Association Between Dipsacus Saponin VI Level and

World Journal of Microbiology and Biotechnology (2019) 35:42
https://doi.org/10.1007/s11274-019-2616-y
ORIGINAL PAPER
Association between dipsacus saponin VI level and diversity

of endophytic fungi in roots of Dipsacus asperoides
Anhui Gong1 · Tao Zhou1,2 · Chenghong Xiao1 · Weike Jiang1,2 · Yongqiang Zhou1 · Jinqiang Zhang1 · Qing Liang1 ·
Changgui Yang1 · Wei Zheng1 · Chenggang Zhang1
Received: 19 July 2018 / Accepted: 6 February 2019 / Published online: 18 February 2019
© The Author(s) 2019
Abstract
Dipsacus asperoides contains multiple pharmacologically active compounds. The principal are saponins. The plant can be
cultivated, but it contains lower levels of bioactive compounds than the plant in the wild. It may be the reason to exploit the
endophytic fungi that colonize the plant roots in order to produce bioactive compounds. However, the endophytic fungi of
D. asperoides have not been analyzed in detail. In this study, we isolated and identified 46 endophytic fungal strains from
the taproots, lateral roots and leaves, and we used morphological and molecular biological methods to assign them into 15
genera: Fusarium sp., Ceratobasidium sp., Chaetomium sp., Penicillium sp., Aspergillus sp., Talaromyces sp., Cladosporium
sp., Bionectria sp., Mucor sp., Trichoderma sp., Myrothecium sp., Clonostachys sp., Ijuhya sp., Leptosphaeria sp. and Phoma
sp. Taproots contained abundant endophytic fungi, the numbers of which correlated positively with level of dipsacus saponin
VI. Primary fermentation of several endophytic fungal strains from taproots showed that Fusarium, Leptosphaeria, Cera-
tobasidium sp. and Phoma sp. can produce the triterpenoid saponin. These results may guide efforts to sustainably produce
bioactive compounds from D. asperoides.
Electronic supplementary material The online version of this

article (https://doi.org/10.1007/s11274-019-2616-y) contains
supplementary material, which is available to authorized users.
* Tao Zhou
taozhou88@163.com
* Weike Jiang
jwk_88@163.com
1
Experimental Center, Guiyang University of Chinese
Medicine, Guiyang 550025, China
2
Department of Molecular Biology Laboratory, Guiyang
University of Chinese Medicine, Guiyang 550025, China
13
Vol.:(0123456789)
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42 Page 2 of 14 World Journal of Microbiology and Biotechnology (2019) 35:42
Graphical abstract
Cladosporium sp. Fusarium sp. Ceratobasidium sp.
Isolation and
identification
endophyc
fungi in roots
Primary
fermentation
Dipsacus saponin VI
Dipsacus asperoides
Keywords Dipsacus asperoides · Dipsacus saponin VI · Diversity · Endophytic fungi · Evolutionary system · Fermentation
Introduction Endophytic fungi can combine with other endogenous

microorganisms and antibacterial compounds to form
Dipsacus asperoides is a well-known medicinal plant used a defense system that produces alkaloids to strengthen
to curing occlusion diseases, punch injury, and rheumatism immunity and maintain growth under stress (Qin et al.
(Niu et al. 2015; Wong et al. 2007). Saponins, the major 2011; Clay and Holah 1999). Some endophytic fungi
bioactive compound in D. asperoides, are isolated primarily produce active proteases helped maintain plant activities,
from the taproots and widely used to treat fractures (Zhang such as pectinase and esterase, which degrade cell walls
et al. 2003; Jung et al. 2012). D. asperoides in the wild has (Zhao et al. 2016). Pathology can result when programmed
diminished as a result of exploitation (Zhang et al. 1997; senescence in the plant or environmental change perturb
Chen et al. 2014; Wang et al. 2016), and the cultivated plant the fungal population in the plant (Stamford et al. 2001).
contains lower levels of dipsacus saponin VI than the plant In addition to supporting the growth and productivity of
in the wild. Therefore, rapid, efficient and environmentally medicinal plants, endophytic fungi can produce bioactive
sustainable methods are needed to obtain this and other sap- metabolites similar to plant hosts, making them a poten-
onins from D. asperoides (Cira et al. 2008; Jiao et al. 2015). tial source of medicinal compounds (Chandra 2012). For
It may be possible to obtain saponins from the endo- example, the endophytic fungus isolated from Taxus chin-
phytic fungi that colonize D. asperoides (Jiao et al. 2015). ensis can be produced the anti-cancer compound paclitaxel
Such fungi colonize the flowers, seeds, taproots, stems (Li et al. 2009b). Other endophytic fungi produce the drug
and leaves of many plant species, without causing visible compounds camptothecin, podophyllotoxin (Eyberger et al.
disease symptoms (Aly et al. 2011). Endophytes establish 2006; Puri et al. 2006), hypericin and emodin (Kusari et al.
a long-term symbiotic relationship with their plant hosts 2009). Endophytic fungi can produce bioactive compounds
(Zuccaro et al. 2011). Some endophytic fungi and their through industrial fermentation (Winter et al. 2011; Walsh
metabolites increase resistance to plant pathogens and and Fischbach 2010) (Kusari and Spiteller 2011). The
tolerance to drought (Redman et al. 2002; Waller et al. research of endophytic fungi may provide new ideas and
2005; Herre et al. 2007; Rodriguez and Redman 2008). methods for developing bioactive compounds in medicinal
13

World Journal of Microbiology and Biotechnology (2019) 35:42 Page 3 of 14 42
plants in ways that sustain the development of traditional including colony shape, height and color of aerial hyphae,
Chinese medicine resources (Zuccaro et al. 2011). base color, growth rate, margin, surface texture, and depth of
Endophytic fungi have been analyzed in at least 145 growth into the agar. At least three cultures were character-
species of medicinal plants, but no such analysis has been ized on each petri dish, and on the attempts equated colony
reported for D. asperoides, to the best of our knowledge. morphologies from different plates of the same plant. Endo-
Here we characterized the taxonomic diversity of D. asper- phytic fungi were preliminarily assigned to genera based on
oides taproots, lateral roots and leaves, and analyzed the spore and culture characteristics.
correlation between the number of fungi and the level of The sequence analysis was also performed to assist in
saponins. Primary fermentation was performed with several specimen identification. Mycelium was gathered directly
endophytic fungal strains to examine the possibility of large- from the surface of 4-day-old agar cultures and ground into
scale development of natural products. a powder in liquid nitrogen. The powder was suspended in
buffer [200 mM Tris–HCl (pH 8.0), 25 mM EDTA (pH 8.0),
250 mM NaCl and 0.5% SDS (pH 7.5)]. DNA was extracted
Materials and methods using phenol and chloroform, and precipitated in ethanol.
DNA integrity was analyzed by agarose gel electrophoresis,
Sample collection and purity was assessed using a Micronuclear Quantifier
(Nanodrop 2000, Thermo Scientific, USA).
Two-year-old D. asperoides from Meihuashan in Wein- Internal transcript spacer (ITS) regions of endophytic
ing County, Guizhou Province (N26°23′10.46′′) was fungi were amplified using polymerase chain reaction (PCR)
planted at the Guiyang University of Chinese Medicine and the universal ITS primers, V9D (5′-TTAAGTCCC
(E106°37′41.64′′). Plant material was washed and soaked TGCCCTTTGTA-3′) and LS266 (5′-GCATTCCCAAAC
in 0.1% SDS for 10 min, then rinsed with double-distilled AACTCGACTC-3′). Reactions (25 µL) contained 100 ng
water. The material was divided into taproots, lateral roots of genomic DNA, 10 µM of each primer, 12 µL of Premix
and leaves, which were stored at 4 °C. Taq™ (Ex Taq™ 2.0 plus dye) and sterile double-distilled
water. Thermal cycling parameters for PCR were as follows:
Isolation of endophytic fungi pre-denaturation at 94 °C for 5 min; 30 cycles of denatura-
tion at 94 °C for 30 s, annealing at 53 °C for 30 s and exten-
The surface of plant material was sterilized by soaking sion at 72 °C for 2 min; and a final extension step at 72 °C
in 0.1% mercuric chloride for 5 min, then in 75% ethanol for 10 min. PCR products were detected on 1.2% (w/v) aga-
for 3 min. The disinfected material was rinsed three times rose gel prepared in 1× TAE buffer and electrophoresed at
(1 min each time) with sterile water. The material was cut 100 V for 45 min.
into pieces measuring 0.5 × 0.5 cm, and incubated at 28 °C Fragments were eluted and sent to be sequenced by King-
on petri dishes containing potato dextrose agar (PDA), tryp- sley Biotech (Nanjing, China). Further information to guide
tone soy agar (TSA), beef extract tryptone agar (NA) and the taxonomic identification of fungal strains came from
Luria–Bertani (LB) medium. Five biological replicates were the Flora of Chinese Mycology. BLAST searches of fungal
prepared for each tissue, and growth was monitored every sequences were conducted to analyze homology with iden-
day. Endophytic fungal strains were inoculated on PDA slant tified sequences in ITS. Moreover, the comparison analy-
culture-medium. After fungal cultures were fully grown, sis of UNITE database to complement GenBank results.
slant culture tubes were closed with tampons wrapped with Tree topologies were evaluated using bootstrap analyses in
oilpaper and stored at 4 °C. MEGA6 (1000 bootstrap replicates). Phylogenetic trees were
inferred using the neighbor-joining method.
Taxonomic identification of endophytic fungi
Analysis of endophytic fungal diversity
Identification of endophytic fungi was accomplished fol-
lowing the methods described by Cannon et al. (Cannon Menhinick’s index (Dmn) was used to quantify species rich-
and Simmons 2002). In this study, we perform its molecular ness among the isolated√ endophytic fungi. Dmn was cal-
reidentification, based on the analysis of internal transcript culated as Dmn = S∕ N , where S refers to the number of
spacer (ITS) regions of endophytic fungi (Ding et al. 2018; different endophytic fungal species, and N refers to the total
Koljalg et al. 2005). Colonial morphology of endophytic number of isolated endophytic fungi. The Shannon diversity
fungi was identified using the point planting method as index (H′) was calculated using H � = − i Pi(LnPi), where
∑k
described (Chen et al. 2012). In brief, fungal spores were Pi = Ni/N, Ni is the number of individuals of the species, and
inoculated onto the center of solid PDA and incubated at k is the number of different endophytic species in a sample.
28 °C. Fungal characteristics were recorded every day, The isolation rate (IR) was calculated by dividing the total
13

number of isolates in a trial by the total number of samples and other properties of the fermentation broth. Samples of
in the trial. IR was used to measure the richness of endo- mycelium (100 mg) were harvested by filtering and ground
phytic fungi in plant tissues. The Sorensen similarity index into powder in liquid nitrogen. DNA was extracted and PCR-
(Cs) was calculated as Cs = 2j/(a + b), where j is the num- amplified as described above (Cannon and Simmons 2002).
ber of endophytic fungi common to the two tissues being
compared, and a and b are the numbers of endophytic fungi
Statistical analyses
in each tissue. Cs was used to quantify species similarity
between different tissues.
All results were expressed as mean ± SEM. Graphs were
prepared using GraphPad Prism 7.0. Differences between
Quantification of dipsacus saponin VI
mean values were assessed for significance using one-way
analysis of variance (ANOVA), followed by the least signifi-
Samples of D. asperoides taproots, lateral roots and leaves
cant difference (LSD) test for post hoc comparisons (equal
were dried and ground into powder. Sample powder were
variances were assumed). Significance was indicated as fol-
soaked in methanol solution, ultrasonicated for 30 min
lows: *P < 0.05, **P < 0.01, and ***P < 0.005.
(power, 100 W; frequency, 40 kHz), allowed to cool,
weighed, and membrane-filtered. The filtered sample was
analyzed for dipsacus saponin VI on a C18 symmetry col-
umn (4.6 × 250 mm, 5 µm) on a Waters HPLC system, with Results
the following chromatography parameters: mobile phase,
acetonitrile–water (30:70); flow rate, 1.0 mL/min; injection Identification of endophytic fungi from D.
volume, 20 µL; detection wavelength, 212 nm; and theo- asperoides roots and leaves
retical plate number, ≥ 3000. HPLC run time was 25 min
(Pharmacopoeia of the People’s Republic of China, 2015). Different tissues of D. asperoides were cultured in PDA,
HPLC was also conducted with standard dipsacus sapo- LB, TSA and NA culture media. A total of 46 strains were
nin VI (purity, 91.3%; JY8R—BINA2), which was obtained isolated and preliminarily assigned based on colony and
from the China Food and Drug Certification Research Insti- hyphal characteristics (Fig. 1). The largest number of endo-
tute (Beijing, China). The standard was dissolved in metha- phytic fungal isolates (40) were found in taproots, followed
nol to a concentration of 0.15 mg/mL. Retention time of the by leaves (4) and lateral roots (2) (Fig. 1A). The isolates in
standard was 18.254 min under our conditions. four media showed that the greatest number was obtained
in PDA (37), followed by NA (4), TSA (3) and finally LB
Fermentation of endophytic fungi medium (2) (Fig. 1B). The IR in taproots (0.40) was sig-
nificantly higher than that in lateral roots (0.02) or leaves
Taproot mycelium were transferred to an Erlenmeyer flask (0.04). Taproots also showed that H′ and Dmn were higher
containing 100 mL liquid medium and cultured at 28 °C for than leaves and lateral roots (Table 1; Fig. 2). These results
5 days with shaking at 160 rev min−1. Fungal characteristics suggest that the taproots may provide the best niche or entry
were recorded every day, including color, viscosity, odor point for colonization and penetration by endophytic fungi.
Fig. 1 Isolation of endophytic fungi from Dipsacus asperoides. Tis- from the taproots, 2 strains (4.35%) were isolated from lateral roots
sues of Dipsacus asperoides were cultured in the culture medium of and 4 strains (8.70%) were isolated from the leaves. B Culture of dif-
PDA, LB, TSA and NA. 46 isolates were identified in 100 taproot ferent endophytes from different D. asperoides tissues on different
segments, 100 lateral root segments and 100 leaf segments based on media: PDA supported growth of 37 strains (80.43%); LB medium,
their morphological characteristics. A Distribution of endophytes in 2 strains (4.35%); TSA, 3 strains (6.52%); and NA, 4 strains (8.70%)
different tissues of D. asperoides: 40 strains (86.96%) were isolated
13

Table 1 Colonisation, isolation, species richness and multiple infec- basis of white cotton-like appearance with some water-like
tion rates of endophytic fungi at each healthy tissue of Dipsacus substances, broom-like branches with long spindle-shaped
asperoides
spore stalks on the branchlets, and small curved elliptical
Parameter Taproots Lateral roots Leaves Total and ovate spores on the spore stalks (Figs. 3 and S1).
The remaining endophytic fungal isolates were assigned
No. of samples 100 100 100 300
to genera based on comparison with known fungi: Clonos-
Isolation rate (IR) 0.40 0.02 0.04 0.46
tachys sp. (daef 28–29), Mucor sp. (daef 30–34), Tricho-
Shannon diversity index (H′) 2.60 0.00 1.40 4.00
derma sp. (daef 35–36), Myrothecium sp. (daef 37), Ijuhya
Menhinick’s index (Dmn) 2.53 0.71 2.00 5.24
sp. (deaf 38–39), Leptosphaeria sp. (daef 40–42), Phoma sp.
Diversity statistical table of endophytic fungi in D. asperoides tap- (daef 43–45) and Heliogales sp. (daef 46).
roots, lateral roots and leaves. Indicated are the number of isolates Comparison of ITS sequences from the 46 isolates with
recovered, isolation rate (IR), Shannon diversity index (H′), and Men-
hinick’s index (Dmn) fungal sequences in GenBank (Table 2) lead to the iden-
tification of 15 genera: Fusarium sp., Ceratobasidium sp.,
Chaetomium sp., Penicillium sp., Aspergillus sp., Talaromy-
ces sp., Cladosporium sp., Bionectria sp., Mucor sp., Tricho-
derma sp., Myrothecium sp., Clonostachys sp., Ijuhya sp.,
Leptosphaeria sp. and Phoma sp. Two strains that could not
be assigned to a genus were identified as Chaetomiaceae sp.
and Helotiales sp. based on GenBank analysis. Taxonomic
identification based on ITS sequencing was consistent with
that based on morphological observation. In addition, the
results of blastn analysis by UNITE database were consistent
with NCBI analysis (Table S1).
The two dominant genera were Fusarium sp. to which
29.09% of isolates, and Ceratobasidium sp. to which 10.91%
of isolates. Myrothecium sp. was isolated only from leaves.
Cs analysis showed that the tissue pair with greatest simi-
larity was lateral roots and leaves (Cs 2.00), followed by
taproots and lateral roots (1.88) and finally taproots and
leaves (1.79). These results suggest the heterogeneity of the
endophyte assemblage.
Fig. 2 Diversity of endophytic fungi from D. asperoides. Statistical A phylogenetic tree based on ITS sequences (Fig. 4)
histogram of the number of different endophytic fungi in the taproots
(green), lateral roots (blue) and leaves (orange) assigned Fusarium sp. isolates to six clusters, three of
which were closely related and clustered with Fusarium
globosum (LT746280.1), two of which clustered with
Microscopic analysis of the 46 endophytic fungi allowed Fusarium tricinctum (MG274296.1) and Fusarium sp.
them to be assigned preliminarily to Fusarium sp. (samples (JF740911.1), and one of which was related to Fusarium
daef 1–14) on the basis of their irregular, round shape and solani (KY484984.2). Clades comprised daef 6, 7, 13 and
hyphae uplift, fast growth, yellow pigment production, and 14; daef 4 and 8; and daef 11 and 12. Fusarium sp. was the
presence of conidia or spores; to Ceratobasidium sp. (daef most frequently isolated fungal genus. The four isolates daef
15–18) on their basis of their loose white hyphae and lack of 30 and 32–34 were grouped into a branch with the refer-
conidia; to Chaetomiaceae sp. (daef 19–20) on the basis of ence taxon Mucor sp. The daef 15, 16 and 18 and Ceratoba-
white colonies and soft hair or cotton with yellow pigment sidium sp. were grouped into a branch with 100% bootstrap
on the back of hyphae; to Penicillium sp. (daef 21–22) on the support, with daef 15 clustering with Ceratobasidium sp.
basis of scattered hyphae, pigment production, and broom- (KC782938.1).
like stem with a string of conidia; to Aspergillus sp. (daef Leptosphaeria isolates formed a cluster with reference
23) on the basis of white, pilose, cotton-like hyphae with taxa Leptosphaeria sp. (KJ934197.1 and AJ317958.1).
erect hyphae, conidiophores and a hemispherical capsule; to Phoma sp. isolates formed a cluster with reference taxa
Talaromyces sp. (daef 24) on the basis of green cotton-like Phoma exigua var. (EU343130.1 and EU343168.1).
hyphae and a small, broom-like stem with conidiophores; Penicillium sp. isolates were grouped into two clus-
to Cladosporium sp. (daef 25) on the basis of green villi- ters: daef 21 clustered with Penicillium janthinellum
like hyphae with small water-like substances, elliptical and (MG938669.1), and daef 22 clustered with Penicillium
round conidia; and to Bionectria sp. (daef 26–27) on the skrjabinii (EU427287.1). Trichoderma isolates formed a
13

A Surface Back 100h Surface Back 100h
Leptosphaeria sp. Cladosporium sp.

Ceratobasidium sp. Fusarium sp.
(daef 11)
(daef 25)
20μm 20μm
(daef 15)
(daef 40)
20μm 20μm
Penicillium sp.
Leptosphaeria sp.
(daef 22 )
(daef 41)
20μm 20μm
Aspergillus sp.
Phoma sp.
(daef 23)
(daef 44)
20μm 20μm
B Surface Back 100h C Surface Back 100h

Myrothecium sp. Ceratobasidium sp.
Fusarium sp.
(daef 5)
(daef 18)
20μm 20μm
Penicillium sp.
(daef 21)
(daef 37)
20μm 20μm
Fig. 3 Morphological characteristics of endophyte fungi. Photo- bar, 20 µm. B Characteristics of endophytic fungi isolated from the
graphs showing typical morphology of endophyte fungi from tap- lateral roots, showing “surface”, “back” and microstructure. These
roots, lateral roots and leaves of D. asperoides. A Characteristics characteristics were observed for daef 5 and 21. Scale bar, 20 µm. C
of endophytic fungi isolated from the taproots, showing “surface”, Characteristics of endophytic fungi isolated from the leaves, show-
“back” and microstructure. These characteristics were observed for ing “surface”, “back” and microstructure. These characteristics were
the following isolates: daef 11, 15, 22, 23, 25, 40, 41 and 44. Scale observed for daef 18 and 37. Scale bar, 20 µm
cluster with Trichoderma hamatum (KM491888.1), Tricho- cluster with Bionectria sp. (KF367470.1) with 100% boot-
derma asperellum (KF723005.1) and Trichoderma konin- strap support. Myrothecium sp. isolates were grouped into
giopsis (GQ229070.1). Clonostachys sp. isolates formed two clusters: two closely related isolates formed a clade
a cluster with Clonostachys rosea f. (HM751081.1), Clo- and one isolate formed a clade with Myrothecium roridum
nostachys sp. (KC806284.1) and Clonostachys pseudoch- (FJ914699.1) and Myrothecium sp. (KY086248.1) with
roleuca (KC806259.1). A Bionectria sp. isolate formed a 99% bootstrap support. Chaetomium sp. isolates formed a
13

Table 2 Similarity between the Strain ID Accession no. Closest (Accession no.) Similarity (%)
isolates and closest species in
GenBank daef1 MH550471 Fusarium oxysporum (KU872828.1) 99
daef2 MH550472 Fusarium globosum (LT746280.1) 99
daef3 MH550473 Fusarium solani (KY484984.2) 98
daef4 MH550474 Fusarium sp. (JF740911.1) 99
daef5 MH550475 Fusarium tricinctum (MG274296.1) 99
daef6 MH550476 Fusarium lateritium (AF310980.1) 99
daef7 MH550477 Fusarium acuminatum (KJ082098.1) 99
daef8 MH550478 Fusarium sp. (LT746244.1) 98
daef9 MH550479 Fusarium sp. (AF310976.1) 98
daef10 MH550480 Fusarium acuminatum (HM068320.1) 98
daef11 MH550481 Fusarium lateritium (AF310980.1) 99
daef14 MH550484 Fusarium proliferatum (LT841264.1) 99
daef15 MH550485 Ceratobasidium sp. (DQ520098.1) 97
daef16 MH550486 Ceratobasidium sp. (KC782938.1) 99
daef17 MH550487 Ceratobasidium sp.(DQ097889.1) 96
daef18 MH550488 Ceratobasidium sp.(AF354091.1) 99
daef19 MH550489 Chaetomiaceae sp. (KC007192.1) 99
daef20 MH550490 Chaetomium megalocarpum (KC109744.1) 99
Chaetomium pseudocochliodes (JN209925.1) 98
daef21 MH550491 Penicillium janthinellum (MG938669.1) 98
daef22 MH550492 Penicillium sp. (KX961210.1) 98
Penicillium skrjabinii (EU427287.1) 99
daef23 MH550493 Aspergillus lentulus (KX903293.1) 99
Aspergillus viridinutans (EF661280.1) 99
daef24 MH550494 Talaromyces apiculatus (JN899375.1) 98
daef25 MH550495 Cladosporium cladosporioides (KP701868.1) 99
Cladosporium pseudocladosporioides (KP701943.1) 99
Cladosporium delicatulum (KP701939.1) 98
daef26 MH550496 Bionectria sp. (KF367470.1) 99
daef27 MH550497 Bionectria sp.(KF367477.1) 99
daef28 MH550498 Clonostachys rosea f. (HM751081.1) 99
daef29 MH550499 Clonostachys sp. (KC806284.1) 99
Clonostachys pseudochroleuca (KC806259.1) 99
daef30 MH550500 Mucor racemosus (KJ911228.1) 99
daef31 MH550501 Mucor sp. (KU060772.1) 98
daef32 MH550502 Mucor fragilis (JQ972062.1) 97
daef33 MH550503 Mucor circinelloides f. (JN205987.1) 96
daef34 MH550504 Mucor fragilis (JQ972063.1) 97
daef35 MH550505 Trichoderma hamatum (KM491888.1) 99
daef36 MH550506 Trichoderma asperellum (KF723005.1) 99
Trichoderma koningiopsis (GQ229070.1) 99
daef37 MH550507 Myrothecium roridum (FJ914699.1) 99
Myrothecium sp. (KY086248.1) 99
Myrothecium verrucaria (KM215639.1) 97
daef38 MH550508 Ijuhya corynospora (KY607539.1) 96
daef39 MH550509 Ijuhya vitellina (KY607531.1) 95
daef 40 MH550510 Leptosphaeria sp. (KJ934197.1) 99
daef41 MH550511 Leptosphaeria sp. (AJ317958.1) 99
daef42 MH550512 Leptosphaeria biglobosa (KY221834.1) 99
13

Table 2 (continued) Strain ID Accession no. Closest (Accession no.) Similarity (%)
daef43 MH550513 Phoma exigua var. (EU343130.1) 99

daef44 MH550514 Phoma exigua var. (EU343168.1) 98
daef45 MH550515 Phoma exigua var.(EU343118.1) 98
daef46 MH550516 Helotiales sp. (FN548161.1) 99
Helotiales sp. (MG066445.1) 99
Fungi were grouped into OTUs defined by 97% internal transcribed spacer (ITS) sequence similarity
The statistical table shows the similarity of the rDNA-ITS sequence of endophytic fungi from D. asper-
oides to the closest fungal sequences in GenBank, based on BLAST alignment. The strain ID has the for-
mat: Latin initials of Dipsacus asperoides, the initial letter of the endophytic fungus and the strain number.
The GenBank accession number is also shown, with “Closest (Accession No.)” indicating the most similar
fungus (and its accession number) from GenBank. Similarity (%) is the Ident value obtained by comparing
the sequences between the two strains
Fig. 4 Phylogenetic identification of endophytic fungi from D. asper- select species showing 95–100% homology with the isolated species.
oides. Phylogenetic tree based on neighbor-joining analysis of ITS Closely related species are labeled with taxonomic names, followed
sequences from the 46 strains of endophytic fungi isolated from tap- by the accession number. Significant bootstrap values are indicated at
roots, lateral roots and leaves. ITS sequences obtained were submit- the branching points
ted to the NCBI database, and BLAST searches were performed to
13

cluster with Chaetomium sp. (KC007192.1), Chaetomium pseudocladosporioides (KP701943.1), Cladosporium deli-
pseudocochliodes (JN209925.1) and Chaetomium meg- catulum (KP701939.1) and Cladosporium cladosporioides
allocarpum (KC109744.1). The Talaromyces sp. isolate (KP701868.1) with 100% bootstrap support. The daef 17
formed a cluster with Talaromyces apiculatus (JN899375.1) and 31 could not be represented in the phylogenetic tree
with 100% bootstrap support. The Aspergillus sp. isolate because of low sequence quality. The daef 46 clustered with
formed a cluster with Aspergillus lentulus (KX903293.1) Helotiales sp. (FN548161.1 and MG066445.1) with 99%
and Aspergillus viridinutans (EF661280.1). The Clad- and 100% bootstrap support.
osporium sp. isolate formed a cluster with Cladosporium
Fig. 5 Dipsacus saponin VI level positively correlated with endo- significant difference test post hoc). F Correlation analysis between
phytic fungi in roots of D. asperoides. Dipsacus saponin VI was dipsacus saponin VI level and the number of endophytic fungi in tap-
quantified in taproots, lateral roots and leaves using HPLC. A Chro- roots and lateral roots. Each isolate is represented by a spot (n = 4,
matogram of the standard dipsacus saponin VI. The y-axis indicates R2 = 0.9035, P = 0.0001). G Correlation analysis between dipsacus
the absorbance of dipsacus saponin VI, and the x-axis indicates the saponin VI level and the number of Fusarium sp. in taproots and lat-
measurement time (min). B Chromatogram of dipsacus saponin eral roots. Each isolate was represented as a spot (n = 4, R2 = 0.9122,
VI in taproots. C Chromatogram of dipsacus saponin VI in lateral P = 0.0001). H Correlation analysis between the dipsacus saponin VI
roots. D Chromatogram of dipsacus saponin VI in leaves. E Quan- level and the number of Mucor sp. in taproots and lateral roots. Each
tification of dipsacus saponin VI content in different tissues. Data isolate was represented as a spot (n = 4, R2 = 0.0875, P = 0.0896)
are mean ± SEM (n = 4). ***P < 0.005 (one-way ANOVA and least
13

Positive correlation between dipsacus saponin VI of the fermented fungi was consistent with that of the origi-
level and number of endophytic fungi in roots nal strains. We also amplified ITS regions from mycelium
of the fermented strains and confirmed that the sequences
Dipsacus saponin VI was quantified in taproots, lateral roots were 100% homologous to the regions sequenced from the
and leaves using HPLC (Fig. 5A–D). Levels differed signifi- original strains (Fig. 6B and S2).
cantly in different tissues (P < 0.05). Levels were highest in
taproots (2.98%), lower in lateral roots (0.87%) and below
the detection limit in leaves (Fig. 5E). Level of dipsacus Discussion
saponin VI positively correlated with the total number of
endophytic fungi (Fig. 5F) and with the number of Fusarium This study begins the process of correlating production of
sp. (Fig. 5G), but independent with the number of Mucor sp. perhaps the most relevant bioactive compound from this
in taproots (Fig. 5H). plant, saponins, with the number and diversity of endo-
phytic fungi in different tissues. Our results help clarify
Primary fermentation of endophytic fungi the biodiversity and phylogenetic relationships of endo-
phytic fungi in D. asperoides, which can begin to shed
Selected endophytic fungal isolates were subjected to light on how endophytic fungi can affect the quality of
primary fermentation tests to identify which strains may traditional Chinese medicinal plants.
produce dipsacus saponin VI. Several endophytic fungi The 46 endophytic fungi were isolated from different
enriched in taproots and from different genera were tested: tissues of D. asperoides. This number is slightly lower
daef 11 (Fusarium sp.), 40 and 41 (Leptosphaeria sp.), 15 than what has been reported with other plants, which may
(Ceratobasidium sp.) and 44 (Phoma sp.). Within 10 min at mean that some strains stopped colonizing D. asperoides
60 °C, all these strains produced foam and showed no fading over time, such as due to inhibition by other rapidly grow-
(Fig. 6A). These strains may produce saponins. In addition, ing strains (Gonzaga et al. 2015).
daef 15 produced red pigment, while daef 40, 41 and 44 Nearly all the fungal isolates in our study were colo-
produced green or deep green pigments. nized in taproots, while only two strains were isolated from
To verify that these fermented fungi were identical to lateral roots and four strains were isolated from leaves.
the strains originally isolated and were not contaminated by This suggests that in this medicinal plant, the taproots are
other microorganisms, we confirmed that the microstructure most likely to be colonized. The much greater abundance
Fig. 6 Primary fermenta- A

tion of endophytic fungi from Fermentaon 100h Fermentaon 100h
Dipsacus asperoides. Five
Fusarium sp.
Ceratobasidium sp.
endophytic fungi that were

(daef 11)
enriched in taproots and came

(daef 15)
from different genera were

subjected to primary fermenta-
tion: daef 11 (Fusarium sp.),
20μm 20μm
40 and 41 (Leptosphaeria sp.),
15 (Ceratobasidium sp.) and 44
(Phoma sp.). A Photographs of
Leptosphaeria sp.
Leptosphaeria sp.
the fermentation of five strains

of endophytic fungi and their
(daef 40)
(daef 41)
microscopic morphology. Scale

bar, 20 µm. B Quantification of
synergistic alignment between
ITS sequences of the foam- 20μm 20μm
ing fungus (ITS’) and the ITS
sequences of the original fungal
B
Genetic similarity vs
isolate 150
primary (%)
Phoma sp.
(daef 44)
100
50
20μm
0
11 f 15 f 40 f 41 f 44
ef e e e e
da da da da da
13

of fungi in taproots reflects that fungi can penetrate host they form specific symbiotic relationships; as a result,
plants via the roots, where they gain access to nutrients in different tissues contain different profiles of secondary
xylem and phloem (Martin et al. 2012; Pfurtscheller and metabolites (Jasinska et al. 2018; Jarvis et al. 1985; Liu
Klimesch 1990; Sheng-Liang et al. 2014). The diversity et al. 2006; Wang et al. 2016).
of endophytic fungi in taproots was more higher than that Changing the environmental conditions of endophytes
in other tissues. We guess that these fungi colonized D. can lead them to produce different secondary metabolites
asperoides as spores that moved from the soil to the roots; (Eaton et al. 2010; Wang et al. 2017), increasing their use-
the lateral roots acted simply as transport bridges to carry fulness as bioactive molecule factories. Many active phar-
the fungi to taproots for storage (Courty et al. 2018). In maceutical compounds have been isolated from filtrates of
contrast to our results, diversity of endophytic fungi was Fusarium sp. cultures (Suzuki et al. 2013). The leptosins
greatest in the leaves of Gossypium hirsutum (Li et al. I and J have been isolated from Leptosphaeria mycelium
2014) and Miscanthus × giganteus (Schmidt et al. 2018). (Takahashi et al. 1994). A cyclic lipodepsipeptide has been
Endophytic fungi in plants are primarily Ascomycetes isolated from Phoma sp. (Herath et al. 2009). When we sub-
and their anamorphs, although they can also be Basidi- jected Fusarium sp., Leptosphaeria sp., Ceratobasidium sp.
omycetes, Zygomycetes, and Oomycetes (Soca-Chafre and Phoma sp. to primary fermentation, we found that all
et al. 2011). In D. asperoides, endophytic fungi included them could produce triterpenoid saponin. In addition, our
many rare species, mainly belong to Deuteromycota, that isolates (Cladosporium sp., Phoma sp., Fusarium sp., and
accounted for 48.94% of isolates; Ascomycetes accounted Penicillium sp.) were produced pigments that may be useful
for only 34.04% of isolates. Some Basidiomycetes and Zygo- in the food, cosmetic and pharmaceutical industries. These
mycetes were observed. results with primary fermentation may facilitate the devel-
Fusarium sp. is the predominant microflora in D. asper- opment of strategies to produce natural products from D.
oides. This genus occurs as an endophyte in various cash asperoides (Bick and Rhee 1966; Zheng et al. 2017; Shah
crops, including Solanum lycopersicum (Aime et al. 2013), et al. 2015).
Drepanocarpus lunatus (Liu et al. 2016), and Dioscorea Our results highlight the diversity of endophytic fungi
zingiberensis (Zhang et al. 2009). Ceratobasidium sp. can in medicinal plants and their ability to synthesize bioactive
cause sheath blight and act as a saprotroph in rice (Mos- secondary metabolites (Gupta et al. 2018). They may also
quera-Espinosa et al. 2013), persimmon (Ceresini et al. guide new approaches to synthesize dipsacus saponin VI
2012) and soybean (Salehi et al. 2005). Aspergillus sp. acts from D. asperoides, permitting sustainable development of
as an endophyte of Opuntia dillenii and several other plants this important traditional Chinese medicine resource.
(Li et al. 2009a). Myrothecium sp. acts as an endophyte of
Calophyllum apetalum and Garcinia Morella (Ruma et al. Acknowledgements This research was financially supported by the
Regional Science Fund of the National Natural Science Foundation of
2015). China (81160501; 81860675), the China Agriculture Research System
Several of the endophytic fungi that were identified (CARS-21), the First-class Discipline Construction Project in Guizhou
in D. asperoides can produce bioactive compounds of Province of China [GNYL (2017) 008], and the Chinese Medicine
medicinal interest. Trichoderma sp., Talaromyces sp., Public Health Program of the National Administration of Traditional
Chinese Medicine [Caishe (2014) No. 76]. We thank Dr. Shenghua Wei
Mucor sp. and Penicillium sp. can produce proteases that for supplying the samples, Prof. Tao Zhou for field knowledge about
degrade cellulose (Zhao et al. 2016; Thongekkaew et al. D. asperoides and microbiology, and Jinqiang Zhang for useful com-
2013), dairy products, and polysaccharides (Inoue et al. ments on the manuscript. We also thank Yongqiang Zhou for guidance
2015). Fusarium sp. can produce triterpenoid saponins during experiments.
(Cira et al. 2008; Jiao et al. 2015), which are the main
secondary metabolites of D. asperoides and used to treat Compliance with ethical standards
osteoporosis, reduce lipids and protect against oxidation
Conflict of interest The authors declare no conflicts of interest.
(Wang et al. 2016). Our results suggest that the main loca-
tion of saponin production in D. asperoides is roots. Lev- Open Access This article is distributed under the terms of the Crea-
els of dipsacus saponin VI were higher in taproot than in tive Commons Attribution 4.0 International License (http://creativeco
other tissues, and the taproot have been also the greatest mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu-
number of endophytic fungi. In contrast, dipsacus saponin tion, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
VI levels in leaves were below the limit of detection, per- Creative Commons license, and indicate if changes were made.
haps due to scarcity of interactions between endophytic
fungi and host, reflected in the relatively low Dmn and
IR. Endophytes can prefer different plant tissues, where
13

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13

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Association Between Dipsacus Saponin VI Level and

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World Journal of Microbiology and Biotechnology (2019) 35:42

Association between dipsacus saponin VI level and diversity

Electronic supplementary material The online version of this

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Cladosporium sp. Fusarium sp. Ceratobasidium sp.

Introduction Endophytic fungi can combine with other endogenous

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A Surface Back 100h Surface Back 100h

Leptosphaeria sp. Cladosporium sp.

B Surface Back 100h C Surface Back 100h

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daef43 MH550513 Phoma exigua var. (EU343130.1) 99

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Fig. 6 Primary fermenta- A

endophytic fungi that were

enriched in taproots and came

from different genera were

the fermentation of five strains

microscopic morphology. Scale

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References Herath K, Harris G, Jayasuriya H, Zink D, Smith S, Vicente F, Bills G,

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