World J Microbiol Biotechnol (2011) 27:495–503
DOI 10.1007/s11274-010-0480-x
ORIGINAL PAPER
Molecular identification of endophytic fungi from medicinal plant
Huperzia serrata based on rDNA ITS analysis
Xu Yu Chen • Yao Dong Qi • Jian He Wei •
Zheng Zhang • De Li Wang • Jin Dong Feng •
Bing Chun Gan
Received: 24 August 2009 / Accepted: 11 June 2010 / Published online: 1 July 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Fifty-two endophytic fungi strains with differ-
ent colony morphologies were isolated from stems, leaves
and roots of Huperzia serrata (Thunb. ex Murray) Trevis.
collected from Bawangling Reserve of Hainan Province in
southern China. They were identified mainly based on
rDNA ITS sequences and phylogenetic analysis. The
results showed that all strains belonged to four classes, i.e.
Sordariomycetes (92.31%), Dothideomycetes (3.85%),
Pezizomycetes (1.92%) and Agaricomycetes (1.92%).
Forty-seven strains were identified at the genus level,
including Glomerella (Colletotrichum), Hypocrea (Trich-
oderma), Pleurostoma, Chaetomium, Coniochaeta (Lecy-
thophora), Daldinia, Xylaria, Hypoxylon, Nodulisporium,
Cazia and Phellinus. As to the other five strains, three were
identified at the order level and two at the family level,
indicating that a great diversity of fungi taxa exists in
H. serrata. Most isolated strains belonged to the genus of
Glomerella (Colletotrichum) and Hypoxylon, twenty-one
from Glomerella and its anamorph Colletotrichum (42.3%
Xuyu Chen and Yaodong Qi contributed equally to this work.
X. Y. Chen  J. H. Wei (&)  D. L. Wang 
J. D. Feng  B. C. Gan
Hainan Branch Institute of Medicinal Plant Development,
Chinese Academy of Medical Sciences & Peking Union Medical
College, 571533 Wanning, China
e-mail: jhwei@implad.ac.cn
X. Y. Chen  J. H. Wei  D. L. Wang  J. D. Feng  B. C. Gan
Hainan Provincial Key Laboratory of Resources, Conservation
and Development of Southern Medicine, 571533 Wanning,
China
Y. D. Qi  Z. Zhang
Institute of Medicinal Plant Development, Chinese Academy of
Medical Sciences & Chinese Peking Union Medical College,
100193 Beijing, China
of total isolated strains) and ten from Hypoxylon (19.2% of
total isolated strains). Pleurostoma, Chaetomium, Conio-
chaeta (Lecythophora), Daldinia, Xylaria, Hypoxylon,
Nodulisporium, Cazia and Phellinus were reported as
endophytic fungi isolated from H. serrata for the first time.
Keywords Huperzia serrata  Endophytic fungi 
Identification  Phylogenetic analysis
Introduction
Huperzia serrata (Thunb. ex Murray) Trevis. (Lycopodia-
ceae) grows at the altitude of 300–2,700 m in damp forests
and rock crevices in China (Yang et al. 2003). The whole
plant has long been used in traditional Chinese medicine to
treat various ailments; including contusions, strains,
swellings, haematuria, schizophrenia and myasthenia gra-
vis (Ma et al. 2007). Huperzine A is an alkaloid first dis-
covered in H. serrata. As a powerful and reversible
inhibitor of acetyl-cholinesterase (AChE), Huperzine A has
been proved to be effective in improving the learning and
memory impairment in Alzheimer’s disease (AD) and
vascular dementia (VaD). It also possesses such protective
function as anti-inflammation (Zhang et al. 2008). Because
chemical synthesis of Huperzine A is not available, it can
be obtained only from H. serrata. However, the over-col-
lection of H. serrata leads to its endangerment. Extensive
propagation and cultivation of H. serrata are urgently
needed (Ma et al. 2005, 2006).
Endophytic fungi spend the whole or part of their life-
cycle colonizing inter-and/or intra-cellular inside the heal-
thy tissue of their host plant, typically causing no apparent
symptoms of disease (Li et al. 2008). They stimulate plant
growth and enhance the host survival against fungal
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pathogens (Perrig et al. 2007; Dai et al. 2008; Shipunov et al.
2008). Some endophytic fungi themselves have been found
to have several biological activities (Rukachaisirikul et al.
2007), such as anticancer and antifungal (Geris dos Santos
et al. 2003; Ding et al. 2007; Gangadevi and Muthumary.
2008; Kour et al. 2008; Wu et al. 2009). Now endophytic
fungi are also considered as a promising source of novel
compounds (Rodrigues et al. 2005).
Shi et al. (2005) isolated four endophytic fungi from H.
serrata and identified them as Cephalosporium sp., Plas-
moparad sp., Saccharomyces sp. and Penicillium sp. based
on their morphological characteristics. Gong et al. (2007)
isolated 180 fungi strains from H. serrata in Anhui Prov-
ince and identified and classified them as belonging to 13
genera. Except for Trichoderma and Colletotrichum
belonging to the identified genera in our study, other strains
were designated to different genera. Li et al. (2007) iso-
lated 17 strains, but failed to make the identification. Xu
et al. (2008) isolated 9 strains from south Hunan Province.
Only one strain was identified as Cladosporium sp., while
the others could not be identified because they did not
sporulate. The quantitative difference of endophytic fungi
distributed in H. serrata is probably due to the climatic and
regional distinction. Up to now, identification of endo-
phytic fungi from H. serrata is mainly based on morpho-
logical characteristics.
With the development of molecular biology, rDNA ITS
(Internal Transcribed Spacer) sequences analysis has been
widely used in identifying fungi. Guo et al. (2000) identi-
fied Mycelia sterilia from Livistona chinensis using this
technique. Sette et al. (2006) identified endophytic fungi
from coffee plants at least at the genus level, and the results
were in accordance with the previous morphological
characterization. According to Lin et al. (2007), 48.9% of
the non-sporulating fungi from Camptotheca acuminata
were identified based on rDNA ITS sequences analysis.
Chen et al. (2008) reported that ITS sequences analysis was
especially effective in non-sporulating fungi identification
and also reduced the impact of subjective judgement. In
addition, Youngbae et al. (1997) proved rDNA ITS to be a
valuable source of evidence to resolve phylogenetic rela-
tionships at lower levels, such as among genera or species.
This paper reports the first isolation of endophytic fungi
from H. serrata grown in Hainan Province of China and
their identification by rDNA ITS sequences analysis.
Materials and methods
Plant materials
The healthy plants of H. serrata were collected in August,
2008 from Bawangling Reserve of Hainan Province, China.
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World J Microbiol Biotechnol (2011) 27:495–503
The taxonomical identification of the plant material was
conducted by Dr. Yaodong Qi from the Institute of
Medicinal Plant Development (IMPLAD), Chinese Acad-
emy of Medical Sciences. The voucher specimens
(Zhu2008001) had been deposited in the same
organization.
Isolation and culture of endophytic fungi
The plant surface was sterilized according to Dobranic
et al. (1995) with some modification. The plant was washed
thoroughly in running tap water and then immerged in 70%
ethanol for 3–5 min and 10% sodium hypochlorite for
5 min, and finally rinsed in sterile distilled water for three
times. The stems and roots were cut into 5 mm segments,
and the leaves were cut into 5 mm 9 5 mm segments by a
flame-sterilized cork borer. The segments were placed in a
90 mm-diameter petri dish containing potato dextrose agar
(PDA, containing (g/l): potato 200; dextrose 20; agar 15)
medium with streptomycin sulfate (200 mg/l) to suppress
bacterium contamination. The petri dishes were sealed by
parafilm ‘‘M’’ and incubated in a light chamber at 12 h
light/dark cycles at 28 °C ± 2 °C. After incubation for five
days, new fungal colonies were monitored or picked out
everyday, and this process lasted at least 2 weeks. Indi-
vidual fungal colony was picked from the edge with a
sterile fine tipped needle and transferred onto PDA. After
subculture, these fungi were stored in the National
Medicinal Plant Gene Bank of IMPLAD.
DNA extraction and PCR amplification
After subculture of fungal strains grown on PDA for
5 days, fresh mycelia were inoculated into a 100 ml
Erlenmeyer flask containing 50 ml of liquid potato dex-
trose (PD) medium and cultured in a shaking incubator at
120 rpm/min for 5–10 days in darkness at 28 °C. Mycelia
from each fungus were obtained by vacuum filtration. The
total genomic fungal DNA was extracted by the plant
genomic DNA kit (TIANGEN, BEIJING). Each fungal
DNA was amplified with primers ITS1 (sequence: 50 -
TCCGATGGTGAACCTGCGG-30 ) and ITS4 (sequence:
50 -TCCTCCGCTTATTGATATGC-30 ). The amplification
was performed in a 25 ll reaction volume which contained
100 ng template DNA, 1 ll of 10 pmoL of each primer,
and 12.5 ll of 29 PCR MasterMix (TIANGEN, BEIJING).
The thermal cycling program was as follows: 5 min initial
denaturation at 94 °C, followed by 30 cycles of 1 min
denaturation at 94 °C, 1 min primer annealing at 50 °C,
1 min extension at 72 °C, and a final 7 min extension at
72 °C. A negative control using water instead of template
DNA was included in the amplification process. From each
PCR reaction 5 ll of PCR products were examined by
World J Microbiol Biotechnol (2011) 27:495–503 497

agarose gel electrophoresis in (0.8% w/v) using ethidium
bromide staining. For the identification, the PCR products
were purified using the DNA fragment purification kit and
sequenced using the primer pairs ITS1 and ITS4 on an ABI
3730 XL sequencer. The partial sequence of the 5.8S gene
and the flanking internal transcribed spacer (ITS1 and
ITS2) of the isolated strains were submitted to GenBank
and the accession numbers of sequences are listed in
Table 1.
Phylogenetic analysis
Strain sequences and their similar sequences obtained by
blasting in GenBank are listed in Table 2. The general
placement of these fungi strains was initially identified by
5.8S gene, because this region is highly conserved. All
isolated strains belonged to four classes, Sordariomycetes,
Dothideomycetes, Pezizomycetes and Agaricomycetes. The
ITS rDNA sequences of each class were aligned with
CLUSTAL X, and then by manual adjustment. The maxi-
mum parsimony, as implemented in PAUP* version
4.0b10, was used to identify the placement of each class.
Heuristic searches were performed with 1,000 random
addition sequence replicates and the tree-bisection-
reconnection (TBR) branch swapping and MULTREES
option. Bootstrap analysis was carried out with 1,000
replicates of the heuristic search with TBR branch
swapping, ACCTRAN optimization, and random taxon
addition. MaxTree was set at 1,000.
Results and discussion
Isolation of 52 strains of endophytic fungi
from H. serrata
Fifty-two strains of endophytic fungi were isolated from
stems, leaves and roots of H. serrata based on different
colony morphologies. The fungal quantity was significantly
different at different organs: stems (50%) [ leaves
(42.3%) [ roots (7.7%). Nineteen strains produced the
sexual structure in culture medium, while the others
(63.5%) failed to sporulate.
Identification of 48 strains belonging
to Sordariomycetes
The maximum parsimony analysis of 106 Sordariomycetes
taxa containing 48 isolated strains as well as the related
species from GenBank was performed with 1,000 bootstrap
replications. A 50% majority rule tree with 1,767 steps was
obtained with CI of 0.4550 and RI of 0.8808. The phylo-
genetic tree of Sordariomycetes was made up of six orders,
including Phyllachorales, Hypocreales, Calosphaeriales,
Sordariales, Coniachaetales and Xylariales (Fig. 1).

Table 1 The codes of the isolated strains and their GenBank accession numbers
Strains GenBank Strains GenBank Strains GenBank
accession No. accession No. accession No.

HSS7 GQ334408 HSS16_HSL5 GQ334422 HSL11 GQ334436

HSL7 GQ334409 HSL23 GQ334423 HSS6 GQ334437
HSS8 GQ334410 HSR1 GQ334424 HSS15 GQ334438
HSS10_HSL9_HSL12*1 GQ334411 HSS26 GQ334425 HSS3 GQ334439
HSS12 GQ334412 HSS21_HSL16 GQ334426 HSS19_HSL8 GQ334440
*2
HSS9_HSS25 GQ334413 HSS23 GQ334427 HSL2_HSS2 GQ334441
HSL7_HSS13*3 GQ334414 HSL15 GQ334428 HSS4 GQ334442
HSR4 GQ334415 HSS5 GQ334429 HSS1 GQ334443
HSL14 GQ334416 HSL24 GQ334430 HSL10 GQ334444
HSL20 GQ334417 HSS11 GQ334431 HSL1 GQ334445
HSL4 GQ334418 HSS2 GQ334432 HSL13 GQ334446
HSS22 GQ334419 HSL3 GQ334433 HSR3 GQ334447
HSL18_HSL19_HSL21_HSS17*4 GQ334420 HSS17 GQ334434 HSS14 GQ334448
HSS20 GQ334421 HSR2 GQ334435
*1
HSS10_HSL9_HSL12 represents three strains. Although having different culture characteristics on PDA, they have the same base site, so
only one sequence was submitted to GenBank
*2, *3
HSS9_HSS25 represents two strains
*4
HSL18_HSL19_HSL21_HSS17 represents four strains

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Table 2 The closely related species from GenBank used for phylogenetic analysis
Closely related species GenBank accession No. Closely related species GenBank accession No.

Colletotrichum sp. IP-56 DQ780416 Coniochaeta ligniaria AY198390

Colletotrichum gloeosporioides AB042314 Coniochaeta nepalica DQ093664
Colletotrichum boninense EU822802 Xylaria enteroleuca AM084368
Colletotrichum gloeosporioides DQ454000 Xylaria sp. S23 EU678654
Colletotrichum sp. VegaE2-38 EF672320 Xylaria allantoidea AY909005
Colletotrichum kahawae AM90330 Xylaria sp. P055 EF423534
Colletotrichum gloeosporioides FJ515005 Xylaria longipes AY909016
Colletotrichum crassipes FJ515007 Xylaria polymorpha AB274817
Colletotrichum gloeosporioides EU326190 Xylaria castorea AF163030
Colletotrichum sp. E5T9 FJ480405 Daldinia eschscholzii DQ322087
Colletotrichum sp. IP_42 DQ780413 Daldinia placentiformis AM749921
Colletotrichum dracaenophilum EU003533 Daldinia eschscholzii AB284189
Glomerella cingulata EU196743 Daldinia clavata AM749931
Glomerella cingulata EF423540 Daldinia caldariorum EF026144
Glomerella graminicola EF608060 Hypoxylon sp. SUT041 DQ322115
Glomerella sp. CBMAI 40 DQ123587 Hypoxylon sp. SUT063 DQ322116
Glomerella truncata FJ172231 Hypoxylon sp. S10 EU780697
Trichoderma ovalisporum EU280118 Nodulisporium sp. MF6315 AF201754
Trichoderma gamsii EF488161 Nodulisporium sp. MF5954 AF201753
Trichoderma gamsii EF488141 Nodulisporium sp. VegaE3-62 EF694672
Hypocrea lixii EF488145 Nodulisporium sp. MF6245 AF201756
Hypocrea rufa AJ230685 Guignardia camelliae FJ462743
Calonectria morganii FJ884692 Guignardia sp. AF374356
Xenocylindrocladium guianense AF317349 Guignardia psidii FJ538351
Cylindrocladiumquin queseptatum FJ601696 Phyllosticta sp. MSR_1 AF532314
Cylindrocladium insulare AM945189 Phyllosticta sp. TACP00K4021 AB179771
Cylindrocarpon obtusisporum AM419064 Chromelosporium carneum FJ791160
Leaf litter ascomycete ITS109 AF502687 Cazia flexiascus EU834194
Pleurostoma ootheca AY725469 Kalaharituber pfeilii AF301422
Chaetomium sp. aurim1182 DQ093660 Peziza succosella DQ200841
Chaetomium hispanicum DQ069021 Phellinus noxius EF065632
Chaetomium globosum EU982829 Inonotus pachyphloeus AY558635
Corynascus sepedonium EF550982 Pyrrhoderma adamantinum FJ481040
Corynascus verrucosus sp. nov AJ224203 Mensularia radiata AY624992
Lecythophora sp. AY219880 Fomitiporia punicata FJ613649
Lecythophora luteoviridis DQ404354 Porodaedalea chrysoloma FJ903363
Lecythophora hoffmannii AY805566 Hymenochaete adusta FJ810171
Fuscoporia cinchonensis AY558613

Within the Phyllachorales clade, 21 isolated strains as
well as Glomerella and its anamorph Colletotrichum of
Phyllachoraceae formed a group with a strong bootstrap
support of 100% (Fig. 1). Strains of HSS12, HSS9_HSS25,
HSL7_HSS13, HSL4, HSR4, HSS10_HSL9_HSL12,
HSS22, HSS20, HSL18_HSL19_HSL21_HSS24, HSL20,
HSL14 and G. cingulata, C. crassipes, C. gloeosporioides
and two unknown Colletotrichum species formed a group
with a bootstrap support of 78%. Strain HSS7,
Colletotrichum sp. IP_42 and C. dracaenophilum were
grouped into one subclade with a bootstrap support of 96%.
Strain HSL6 clustered together with three representative
species of Colletotrichum (C. gloeosporioides, C. bonin-
ense, G. graminicola) with a bootstrap support of 100%.
Strain HSS8 as well as Glomerella sp. CBMAI 40 and
Colletotrichum sp. VegaE2_38 formed a group with a
bootstrap support of 88% (Fig. 1). Phylogenetic analysis
indicated that HSS12, HSS9_HSS25, HSL7_HSS13,

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HSS12
MajRule Colletotrichum sp. IP_56 (DQ780418)
Colletotrichum sp. E5T9 (FJ480405)
Colletotrichum crassipes (FJ515007)
Glomerella cingulata (EF423540)
HSS9_HSS25
62 HSL7_HSS13
HSL4
HSR4
78 60 HSS10_HSL9_HSL12
HSS22
Glomerella cingulata (EU196743)
Colletotrichum gloeosporioides(EU326190)
Colletotrichum gloeosporioides(DQ454000)
HSS20 Phyllachorales
Colletotrichum gloeosporioides(FJ515005)
HSL18_HSL19_HSL21_HSS24
HSL20
HSL14
Colletotrichum kahawae (AM903330)
96 99 HSS7
Colletotrichum sp. IP_42 (DQ780413)
Colletotrichum dracaenophilum(EU003533)
53 HSL6
100 100 Colletotrichum gloeosporioides(AB042314)
Colletotrichum boninense(EU822802)
Glomerella graminicola(EF608060)
88 HSS8
52 Glomerella sp. CBMAI 40 (DQ123587)
Colletotrichum sp. VegaE2_38 (EF672320)
Glomerella truncata (FJ172231)
Cylindrocladium insulare (AM945189.1)
96 Cylindrocladium quinqueseptatum (FJ601698 )
Calonectria morganii (FJ884692)
77 Xenocylindrocladium guianense (AF317349)
90 99 HSS16_HSL5
HSL23
Cylindrocarpon obtusisporum (AM419064) Hypocreales
Trichoderma ovalisporum (EU280118)
Hypocrea rufa (AJ230685)
100 Trichoderma gamsii (EF488161)
88 Trichoderma gamsii (EF488141)
HSR1
Hypocrea lixii (EF488145)
HSS26
82 100 HSS21_HSL16
Leaf litter ascomycete strain its109 (AF502687) Calosphaeriales
71 Pleurostoma ootheca (AY725469)
100 HSS23
Chaetomium sp.aurim1182(DQ093660)
99 Chaetomium hispanicum (DQ069021)
Corynascus sepedonium(EF550982) Sordariales
Corynascus verrucosus sp.nov(AJ224203)
55 Chaetomium globosum(EU982829.1)
8098 Coniochaeta ligniaria (AY198390.1)
Lecythophora hoffmannii (AY805566)
100
61 Coniochaeta nepalica (DQ093664.1) Coniochaetales
Lecythophora luteoviridis (DQ404354.1)
Lecythophora sp. (AY219880.1)
HSL15
96 HSS3
93 Daldinia eschscholzii (DQ322087.1)
89 Daldinia eschscholzii (AB284189.1)
90 Daldinia placentiformis(AM749921 )
Daldinia clavata (AM749931.1)
99 Daldinia caldariorum(EF026144)
100 HSL11
HSS6
HSS15
100 HSS1
Xylaria sp. P055( EF423534.1)
69 Xylaria longipes (AY909016.1 )
Xylaria castorea (AF163030 )
100 Xylaria polymorpha (AB274817.1)
HSL10
89 Xylaria enteroleuca (AM084368.1)
Xylaria sp. S23 (EU678654.1)
Xylaria allantoidea (AY909005)
93 6470 HSS2 Xylariales
Hypoxylon sp. SUT041( DQ322115.1)
9769 HSS4
HSS11
99 Hypoxylon sp. SUT063 (DQ322116.1)
HSL3
51 HSR2
86 HSS17
HSS19_HSL8
HSL2_HSS18
99 Hypoxylon rubiginosum (DQ223758)
62 HSS5
HSL24
Nodulisporium sp. MF5954 (AF201753.1)
100 Nodulisporium sp. VegaE3_62 (EF694672.1)
Nodulisporium sp. MF6315 (AF201754.1)
Nodulisporium sp. MF6245(AF201756.1)
Chromelosporium carneum (FJ791160.1)

Fig. 1 The Sordariomycetes phylogenetic tree of the strains isolated length = 1,767, CI = 0.4550, RI = 0.8808). The bootstrap percent-
from H. serrata and the related species based on partial ITS1-5.8S- ages of more than 50% are shown above each branch. Chromelos-
ITS2 sequences by the maximum parsimony analysis (tree porium carneum was used as the outgroup

HSL4, HSR4, HSS10_HSL9_HSL12, HSS22, HSS20,
HSL18_HSL19_HSL21_HSS24, HSL20, HSL14, HSS7,
HSL6 and HSS8 belong to the genus of Glomerella and its
anamorph Colletotrichum. Species of Glomerella (Collet-
otrichum) are most frequently isolated from H. serrata.
Isolation of the same species is also common in other
plants (Pereira et al. 1999; Hata et al. 2002). Colletotri-
chum species are commonly known as foliar pathogens,
and C. gloeosporioides is even considered a worldwide
plant pathogen because it secretes a phytotoxic compound
that causes anthracnose, thus infecting many plant species
(Wang et al. 2008).
Order Hypocreales showed that HSS16_HSL5 and
HSL23 formed a clade with five representative species
(C. insulare, C. quinqueseptatum, C. morganii, C. obtusi-
sporum, X. guianense) of Hypocreales, with a strong
bootstrap support of 99%. HSS16_HSL5 and HSL23 clus-
tered together with three genera, Calonectria, Cylindroc-
ladium and Xenocylindrocladium of Hypocreales. HSR1
and HSS26 clustered together with Hypocrea and its ana-
morph Trichoderma of Hypocreaceae, with a strong boot-
strap support of 100%. Trichoderma species are most
frequently isolated from soils and have been extensively
studied due to their remarkable biocontrol and plant-growth
promoting capacity. In this study, Trichoderma species was
isolated as an endophytic fungus from H. serrata.
Order Calosphaeriales showed that HSS21_HSL16 and
the unidentified leaf litter ascomycete strain ITS109

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formed a clade with a bootstrap value of 100%. Moreover,
HSS21_HSL16 had a higher sequence similarity with the
species Pleurostoma ootheca than any other sequence and
clusters with Pleurostoma ootheca (88% blast similarity
and 82% bootstrap). Therefore, HSS21_HSL16 belongs to
Pleurostoma (Calosphaeriaceae).
Order Sordariales showed that HSS23 and five repre-
sentative species (Chaetomium sp._aurim1182, C. hispan-
icum, C. globosum, C. sepedonium, C. verrucosus sp. nov)
clustered together with a bootstrap support of 99%. Within
this clade, HSS23 and Chaetonium (Chaetonium sp._au-
rim1182, C. hispanicum) clustered together with a boot-
strap support of 100%. The results of the phylogenetic
analysis strongly suggest that HSS23 belongs to the genus
of Chaetonium. Chaetomium sp. is an endophytic fungus
which produces biologically active metabolites. Huang
et al. (2007) selected fungal strain Chaetomium sp. NoS3
from Nerium oleander L. with strong antioxidant activity.
Sun et al. (2006) reported the antifungal activity of
Chaetomium sp. and isolated from it an extracellular b-1,
3-glucanase.
Order Coniochaetales showed that HSL15 clustered
together with Coniochaeta (C. ligniaria, C. nepalica) and
its anamorph Lecythophora (L. luteoviridis, Lecythophora
sp., L. hoffmannii), with a strong bootstrap support of
100%. The results suggest that HSL15 belongs to the genus
Coniochaeta and its anamorph Lecythophora.
Order Xylariales showed that HSS3, HSL11, HSS6 and
HSS15 clustered together with five reference species of
Daldinia (D. eschscholzii, D. caldariorum, D. eschscholzii,
D. placentiformis, D. clavata), with a bootstrap support of
99%. HSS1 and HSL10 clustered together with seven
species of Xylaria (X. enteroleuca, Xylaria sp. S23, X.
allantoidea, Xylaria sp. P055, X. longipes, X. castorea, X.
polymorpha), with a bootstrap value of 100%. Within this
clade, strain HSS1 and Xylaria sp. P055 formed a mono-
phytic clade, with a bootstrap support of 100%. HSS2,
HSS4, HSS11, HSL3, HSR2, HSS17, HSS19, HSL8, HSL2
and HSS18 clustered together with three representative
species (Hypoxylon sp. SUT041, Hypoxylon sp. SUT063,
H. rubiginosum), with a bootstrap support of 86%. HSS5
and HSL24 formed a monophyletic clade with the genus of
Nodulisporium (Nodulisporium sp. MF6315, Nodulispori-
um sp. MF5954, Nodulisporium sp. VegaE3_62, Nodulis-
porium sp. MF6245), with a bootstrap support of 100%.
The results of the phylogenetic analysis strongly suggest
that HSS3, HSL11, HSS6 and HSS15 belong to the genus
of Daldinia, HSS1 and HSL10 belong to Xylaria, HSS2,
HSS4, HSS11, HSL3, HSR2, HSS17, HSS19, HSL8, HSL2
and HSS18 belong to Hypoxylon, and HSS5 and HSL24
belong to Nodulisporium.
Xylaria and Hypoxylon are usually isolated from tropical
hosts. They frequently cause root rot of angiosperms, and
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World J Microbiol Biotechnol (2011) 27:495–503
have a great capacity to degrade cellulose and lignin
(Roger 1979). Xylaria has been proved to be a good source
of bioactive compounds. Wei et al. (1992) screened twelve
Xylaria species and five Hypoxylon species which produce
cellulolytic enzymes. Liu et al. (2008) isolated an endo-
phytic Xylaria. sp from Ginkgo biloba L., which has a
broad-spectrum antimicrobial activity.
Identification of two strains (HSL1 and HSL13)
belonging to Dothideomycetes
The maximum parsimony analysis of the 20 taxa containing
600 sites was performed with 1,000 bootstrap replications. A
strict tree with 880 steps was obtained with CI of 0.7330 and
RI of 0.7978. The Dothideomycetes phylogenetic tree
showed that HSL1 and HSL13 clustered together with five
representative species (Phyllosticta sp. TACP00K4021,
Phyllosticta sp. MSR_1, G. psidii, Guignardia sp., G. ca-
melliae) of Botryosphaeriaceae with a bootstrap support of
100% (Fig. 2). Because HSL1 and HSL13 were closely
related to two genera (Phyllosticta and Guignardia) of Bot-
ryosphaeriaceae, HSL1 and HSL13 are considered to belong
to Botryosphaeriaceae.
Identification of HSR3 belonging to Pezizomycetes
The maximum parsimony analysis of the 14 taxa contain-
ing 613 sites was performed with 1,000 bootstrap replica-
tions. A strict tree with 1,207 steps was obtained with CI of
0.6678 and RI of 0.6498. The Pezizomycetes phylogenetic
tree showed that HSR3, C. flexiascus, C. carneum,
K. pfeilii, and P. succosella of Pezizaceae clustered toge-
ther with a bootstrap value of 100%. Within this clade,
HSR3 and C. flexiascus clustered together with a bootstrap
support of 82% (Fig. 3). Therefore, HSR3 was classified to
the genus of Cazia.
Identification of HSS14 belonging to Agaricomycetes
The maximum parsimony analysis of the 14 taxa contain-
ing 610 sites was performed with 1,000 bootstrap replica-
tions. A strict tree with 1,416 steps was obtained with CI of
0.6949 and RI of 0.6737. The Agaricomycetes phylogenetic
tree showed that HSS14 formed a monophyletic clade with
P. noxius, I. pachyphloeus, P. adamantinum, M. radiata,
F. punicata, P.chrysoloma, H. adusta and F. cinchonensis
of Hymenochaetaceae, with a strong bootstrap of 98%.
Within this clade, HSS14 clustered together with P. noxius,
I. pachyphloeus and P. adamantinum, with a bootstrap
value of 100%. HSS14 formed a top clade with P. noxius
(99% blast similarity and 100% bootstrap) (Fig. 4).
Therefore, HSS14 belongs to the genus of Phellinus.
World J Microbiol Biotechnol (2011) 27:495–503 501

Fig. 2 The Dothideomycetes Strict 90 Neofusicoccum parvum (FJ919389)

phylogenetic tree of strains 89 Dichomera eucalypti (EF591913)
HSL1 and HSL13 isolated from
H. serrata and the related 93 Dothiorella sp. MUCC509 (EF591924)
species based on partial ITS1- 88 Endomelanconiopsis endophytica (FJ799942)
5.8S-ITS2 sequences by the 99 Macrophomina phaseolina (FJ960441)
maximum parimony analysis
(tree length = 880, 99 Botryosphaeria rhodina (FJ941882)
CI = 0.7330, RI = 0.7978). 100 Lasiodiplodia theobromae (FJ882072)
The bootstrap percentages of Sphaeropsis sapinea (AY159255)
more than 50% are shown above 100
HSL1
each branch. Inonotus
pachyphloeus was used as the Phyllosticta sp. TACP00K4021 (AB179771)
outgroup HSL13
100
Phyllosticta sp. MSR_1(AF532314 )
Guignardia psidii (FJ538351 )
Guignardia sp.(AF374356)
Guignardia camelliae (FJ462743)

100 Nodulisporium sp.JP807 (AF280629)

Daldinia eschscholzii (DQ322087)
89 Hypoxylon sp. SUT041( DQ322115)
Xylaria longipes (AY909016)

Inonotus pachyphloeus (AY558635)

Fig. 3 The Pezizomycetes Strict HSR3

82
phylogenetic tree of strain
HSR3 isolated from H. serrata Cazia flexiascus (EU834194.1)
100
and the related species based on
partial ITS1-5.8S-ITS2 Chromelosporium carneum (FJ791160.1)
65
sequences by the maximum 72
parsimony analysis (tree Kalaharituber pfeilii (AF301422.1)
length = 1,207, CI = 0.6678,
RI = 0.6498). The bootstrap Peziza succosella (DQ200841.1)
percentages of more than 50%
Guignardia camelliae (FJ462743.1)
are shown above each branch.
Inonotus pachyphloeus was
Phyllosticta sp. TACP00K4021 (AB179771 )
used as the outgroup 100
Phyllosticta sp. MSR_1(AF532314 )

Guignardia mangiferae (EU273524.1)

Nodulisporium sp._JP807(AF280629.1)
99
100 Daldinia eschscholzii (DQ322087.1)

98 Xylaria longipes(AY909016.1 )

Hypoxylon sp. SUT041( DQ322115.1)

Inonotus pachyphloeus (AY558635.1)

To sum up, to screen out those fungi strains capable of
promoting the active component accumulation of H. ser-
rata and its growth. In this study, 52 strains of endophytic
fungi with different colony morphologies were isolated
from H. serrata and identified by the rDNA ITS sequences
comparison and phylogenetic analysis. All the isolated
strains belong to 4 classes, Sordariomycetes, Dothideo-
mycetes, Pezizomycetes and Agaricomycetes. One strain is
distributed in Agaricomycetes of Basidiomycota, and the
others are distributed in Sordariomycetes, Dothideomycetes
and Pezizomycetes of Ascomycota, including nine orders of
Phyllachorales, Hypocreales, Calosphaeriales, Sordari-
ales, Coniochaetales, Xylariales, Botryosphaeriales, Pez-
izales and Hymenochaetales. Of the 52 strains, 46 were
identified at the genus level, including Glomerella (col-
letotrichum), Hypocrea (Trichoderma), Pleurostoma,

123
502
Fig. 4 The Agaricomycetes
Strict
phylogenetic tree of strain
HSS14 isolated from H. serrata
and the related species based on
ITS1-5.8S-ITS2 partial
sequences by the maximum
parsimony analysis (tree
length = 1,416, CI = 0.6949,
RI = 0.6737). The bootstrap
percentages of more than 50%
are shown above each branch.
Glomus mosseae was used as 98
the outgroup
100
Chaetomium, Coniochaeta (Lecythophora), Daldinia, Xy-
laria, Hypoxylon and Nodulisporium, Cazia and Phellinus,
3 at the order level, and 2 at the family level, indicating that
a great diversity of taxa exists in H. serrata.
This work shows that the majority of fungi isolated from
H. serrata belong to Sordariomycetes (92.3%). A sum of
21 strains belong to the genus of Glomerella and its ana-
morph Colletotrichum, and 10 strains belong to the genus
of Hypoxylon (Fig. 1). Here Pleurostoma, Chaetomium,
Coniochaeta (Lecythophora), Daldinia, Xylaria, Hypoxy-
lon, Nodulisporium, Cazia and Phellinus are reported as
endophytic fungi from H. serrata for the first time.
For the 52 strains of endophytic fungi isolated from
H. serrata, the function of the strains on promoting the
growth and active component accumulation in H. serrata
will be the further work.
Acknowledgments We are grateful to Dr. J. PAN from the State
Key Laboratory of Systematic and Evolutionary Botany, the Institute
of Botany, Chinese Academy of Sciences, and Dr. L.XIE from the
Kunming Institute of Botany, Chinese Academy of Sciences, for their
assistance in sequence data analysis. This research was supported by
the National Key Project of Scientific and Technological Supporting
Programs Funded by the Ministry of Science & Technology of China
(2007BAI27B01), and the Special Fund in Basic Scientific Research
for Non-Profit Research Institutes Financed by the Ministry of
Finance of China (2007HNB003).
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