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Horticulturae 2022, 8, 1213. https://doi.org/10.3390/horticulturae8121213 www.mdpi.com/journal/horticulturae
Article
Correlations between the Phylogenetic Relationship of 14
Tulasnella Strains and Their Promotion Effect on Dendrobium
crepidatum Protocorm
Jiayi Zhao, Zhenjian Li, Siyu Wang, Fu Yang, Lubin Li and Lei Liu *
Key Laboratory of Silviculture of the State Forestry Administration, Research Institute of Forestry,
Chinese Academy of Forestry, Beijing 100091, China
* Correspondence: liulei519@caf.ac.cn
Abstract: The compatibility of mycorrhizal fungi with the early growth stage of orchids is essential
for their growth. In this study, the compatibility and promotion effects of 14 Tulasnella strains from
different hosts were studied by co-culturing them with the protocorms of Dendrobium crepidatum,
which has high ornamental and economic value in China. The ITS–LSU–SSU–TEF combined se-
quence analysis divided the 14 strains into three clades belonging to Tulasnella calospora (clades A
and B) and Tulasnella asymmetrica (clade C). All the strains were compatible with D. crepidatum pro-
tocorms within 90 d of the co-culture. Strain T12 in Clade A had a significantly higher (p < 0.05)
effect on the biomass and morphology of D. crepidatum, and strain T13 in Clade C had a significantly
lower (p < 0.05) effect than the other strains. Through morphological principal component analysis,
we constructed a hierarchical cluster analysis tree, which was consistent with the phylogenetic tree
of these 14 strains at the clade level. Orthogonal partial least squares-discriminant analysis showed
that these strains have an important effect on the plant height, root number, and length of D. crepi-
datum. The findings of this study will contribute to the identification of Tulasnella strains, conserva-
tion of D. crepidatum resources, and commercial utilization of mycorrhizal technology.
Keywords: Dendrobium crepidatum; mycorrhizal fungi; Tulasnella spp.; molecular identification;
symbiosis
1. Introduction
Orchid mycorrhizal (ORM) is a mutualistic symbiosis established between orchids
and ORM fungi [1]. Orchid seeds are tiny and lack sufficient nutrient reserves, as they are
not surrounded by an endosperm [2–4]. Therefore, they obtain carbon, mineral nutrients,
and vitamins from their symbiotic partners, which enables them to germinate and de-
velop into a unique seedling structure composed of parenchyma cells called protocorms
[5–7]. Subsequently, ORM fungal hyphae enter the protocorm to form coiled complexes
called pelotons, and orchids obtain nutrients by digesting the pelotons [5,8,9]. Addition-
ally, the further development of orchids from protocorms to plants also requires the sup-
port of ORM fungi [5,10].
Most ORM fungi belong to the ‘Rhizoctonia’ species complex. Among them, Tu-
lasnella, which belongs to the fungal family Tulasnellaceae, is the most predominant ORM
fungus found in temperate and tropical regions and has been extensively studied because
of its abundant and widespread distribution [11–13]. Therefore, studies on the identifica-
tion of Tulasnella species can better exploit and utilize mycorrhizal resources [14]. With
numerous Tulasnella species, molecular sequencing provides a powerful method for Tu-
lasnella species identification [15,16]. The most important and commonly sequenced frag-
ments are the internal transcribed spacers (ITS) of fungal ribosomal DNA [14,17–20]. In
addition, several studies have reported the use of multilocus species delineation to further
Citation: Zhao, J.; Li, Z.; Wang, S.;
Yang, F.; Li, L.; Liu, L. Correlations
between the Phylogenetic
Relationship of 14 Tulasnella Strains
and Their Promotion Effect on
Dendrobium crepidatum Protocorm.
Horticulturae 2022, 8, 1213.
https://doi.org/10.3390/
horticulturae8121213
Academic Editor: Michelle L Jones
Received: 18 November 2022
Accepted: 15 December 2022
Published: 17 December 2022
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Copyright: © 2022 by the authors. Li-
censee MDPI, Basel, Switzerland.
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distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://cre-
ativecommons.org/licenses/by/4.0/).
Horticulturae 2022, 8, 1213 2 of 16
improve the resolution of Tulasnella species identification [21]. Species delimited by Tu-
lasnella mainly include the following loci: nuclear rDNA internal transcribed spacer region
(ITS), large subunit region (nrLSU) [22–24], small ribosomal subunit (nrSSU) [25], and mi-
tochondrial large rDNA gene (mtLSU) [26,27]. Furthermore, three protein-coding genes
are commonly used in the phylogeny of the fungal phylum Basidiomycota: RNA poly-
merase II largest subunit (RPB1), RNA polymerase II second largest subunit (RPB2), and
translation elongation factor 1α (TEF1) [28–30]. Multilocus sequence analysis (MLSA) has
broadened previous knowledge of Tulasnella species identification.
Notably, not all ORM fungi can promote orchid growth, and fungi that promote or-
chid growth may not be long-lasting. The compatibility between the orchid and the ORM
fungi may change with the growth of orchids [1,3,9,31–33], and only orchid-compatible
mycorrhizal fungi can support the development of orchids into seedlings [3]. This symbi-
otic pattern is particularly evident in Dendrobium plants. Dendrobium is one of the largest
genera in the orchid family and has been the focus of the cut flower and pharmaceutical
industries for decades [34–36]. Several symbiotic studies have been conducted on Den-
drobium plants and ORM fungi. In previous co-culture studies, Tulasnella strains compati-
ble with Dendrobium plants rapidly promoted seed germination, protocorm growth, and
development into seedlings. In contrast, Tulasnella strains incompatible with Dendrobium
plants stimulated the seeds to germinate, but the protocorms did not grow and develop
into seedlings [32]. For example, the Tulasnella sp. SSCDO-5 can promote the development
of D. officinale from the protocorm to the seedling stage, whereas Tulasnella sp. FDd1 can
only promote the germination of D. officinale seeds and cannot form protocorms [3]. Sim-
ilar findings have been reported for other Dendrobium plants, such as D. exile [37], D. mo-
niliforme [38], and D. chrysotoxum [39]). The compatibility between Tulasnella strains and
Dendrobium plants changes with the growth of Dendrobium, therefore, effective strains of
Dendrobium at different growth stages can be specifically screened [33,40]. In addition,
determining the effects of different Tulasnella species on Dendrobium plant growth will
help meet the needs of artificial propagation of Dendrobium and promote the development
of the Dendrobium industry [37].
Dendrobium crepidatum, a traditional Chinese medicine in Yunnan Province, is an ep-
iphytic orchid medicine (TCM) in South Asia [41]. The active components of D. crepidatum
mainly include indole–azine alkaloids [42,43], flavonoids [44], and bioactive secondary
metabolites [45]. Furthermore, D. crepidatum has recently attracted attention because of its
high medicinal value. However, compared to studies on the active ingredients of D. crep-
idatum, there have been few studies on its symbiotic relationship with ORM fungi [46]. In
this study, 14 Tulasnella strains were co-cultured with D. crepidatum protocorms to estab-
lish a symbiotic relationship. We aimed to answer the following questions: (1) Will poly-
genic MLSA identification of Tulasnella strains improve resolution compared to using ITS
sequences alone? (2) What is the effect of the 14 Tulasnella strains on the growth of D.
crepidatum protocorms? (3) What is the association between the 14 Tulasnella strains’ phy-
logenetic relationship and the promotion efficiency of D. crepidatum?
2. Materials and Methods
2.1. Fungal Isolation and Identification
Purified fungal strains were obtained from previous studies, and the sources of the
plant samples are Cymbidium mannii, Epidendrum radicans, Cymbidium faberi, Cymbidium
goeringii, and Epidendrum radicans (Table 1). The fungal strains were cultured on potato
dextrose agar (PDA; BD Difco 213400, NY, USA). The colony morphology was recorded,
and the hyphae of the fungi were cultured on PDA for 8 days at 28 °C. The fungal mycelia
were frozen using liquid nitrogen, and DNA was extracted using the E.Z.N.ATM Fungal
DNA Miniprep Kit (D3390-01, Omega, Guangzhou, China), according to the manufac-
turer’s instructions. This study used the primers ITS1 and ITS4 [47] for the nuclear ITS
region and the primers LROR and LR5 [30] for the nuclear LSU-rDNA region. The primers
Horticulturae 2022, 8, 1213 3 of 16
for the SSU and TEF regions are listed in Table S1. The ITS, LSU, SSU, and TEF sequences
were compared with those in the NCBI GenBank database using the basic local alignment
search tool (BLAST). Subsequently, the ITS, LSU, SSU, and TEF sequences of the 14 strains
were registered in GenBank (Table 1).
Table 1. NCBI registration number of fungal gene and orchid source of isolated fungus.
Isolates Species Original Plants NCBI Accession Number
ITS LSU SSU TEF
T1 T. calospora C. mannii OM672252 OM728181 OP537825 OP819646
T3 T. asymmetrica E. radicans OM672253 OM728182 OP537826 OP819647
T4 T. calospora C. mannii OM672254 OM728183 OP537827 OP819648
T5 T. calospora C. faberi OM672255 OM728184 OP537828 OP819649
T6 T. calospora C. faberi OM672256 OM728185 OP537829 OP819650
T7 T. asymmetrica C. mannii OM672257 OM728186 OP537830 OP819651
T9 T. calospora C. mannii OM672258 OM728187 OP537831 OP819652
T10 T. calospora C. goeringii OM672259 OM728188 OP537832 OP819653
T11 T. calospora C. goeringii OM672260 OM728189 OP537833 OP819654
T12 T. calospora C. goeringii OM672261 OM728190 OP537834 OP819655
T13 T. asymmetrica C. goeringii OM672262 OM728191 OP537835 OP819656
T14 T. calospora E. radicans OM672263 OM728192 OP537836 OP819657
T24 T. asymmetrica E.radicans OM672264 OM728193 OP537837 OP819658
T25 T. asymmetrica C. mannii OM672265 OM728194 OP537838 OP819659
For phylogenetic analysis, 21 strains of Tulasnella sp. were constructed using ITS se-
quences, with Sebacina vermifera isolate K251 and Sebacina vermifera isolate FFP337 classi-
fied as outgroups. Phylogenetic trees were constructed using five genome sequences and
ITS–LSU–SSU–TEF combined sequences, with the Trichoderma reesei genome as an out-
group.
Finally, 14 polygenic combination sequences were used to construct an evolutionary
tree to compare the effects of the relationships between the fungi and the growth of pro-
tocorms. BioEdit 7.2 (https://bioedit.software.informer.com/7.2/, accessed on 8 November
2022) was used to calculate the sequence identity and sequence concatenation. All of the
sequences were analyzed using maximum likelihood, applying the rapid bootstrapping
algorithm for 1000 replications in MEGA7 [48]. Clades with bootstrap values (BS) ≥ 50%
were considered significantly supported [49].
2.2. Protocorm Formation
Dendrobium crepidatum seed capsules were collected from the greenhouse of the Chi-
nese Academy of Forestry (Beijing, China) at day/night temperatures of 24 °C/18 °C, rela-
tive air humidity of 65–75%, and 8-h day/16-h night photoperiod. Four D. crepidatum ma-
ture capsules were collected from the greenhouse prior to dehiscence (Figure 1). After
collecting the seed capsules, the first scrub was placed using a soft brush under running
tap water to remove dirt and other stains. Each capsule was then cleaned thrice with 75%
ethanol, sterilized with 5% NaClO for 5–10 min, and rinsed thrice in sterile distilled water.
Finally, the capsules were cut lengthwise, and dusty ripe seeds were picked [50]. After
sterilization, as described above, approximately 100–150 seeds were added to a culture
flask (glass bottles) containing Murashige and Skoog (MS) medium [51]. The cultures were
placed in a growth room under a 12/12-h photoperiod at 25 ± 1 °C with a relative air hu-
midity of 65–75%.
Horticulturae 2022, 8, 1213 4 of 16
Figure 1. Plant material of D. crepidatum. (a) The flowers of D. crepidatum; (b) The capsules of D.
crepidatum; (c) Seeds swollen after absorbing water; (d) The protocorms of D. crepidatum. Bar: (a) 2
cm, (b) 1 cm, (c) 0.1 mm, (d) 1mm.
2.3. Symbiotic Culture of Protocorms
Protocorms are used for co-culture when they develop to stage 2 [39] (rupture of
testa, appearance, and extension of the protomeristem). Protocorms with consistent
growth were inoculated in 2.0 g/L OA medium [50], and an approximately 1 cm3 cube of
each fungal culture was inverted onto the surface of the OA medium using an inoculating
needle. The control treatment group contained no fungal inoculum. The Petri dishes were
sealed with Parafilm (Bemis, Neenah, WI, USA) and incubated at 24 °C/18 °C day/night
temperatures and 65–75% relative humidity. Each treatment comprised 10 replicates.
Symbiotic cultures were placed in a growth room under a 12/12-h photoperiod at 25 ± 1
°C, with a relative air humidity of 65–75%, as described previously. The plates were sealed
with Parafilm (Bemis, Neenah, WI, USA) and maintained at day/night temperatures of 24
°C/18 °C and relative air humidity of 65–75%.
2.4. Assessment of the Fungal Capacity to Promote Protocorm and Seedlings Growth
Protocorm growth was observed daily under a stereomicroscope (Nikon HFX, To-
kyo, Japan) and light microscope (OLYMPUS BX51, Tokyo, Japan). Trypan blue staining
was performed to examine fungal colonization [52]. A concentration of 0.4% trypan blue
stain was used for the study. Data were subsequently collected on days 30, 60, and 90 after
sowing to determine the average fresh and dry weight growth rates. When measuring
fresh weight, 30 symbiotic materials were randomly selected for each sampling, and the
average value was calculated three times. To measure the dry weight, 30 symbiotic mate-
rials were randomly selected and dried at 60 °C until the weight remained constant. The
samples were recorded, which was repeated three times. The average growth rate was
determined as follows: (weight after inoculation − weight before inoculation
amount)/weight before inoculation × 100% [53].
At 90 days of co-cultivation, the plant height, stem diameter, root length, leaf length,
and leaf width were measured using a Vernier caliper, and the root number and leaf num-
ber were recorded [54]. Ten symbionts were randomly selected during the measurement,
and the average value was obtained and the measurement was repeated three times. The
relative leaf area is a product of the leaf length and width [55]. SIMCA 17 software
(https://www.sartorius.com/en/products/process-analytical-technology/data-analytics-
software/mvda-software/simca, accessed on 8 November 2022) was used to perform prin-
cipal component analysis (PCA), hierarchical clustering analysis (HCA), and orthogonal
partial least squares-discriminant analysis (OPLS-DA) of the morphological parameters.
In addition, HCA was performed on the PCA results, and the HCA tree of principal com-
ponent 1 (PC1) and principal component 2 (PC2) was constructed via sorting by index
using Ward calculation.
Horticulturae 2022, 8, 1213 5 of 16
2.5. Statistical Analysis
All the experiments were performed in a completely randomized design. One-way
ANOVA was used, and the significant differences between the treatments were assessed
using least significant difference (LSD) multiple comparison tests (p < 0.05). All the statis-
tical analyses were performed using IBM SPSS Statistics 25.0 (IBM Corporation, Armonk,
NY, USA). GraphPad Prism 9 (www.graphpad.com, accessed on 26 September 2022)and
R Studio software (https://posit.co/downloads/, accessed on 9 November 2022).
3. Results
3.1. Tulasnella Identification and Phylogenetic Analyses
The BLAST results of the ITS sequences in the GenBank database showed that all 14
strains were Tulasnella (Table S2). The phylogenetic trees constructed based on ITS se-
quences showed that the nine strains were clustered with T. calospora and divided into
two branches. The first branch (Clade A) consisted of five strains (T5, T12, T10, T4, and
T1), and the second branch (Clade B) consisted of four strains (T14, T6, T9, and T11) (Fig-
ure 2). Meanwhile, five strains (T24, T25, T3, T7, and T13) formed a separate phylogenetic
lineage within T. asymmetrica, named Clade C (Figure 2).
Figure 2. Maximum likelihood trees of ITS sequences of Tulasnella sp. The sequence of Sebacina ver-
mifera isolate FFP337 and Sebacina vermifera isolate K251 was used as an outgroup. Only bootstrap
values (based on 1000 replications) ≥50 are shown. Scale, 1.0 nt substitutions per site.
Horticulturae 2022, 8, 1213 6 of 16
The comparison results of t
he
ITS sequence consistency showed that the sequence
identity of the 14 strains ranged from 56.5–100% (Figure S1). Notably, the sequence iden-
tity of strains T3 and T7, and T6 and T9 was 100% (Figure S1). Further identification using
ITS–LSU–SSU–TEF combined sequences (Figure S2) showed that the sequence identities
of strains T3 and T7, and T6 and T9 changed to 99.7% and 99.9%, respectively (Figure S1).
These results suggest that polygenic analysis alters the resolution compared to ITS se-
quences alone. Furthermore, the colony and mycelium morphology showed no significant
difference in the colony and hyphae of the three strains (strain T12 in Clade A, T9 in clade
B, and T13 in Clade C). The mycelia of these three strains near the inoculated agar blocks
had distinct concentric circles and rectangular branches in the mycelia (Figure 3).
Figure 3. The mycelium morphological characteristics of three clades strains. (a) Community mor-
phology of T12 (Clade A); (b) Community morphology of T9 (Clade B); (c) Community morphology
of T13 (Clade C); (d) Mycelium morphology of T12 (Clade A); (e) Mycelium morphology of T9
(Clade B); (f) Mycelium morphology of T13 (Clade C). RB: Rectangular branch; S: Septum. The Petri
dishes are 9 cm in diameter.
3.2. Compatibility between Tulasnella Stains and D. crepidatum Protocorms
After co-culturing the fungi with D. crepidatum protocorms, all D. crepidatum–Tu-
lasnella symbiotic protocorms grew and developed into seedlings. At 30 days of co-culture,
the protocorm in symbiosis with Tulasnella began differentiating into its first leaf (Figure
4a). After 60 days of co-culture, the symbiotic protocorms developed into seedlings with
two leaves and one root (Figure 4b). Finally, after 90 days of co-culture, the symbiotic
protocorms further developed into seedlings with an average of four leaves and two roots
(Figure 4c). These results indicate that the 14 Tulasnella strains used in this study and D.
crepidatum protocorms are compatible (Figure 4a–c). Meanwhile, successful colonization
of the symbiotic protocorms by the 14 Tulasnella strains was observed under stereomicros-
copy (Figure 4d–f) and light microscopy (Figure 4d–j). Furthermore, the results of trypan
blue staining showed that the strains colonized the parenchyma cells of D. crepidatum pro-
tocorms, and the pelotons were apparently visible when co-cultured for 30, 60, and 90
days (Figure 4d–j). Furthermore, all 14 strains belonging to T. calospora (in Clade A and
Clade B) and T. asymmetrica (in Clade C) were compatible with D. crepidatum protocorms.
Horticulturae 2022, 8, 1213 7 of 16
Figure 4. The colonization of strain T12 in D. crepidatum protocorms during 90 days of co-culture.
The growth of the D. crepidatum protocorms into seedlings in co-culture at 30 days (a), 60 days (b),
and 90 days (c). Pelotons were observed in a stereomicroscope using trypan blue staining at 30 days
(d), 60 days (e), and 90 days (f) of co-culture; Cross-section of the symbionts that were observed in
the light microscope using trypan blue staining at 30 days (g), 60 days (h), and 90 days (i) of co-
culture. Bar: (a–f) 1 mm, (g–i) 0.2 mm. The red arrows point to the pelotons.
3.3. Effects of different Tulasnella Strains on the Biomass of the Symbionts
The biomass parameters of the fungus–protocorm symbiont, including the average
fresh weight growth rate and the average dry weight growth rate, represent the growth
status of fungus–protocorm symbiont. The average fresh weight growth rate and average
dry weight growth rate of the 14 symbionts were significantly higher (p < 0.05) than those
of the control group on days 30, 60, and 90 after co-culture, and this growth-promoting
effect lasted for 90 days (Figures 5 and S3, Tables S3 and S4). This result indicates that the
14 Tulasnella strains are consistently and stably compatible with D. crepidatum protocorms
and promote their growth.
Meanwhile, the 14 compatible Tulasnella strains differed in their promotional effect
on D. crepidatum protocorms (Figure 5). In detail, during the 90 days of co-cultivation,
strain T12 in Clade A had the strongest effect (p < 0.05) on promoting the fresh weight (the
average growth rates were 116.67 ± 7.02%, 288.02 ± 7.55%, and 622.16 ± 19.06% on days 30,
60, and 90 of co-culture, respectively) and dry weight (206.00 ± 10.15%, 462.67 ± 13.32%,
and 895.37 ± 26.27% on days 30, 60, and 90, respectively) of D. crepidatum protocorm,
whereas strain T13 in Clade C had the weakest effect (p < 0.05) on promoting the fresh
weight (only 54.01 ± 4.58%, 116.33 ± 8.08%, 210.28 ± 18.78% on days 30, 60, and 90 after co-
culture, respectively) and dry weight (74.33 ± 8.62%, 173.02 ± 11.53%, and 327.67 ± 25.88%,
on days 30, 60, and 90 after co-culture, respectively) among all 14 strains (Figure 5).
Overall, when cultured for 30 days, three strains of Clade A (T12, T10, and T1) had a
significantly higher effect on the fresh weight (116.67 ± 7.02%, 120.67 ± 8.51%, and 120.67
± 5.03% in T12, T10, and T1, respectively) and dry weight (206.00 ± 10.15%, 198.00 ± 11.53%,
Horticulturae 2022, 8, 1213 8 of 16
and 186.67 ± 8.08% in T12, T10, and T1, respectively) of the protocorms than the other
strains. Furthermore, on days 60 and 90 of the co-culture, the growth-promoting effect of
the Tulasnella strain in clade A was evidently higher than that of other strains in Clade B
and Clade C, whereas the five strains in Clade C were significantly lower than those of
the other strains. Overall, these results indicate that the growth-promoting effects of the
strains may be related to their evolutionary relationship.
Figure 5. Comparison of the average fresh and dry weight growth rates of 14 fungi on the 30th, 60th,
and 90th days of symbiosis. (a) The average fresh weight growth rate on day 30 after co-culture. (b)
The average fresh weight growth rate on day 60 after co-culture. (c) The average fresh weight
growth rate on day 90 after co-culture. (d) The average dry weight growth rate on day 30 after co-
culture. (e) The average dry weight growth rate on day 60 after co-culture. (f) The average dry
weight growth rate on day 90 after co-culture. Strains with different lowercase letters are signifi-
cantly different (p < 0.05) based on the LSD test.
3.4. Effects of Different Tulasnella Strains on the Morphology of the Symbionts
Trypan blue staining results showed that pelotons were detected in all 14 symbionts
after 90 days of co-culture, and the symbionts grew from protocorms to seedlings (Figure
4f,j). Therefore, to further understand the role of various strains in protocorm growth, we
investigated phenotypic data from six 90-day symbionts. On day 90 of symbiosis, the mor-
phology of the symbionts was significantly better than the non-symbiotic control group
(Figure 6). Further morphological measurements showed that, compared with that of the
control group, the plant height, stem diameter, root length, root number, and leaf number
of all 14 symbionts were significantly increased (p < 0.05) (Figure 7a–e, Table S5). How-
ever, the relative leaf areas of the four symbionts (strains T24, T3, T7, and T13) were not
significantly different from that of the control (Figure 7f, Table S5). Similar to the previous
results on biomass, among different Tulasnella strains, strain T12 was significantly better
(p < 0.05) than the other strains in promoting the plant height (8.68 ± 0.16 mm), stem di-
ameter (2.39 ± 0.05 mm), root length (15.14 ± 0.46 mm), root number (2.22 ± 0.19), leaf
number (5.00 ± 0.33), and relative leaf area (7.26 ± 0.72 mm
2
) of symbionts (Figure 7). By
contrast, strain T13 had the weakest (p < 0.05) promotional effects on plant height (5.55 ±
0.37 mm), stem diameter (1.89 ± 0.04 mm), root length (2.97 ± 0.28 mm), root number (0.89
± 0.19), and leaf number (2.45 ± 0.16) (Figure 7). Compared with that of other strains, the
promotional effect of strain T5 on symbiont plant height (8.50 ± 0.27 mm) and stem
Horticulturae 2022, 8, 1213 9 of 16
thickness (8.50 ± 0.27 mm) (Figure 7a,c) was not significantly different (p>0.05) from that
of the dominant strain T12, whereas its promotional effect on the root length (8.97 ± 0.67
mm) and root number (1.56 ± 0.19 mm) was significantly different (p < 0.05) from that of
the dominant strain T12 (Figure 7b,e).
Notably, strains T12 and T10 (in Clade A) promoted the six morphological parame-
ters to a larger extent than the other strains. In contrast, strains T3, T7, and T13 (in Clade
C) had a weaker promotion effect. These results further suggest that the promoting effi-
ciency may be related to the genetic relationship among Tulasnella strains.
Figure 6. Morphology of the symbionts on day 90. (a) Strain T12 in Clade A; (b) Strain T9 in Clade
B; (c) Strain T13 in Clade C; (d). Control. The Petri dishes are 9 cm in diameter.
Figure 7. The growth of different fungi and D. crepidatum protocorm on day 90 of symbiosis. (a) The
average plant height of symbionts; (b) The average root length of symbionts; (c) The average leaf
number of symbionts; (d) The average stem diameter of symbionts; (e) The average root number of
Horticulturae 2022, 8, 1213 10 of 16
symbionts; (f) The relative leaf area of symbionts. Strains with different lowercase letters are signif-
icantly different (p < 0.05) based on the LSD test.
3.5. Association between Relationship and Symbiotic Effects among Different Tulasnella Strains
Since previous results have found that the promoting efficiency of symbiont growth
by different Tulasnella strains may be related to the relationship between the strains, PCA
and OPLS-DA methods were used to further analyze the association between them. The
PCA for the six morphological parameters showed that the contribution rate of PC1 and
PC2 was 90.6% and 4.49%, respectively, and the sum of the two principal components was
greater than 95% (Figure 8d). Simultaneously, the 14 Tulasnella strain symbionts were di-
vided into three clades in PC1. The fungi within the three clades in the PCA results are
consistent with those within the three clades in the phylogenetic tree constructed using
ITS (Figure 2) and polygenes (Figure S2). Four symbionts (strains T12, T10, T4, and T1) in
Clade A were distinguished in PC1, strain T5 in Clade A was distinguished in PC2, and
strain T12 had the highest contribution rate in PC1 (Figure 8d). The four symbionts (strains
T6, T9, T11, and T14) in Clade B were distinguished in PC2. The other symbionts (strains
T24, T25, T3, T7, and T13) in Clade C were distinguished by PC1 and PC2 (Figure 8d).
Furthermore, HCA was performed on the PCA results, and the HCA tree divided the
strains into three clades. Compared with the phylogenetic tree, the HCA tree was con-
sistent with the phylogenetic tree at the clade level (Figure 8a,b). Further analysis of PC1
in the PCA revealed that the contribution rate of different fungi showed a downward
trend from clade A to clade C, and strain T12 also had the highest contribution rate (Figure
8c).
Horticulturae 2022, 8, 1213 11 of 16
Figure 8. PCA and OPLS-DA of different fungal symbionts. (a) Maximum likelihood trees of ITS
sequences of Tulasnella sp.; (b) Hierarchical clustering analysis of six morphological parameters; (c)
PC1 analysis in a line plot; (d) Principal component analysis of two principal components (PC1 and
PC2); (e) VIP values of six parameters in orthogonal partial least squares-discriminant analysis.
In addition, the OPLS-DA model was used to analyze the contribution of the 14
strains to six morphological parameters. To verify the reliability of the OPLS-DA model,
200 permutation tests were performed on two significant components. The R2Y intercept
was 0.117 (<0.2) and the Q2 intercept was −0.566 (<0.05), indicating that the OPLS-DA
model used was reliable (Figure S4). Furthermore, using the OPLS-DA model, the VIP
values of the plant height (1.08), root number (1.02), and root length (1.01) were greater
than 1.0, indicating that these three parameters were the key variables (Figure 8e).
4. Discussion
Orchids are heterotrophic in the early stages of development and require ORM fungi
to provide nutrients that are likely extracted from soil organic matter for their growth and
development [7,56,57]. Dendrobium crepidatum is a perennial epiphyte distributed in south-
ern China, where it is known as “Shi-Hu”. Dendrobium crepidatum can moisten the lungs,
relieve cough, clear deficiency heat, and nourish the stomach [41]. Tulasnella is extremely
important for orchid mycorrhizas as ORM fungi. Therefore, it is vital to study the symbi-
osis between the Tulasnella species and D. crepidatum to conserve D. crepidatum resources.
Previous studies have shown that Tulasnella species fruiting bodies are usually absent
and difficult to induce in culture, which are less helpful for Tulasnella species identification
[58–62]. Therefore, recent studies have focused on using molecular sequencing to identify
Tulasnella species [15,16,23,63–68]. Polygenic analysis has a higher resolution than ITS sin-
gle gene analysis, and fungal species taxonomy should be based on the phylogeny of core
genes with strong phylogenetic signals, such as ITS regions and at least one protein gene,
including TEF or RPB2 [21,22,24,28,29,68]. Overall, multilocus DNA sequence datasets
typically contain two to six genes depending on the research needs [16,21,69]. However,
recent polygenic analyses of Tulasnella have focused on the identification of new species
of Tulasnella fungi and have been used less for subspecies-level identification [15,16,24].
Our study successfully divided the 14 strains into two species, T. calospora and T. asym-
metrica, using ITS sequences. We used four genes for MLSA identification for each intra-
specific strain and further altered the sequence consistency of T3, T7, T6, and T9 (Table
S3). These results are consistent with those of previous changes in resolution through
MLSA [29,30] and provide a reference for the identification of Tulasnella subspecies levels.
The next step should be to increase the number of genes to further improve the resolution
and, thus, better exploit mycorrhizal resources.
Not all mycorrhizal fungi are compatible with Dendrobium, and the compatibility of
the fungi may change with the growth of mycorrhizal symbionts [3,39,70]. Moreover, per-
sistent observation of pelotons is a sign of fungi–Dendrobium compatibility [3]. For exam-
ple, a study in which six Tulasnella species were co-cultured with D. moniliforme seeds
showed that only two Tulasnella species symbionts were still alive after 120 days of co-
culture because other Tulasnella species symbionts could not develop into seedlings from
protocorms [38]. In other species of Dendrobium, mycorrhizal fungi (e.g., Tulasnella spe-
cies) can live symbiotically with Dendrobium seeds until the formation of protocorms;
however, they often change, and either the compatible fungi continue to promote proto-
corm differentiation to form seedlings or the protocorms stop growing or even die [3,4,37–
39]. The same phenomenon occurs in the symbiosis between fungi and other orchids, such
as Arundina graminifolia [52] and Serapias vomeracea [17]. In our study, the 14 Tulasnella
strains, belonging to T. calospora and T. asymmetrica, are compatible with D. crepidatum
protocorms. This is consistent with previous studies, thereby showing that compatible
fungi can successfully differentiate protocorms into seedlings and can continuously ob-
serve pelotons (Figures 5 and 6).
Horticulturae 2022, 8, 1213 12 of 16
In response to these phenomena, it has been hypothesized that during the transition
from protocorms to seedlings, protocorms may require a higher level of carbon sources in
terms of quantity and quality. Therefore, a better functional match with the fungus may
result in enhanced carbon flow, thereby producing more robust seedlings [38]. Further-
more, under natural conditions, changes in mycorrhizal fungal compatibility can allow
orchids to adapt to various physiological changes during seedling growth, including par-
tial or complete autotrophy, increased transpiration, or environmental fluctuations [71].
In addition, compatibility between fungi and their hosts probably depends on the devel-
opmental stage, which may contribute to narrowing the host distribution range [70].
Therefore, the dynamic change in compatibility between Tulasnella species and Den-
drobium can be regarded as an advanced survival strategy, and it is more intuitive and
effective to study this compatibility during the protocorm period.
Among compatible Tulasnella strains, different strains have varied effects on seed
germination, protocorm formation, and seedling development of Dendrobium species
[31,32,37,40,72]. Moreover, the evaluation indicators include biomass (fresh and dry
weight) and various parameters related to the morphology (plant height, root length, and
leaf number) of symbionts [33,34,40,73–75]. For example, T. calospora strains S6 and S7 can
support the development of D. officinale protocorms into seedlings, unlike S4. Meanwhile,
for the protocorm formation rate with the second leaf, the symbionts inoculated with T.
calospora strain S7 (59.17 ± 8.78%) were higher than those inoculated with S6 (1.35 ± 1.09%).
Furthermore, S7 was better than S6 in promoting the fresh weight and root number of
symbionts. In contrast, S6 was better than S7 regarding the number of tillers of the sym-
bionts and the total crude polysaccharide content in the stem [33]. The same phenomenon
has been observed in other studies; for example, among the three strains of T. calospora,
JM, TG1, and TG3, strain JM had the highest promotion efficiency (p < 0.05) on D. officinale
protocorm formation and seedling development, followed by TG3, and TG1 was not sig-
nificantly different (p < 0.05) from the control group [32]. Furthermore, the phylogenetic
tree of the three strains based on ITS-5.8S sequences revealed that JM was on one branch,
whereas TG1 and TG3 were in two clades of another branch [32]. These studies suggest
that the effectiveness of fungi in promoting Dendrobium growth is related to the species
and intraspecific phylogenetic relationships of the fungi.
Some studies have explained the reason why different Tulasnella strains have varied
effects on orchid germination and development may be because Tulasnella species are dif-
ferent at the species level and this phenomenon must be studied via precise molecular
identification [17]. Meanwhile, the inherent differences between fungi in terms of symbi-
otic compatibility have a greater impact on mycorrhizal symbiosis than previously spec-
ulated. The differences in symbiotic compatibility between Tulasnella strains did not re-
flect their phylogenetic relationships based on fungal ITS sequences because Tulasnella
ribosomal DNA sequences are extremely variable, and the phylogenetic relationships they
present may be inaccurate [75].
In our study, among the 14 Tulasnella strains, T. calospora strain T12 was the most
effective symbiont for promoting the growth of D. crepidatum protocorm, whereas T. asym-
metrica strain T13 had the lowest efficacy (Figures 5–8). The PCA of six morphological
parameters revealed that the contribution rates of the 14 strains were different and could
be well distinguished, and the names of the strains in the three clades of the hierarchical
cluster were consistent with those of the phylogenetic tree (Figure 8). The growth promo-
tion efficiency of the 14 strains decreased from strain T12 in Clade A to strain T13 in Clade
C, which proves that for Tulasnella and D. crepidatum, the phylogenetic relationship be-
tween fungi was closely related to the promotion efficiency of symbiotes; and the growth
promotion efficiency of T. calospora strains was better than that of T. asymmetrica for D.
crepidatum. In addition, although the host plants are different, the two pairs of strains, T3
and T7 and T6 and T9, have a close genetic relationship in terms of identifying the ITS
sequence and ITS–LSU–SSU–TEF polygenic sequence and also have similar symbiotic ef-
fects for D. crepidatum. Although the mechanism of compatibility and the varied symbiotic
Horticulturae 2022, 8, 1213 13 of 16
effectiveness between Dendrobium plants and Tulasnella strains are still unclear, they may
be related to hormones and lignin degradation activity [1,70,76]. Therefore, transcriptomic
and secretory tools may be used to further investigate compatibility patterns and symbi-
otic mechanisms.
5. Conclusions
This study identified 14 strains from different hosts as T. calospora and T. asymmetrica,
where T. calospora contains clade A and clade B, and T. asymmetrica contains clade C. ITS–
LSU–SSU–TEF polygene analysis can further improve identification resolution compared
to using ITS sequences alone. All 14 strains of T. calospora and T. asymmetrica were com-
patible with the D. crepidatum protocorm. The protocorms inoculated with T. calospora
strain T12 in clade A showed optimal fresh and dry matter biomass and morphological
parameters. The T. asymmetrica strain T13 in clade C had a significantly lower (p < 0.05)
promotional effect than the other strains. These fungal strains tested in co-culture have
different effects on the growth and development of protocorms, and the phylogenetic tree
of 14 strains is consistent with the morphological hierarchical clustering tree at the branch-
ing level, suggesting that the phylogenetic relationship of 14 Tulasnella strains correlates
with their promotion effect on the D. crepidatum protocorm. These results may pave the
way for further research on the relationship between mycorrhizal fungi and Dendrobium
plants and help to protect D. crepidatum resources.
Supplementary Materials: The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/horticulturae8121213/s1, Figure S1: Sequence consistency
across different strains; Figure S2: Maximum likelihood trees of ITS–LSU–SSU–TEF concatenated
sequences of Tulasnella sp.; Figure S3: Average fresh weight growth rate and dry weight growth rate
of symbionts on days 30, 60, and 90 of symbiosis; Figure S4: 200 permutation tests of the OPLS-DA
model. Table S1: Primer sequence; Table S2: Blast results of mycorrhizal fungi ITS sequences in the
GenBank database; Table S3: The average fresh weight growth rate of protocorms on days 30, 60,
and 90 of symbiosis; Table S4: Average dry weight growth rate of protocorms on days 30, 60, and
90 of symbiosis; Table S5: Average growth parameters of protocorms on day 90 of symbiosis.
Author Contributions: Conceptualization, L.L. (Lei Liu) and L.L. (Lubin Li); methodology, L.L. (Lei
Liu) and J.Z.; overall conception, L.L. (Lubin Li); validation, S.W. and F.Y.; formal analysis, S.W. and
F.Y.; investigation, J.Z.; sample resources, Z.L.; data curation, J.Z.; writing—original draft prepara-
tion, J.Z.; writing—review and editing, L.L. (Lei Liu); supervision, L.L. (Lei Liu); funding acquisi-
tion, L.L. (Lei Liu). All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by the National Natural Science Foundation of China, grant num-
ber No. 31800522 and the Fundamental Research Funds for the Central Non-profit Research Institu-
tion of Chinese Academy of Forestry CAFYBB2020SZ006.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: The authors are grateful to Lu Xu (Hunan Mid-Subtropical Quality Plant Breed-
ing and Utilization Engineering Technology Research Center, College of Horticulture, Hunan Agri-
cultural University) for her help with the strain materials and suggestions on writing paper and
Yanxia Cheng (State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry)
for her suggestions on experimental methods and data analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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