ITS region. This species is known to form mycorrhiza with wide range of orchid hosts (Roberts
1998). Similar sequences were revealed in association with various orchids in China, Japan and South Africa which assumes wide geographical range of this
species as orchid mycobiont.
Therefore, revealed mycobionts may possess different ecological ranges.
Ceratobasidium
sp1, 5 and 6 are presumably specic for conifer forests in northern
hemisphere, whereas
Ceratobasidium
sp7 and
T. ochraceus
possess a wide geographic range in association with orchid roots which is also proved by their
close relationship with mycobionts of
G. schlechtendaliana
and
G. procera
in Japan. Studying evolutionary specicity of Ceratobasidiaceae is challenging
due to uncertain phylogeny and diculty of ecological preference estimation. Nonetheless, revealing geographic and ecological specicity of ceratiobasidioid
isolates would elucidate evolution perspectives for emergence of clades specic for certain orchid taxa.
Specicity of Goodyerinae hosts
Previous extensive study on
Goodyera
mycorrhizal specicity assumed narrow specicity of
G. repens
based on phylogenetic distance between revealed
mycobionts (Shefferson et al. 2010). Biodiversity of mycobionts revealed in the current study indicates ability of
G. repens
to form OM with broad range of
distantly related mycoiont taxa. Breadth of involved mycobionts is revealed down to regional scale where in each studied region 2 putative ceratobasidioid
mycobionts are shared. Re-qualication of
G. repens
to generalist species along with
G. foliosa, G. velutina
and
G. procera
(according to Shefferson et al.
2010) illustrates necessity to provide extensive studies on mycorrhizal specicity of certain orchid species to reveal evolutionary traits and generalize
information on a scale of tribes and subfamilies.
Phylogeny of Goodyerinae mycobionts suggests that conservative evolution lines of European and tropical mycobionts are diverse. That illustrates specicity
of mycorrhizal Ceratobasidiaceae on a global scale regarding region and plant host. Extensively studied tropical orchid
Anoectochilus formosanus
shows
broad mycobiont range with no common clades in Atlantic region. Orchids in Pacic region, by contrast, tend to associate with phylogenetically diverse
mycobiont taxa which are present on mixed clades sharing them with either Atlantic or tropical orchids.
Orchid mycobionts and ectomycorrhizal fungi

Page 8/11
Various orchid species are known to form mycorrhizal association with ectomycorrhizal fungi. In the current research we spotted a curious tendency that
G.
repens
clones distant from conifer trees contain the highest share of Ceratobasidiaceae fungi. Sites L1, L2 and T4 simultaneously possess low shares of
ectomycorrhizal fungi from Agaricales, Russulales and Atheliales, while sites M2 and T3 with lowest Ceratobasidiaceae share possess the highest share of
ectomycorrhizal fungi (see supplementary materials, Figure S4). These data assume a competition between these two symbiotic groups in orchid roots and a
potential for mycorrhizal networks formation between
G. repens
and surrounding conifers. However, a more extensive study is needed to elucidate an
interaction between orchid mycobionts and ectomycorrhizal fungi.
Cultural characteristics and pathogenicity
In the current study we made an attempt to establish possible connection between ceratobasidioid isolates nutritional modes and morphological
characteristics. Polysaccharide sheath was measured due to ability to prevent decomposition by plant β-glucanases. Signicant difference between
mycorrhizal and pathogenic strains has not been revealved, but sheath thickness differs between
Ceratobasidium
and
Thanatephorus
isolates. Variability of
these two parameters may indicate different adaptations of Ceratobasidiaceae to plant-associated life style. Massive sheath might be benecial for enduring
existence inside living plant cells that is typical for OM symbionts, whereas weak sheath may be a trait of necrotrophic pathogens that aim on quick
decomposition of plant cells. However, large variety of Ceratobasidiaceae nutritional modes from pathogens to mutualists assumes existence of transition
states of these characteristics.
Pathogenicity of Ceratobasidiaceae isolates was studied towards potato tubers. Ability of both mycorrhizal and pathogenic isolates to decompose tubers
under 24oC may witness that even OM strains may show pathogenic activity under certain conditions. Moreover, under favorable conditions (24oC) no
signicant differences between OM and pathogenic isolates were found. Inability of three strains to grow on potato tuber under 12°C may be explained by
their distribution area: tropical China and Astrakhan region of Russia.
Conclusion
Studies of biodiversity and phylogenetic relations of ceratobasidioid mycobionts shed light on OM evolution and specicity. It the current study with the
example of
Goodyera repens
we prove that orchids may be associated with a wide range of distantly related Ceratobasidiaceae embracing both generalist
and narrowly specialized taxa. This diversity is shown on regional and global scales illustrating generalism of
G. repens
.
Cultural isolation of mycobionts with further identication should be supplied with at least indirect evidence towards their ecological role as far as it remains
the most accurate way to identify the certain isolates which form mycorrhizas with orchids.
Competition between orchid mycobionts and ectomycorrhizal fungi is still to be studied in context of preferences given by orchid and potential for assumable
mycorrhizal network establishment. Our data assume that
G. repens
plants located in the area that is accessible for conifer roots may partly substitute
ceratobasidioid mycobionts with ectomycorrhizal fungi. This fact points at possibility of mycorrhizal formation between
G. repens
and ectomycorrhizal fungi
and assumes ectomycorrhizal plant roots accessibility as an important factor that shapes community of orchid mycobionts. Further studies on anatomy and
physiology of this association are required to understand this peculiar interaction.
Morphological characteristics may partly reect the nutritional mode of Ceratobasidiaceae isolates, but wide range of trophic states might generate a variety
of transitional stages with ambiguous delimitation.
Ability of mycorrhizal strains to decompose potato tubers with rates comparable to those of pathogenic isolates assumes ability of OM Ceratobasidiaceae to
show pathogenicity under certain conditions.
Declarations
Author contribution
NB, EV and AK designed experiment, NB and AK performed sample collection, NB performed culture isolation and metagenome data analysis, EV wrote the
main part of the manuscript, OK performed electron microscopy, MY performed pathogenicity tests. All authors reviewed manuscript.
Funding
The study was nancially supported by Russian Federation Ministry of Science and Higher Education (project # 075-15-2021-1396).
Competing interests.
The authors declare no competing interests.
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Figures
Figure 1
Shares of Ceratobasidiaceae members OTUs in
G. repens
root samples

Page 11/11
Figure 2
ITS2 Maximum likelihood tree of
Ceratobasidiaceae
fungi connected to their Goodyerinae hosts. Identical sequences from similar hosts and habitats are
united and displayed as triangles. Purple – Pacic region; green – Atlantic region; red – tropical region; grey – mixed group. Numbers of sequences obtained
in current research are marked in bold. Non-OM sequences are marked in italic.
Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.
Supplementarymaterials.docx