Botanical Studies (2008) 49: 39-43.

*

Corresponding author: E-mail: phwang@thu.edu.tw; Tel:

+886-4-23592026; Fax: +886-4-23590296.

INTRODUCTION

The term "endophyte" was introduced by De Bary

(1866) and was initially applied to any organism found

within a plant that causes asymptomatic infections entirely

within plant tissues but no symptoms of disease (Wilson,

1995). Endophytic fungi have been examined in conifers

(Petrini et al., 1992), including Pinus spp. (Sieber et al.,

1999), Taxus spp. (Fisher and Petrini, 1987), and Juniperus

spp. (Petrini and Muller, 1979; Petrini and Carroll, 1981),

and they are presumed to be ubiquitous.

Endophytic fungi have been described as playing a

protective role against insect herbivory not only in grasses

(Clay, 1990) but also in conifers (Carroll, 1991). Taxus

mairei is important due to its production of taxol and the

production of taxol by at least some of its endophytes.

Wang et al. (2000) screened 45 endophytic fungi

isolated from the inner bark of T. mairei and found that

Tubercularia sp. strain TF5 produced taxol. In another

study that examined hundreds of endophytic fungi from

T. mairei, Cephalataxus fortunei, and Torreya grandis

(Wang et al., 2000), the cytotoxin brefeldin A was found

to be produced by Paecilomyces sp. and Aspergillus

clavatus. Caruso et al. (2000) screened 150 fungal strains

isolated from Taxus baccata and Taxus brevifolia, 15

strains produced taxanes. Huang et al. (2001) screened

172 endophyte isolates from T. mairei, C. fortunei, and T.

grandis to analyze the antitumor and antifungal activities.

Most previous reports on the endophytic fungi of

Taxus mairei focus on their ability to produce important

anticancer agents, such as taxol. In the present study, we

investigate the diversity of the endophytic fungi in T.

mairei from Taiwan.

MATERIALS AND METHODS

Plant materials and fungal isolates

Thirteen symptomless leaf samples were randomly

collected from nine T. mairei trees from Fu-Shan Nature

Reserve, Ilan (24¢X34¡¦ N, 121¢X34¡¦ E, 750 m elevation),

Taiwan. Samples were collected and processed within 24 h

after collection.

The method of sterilization was modified from Guo et

al. (2001). The leaves were surface-sterilized by soaking

for 3 min in a solution of 1% sodium hypochlorite

and then for 30 s in sterile water. Specimens were cut

aseptically into 5-mm-long segments, blotted dry on

sterile paper towels, and placed onto 2% water agar.

Cultures were incubated at room temperature (20-24¢XC)

and microscopically observed daily for the emergence of

fungal mycelium and to check whether hyphae grew from

the inner tissue, not on the outside. After 1-3 days, the

inner hyphal tips were removed and transferred to potato

dextrose agar. Initial identification of these isolates was

achieved based on conidial and colony morphology by

some identification keys. Different morphological isolates

were identified, grouped, and then randomly selected for

DNA analyses.

Endophytic fungi from Taxus mairei in Taiwan: first report

of Colletotrichum gloeosporioides as an endophyte of

Taxus mairei

Yen-Ting WANG

1

, Hui-Shan LO

2

, and Pi-Han WANG

3,

*

1

Department of Medical Research and Education, Cheng Hsin Rehabilitation Medical Center, Taipei 112, Taiwan, ROC

2

Department of Microbiology, Soochow University, Taipei 111, Taiwan, ROC

3

Center for Tropical Ecology and Biodiversity, Department of Life Science, Tunghai University, Taichung 407, Taiwan,

ROC

(Received June 7, 2006; Accepted August 30, 2007)

ABSTRACT.

Forty endophytic fungal isolates were obtained from symptomless leaf samples of nine Taxus

mairei trees on Fu-Shan, Taiwan. Identification was based on morphological characters and comparison of

rDNA ITS sequences to those in GenBank. Colletotrichum and Fusarium were the endophytic fungi most

frequently isolated from leaves of T. mairei. Colletotrichum gloeosporioides was newly recorded as an

endophyte of T. mairei.

Keywords: Colletotrichum gloeosporioides; Endophytes; Fusarium; Taxus mairei.

MICROBIOLOgy

40

Botanical Studies, Vol. 49, 2008

DNA extraction, PCR, and sequencing

DNA from mycelium was extracted by the CTAB

method (Doyle and Doyle, 1990). The PCR amplification

of the rDNA ITS region was undertaken using the

universal ITS5 and ITS4 primers (White et al., 1990). PCR

products were purified using minicolumns (Wizard PCR

Preps DNA purification System, Promega) according to

the manufacturer¡¦s protocol and directly sequenced in ABI

PRISM 3100 Genetic Analyzer (PE Applied Biosystems,

Foster City, CA, USA). Primers ITS5 and ITS4 were

used in the bi-directional sequencing reaction, which was

repeated at least thrice to confirm the sequence data. Using

BLAST, we searched GenBank for sequences that were

similar to those isolated in our study. The most similar

reference sequences were recorded.

RESULTS

Leaf samples of the nine T. mairei trees yielded 40

endophytic fungal isolates. They did not produce sexual

reproductive structures in cultures. Based on a microscopic

observation of mycelia and conidia, these endophytes were

identified as Colletotrichum gloeosporioides, Fusarium

solani, Aspergillus nidulan, and several isolates identified

by genus, including Colletotrichum sp., Phanerochaete sp.

and Rhizoctonia sp. Besides these, three species had sterile

mycelia only.

The rDNA internal transcribed spacer (ITS) region

sequences are now used in the identification and detection

of fungi (Pryor and Gilbertson, 2000; Anderson et al.,

2001; Guo et al., 2001). ITS sequences of about 600

bp were obtained for 17 isolates representing nine taxa

identified by morphological characters. Using the program

FASTA, the GenBank and EMBL databases were screened

for ITS sequences of fungal taxa that closely matched

ours. Eleven reference sequences of nine fungal species

were obtained (Table 1).

In Fu-Shan Nature Reserve, nine species of

endophytic fungi in five genera and three unidentified

fungal endophytes were isolated from nine T. mairei

trees (Table 1). The most frequently isolated genera

were Colletotrichum and Fusarium. Colletotrichum

gloeosporioides was isolated from leaf samples of seven

trees, and thus the isolation frequency was 77.8%. The

isolation frequency of the Fusarium solani was 66.7%.

Colletotrichum gloeosporioides a nd a Fusarium

solani were both present in five leaf samples of T. mairei

collected from Fu-Shan. In fact, one sample had four

endophytes species, Colletotrichum sp., Fusarium sp.,

and two unidentified endophytes, sp.1 and sp.3, which

demonstrates the diversity of fungal endophytes in T.

mairei leaves.

Tm1-5 and Tm6-3-2 were Fusarium solani and Tm-6

was Aspergillus nidulans. They all showed at least a 98%

similarity to reference sequences (Table 1). Tm1-1 and

Tm1-4 were Phanerochaete sordida, and Rhizoctonia

solani, respectively. They showed a 97% similarity to

reference sequences (Table 1).

Endophyte sp.1 Tm4-6 showed a 96% similarity to the

reference EF4200019 fungal endophyte. Endophyte sp.2

(Tm3-5 and Tm8-1) and sp.3 were Basidiomycetes. They

were identical at the ITS region and showed at least a 98%

similarity to reference sequences.

DISCUSSION

We surveyed the endophytic fungi diversity of T.

mairei leaves. We used conidia and mycelia morphology

Table 1. Fungal endophytic isolates recovered from 9 Taxus mairei on Fu-Shan, Taiwan. GenBank Accession numbers of the rDNA

ITS sequence of isolates used in this study and the similarity with their most closely related fungal ITS sequences in GenBank.

Endophytic isolates in this study

GenBank sequences

Endophyte taxa

Isolate no. Tree no. Isolate code Accession no. Blast result

Accession no. Similarity

Colletotrichum sp.

1

1 Tm3-2 AY452985 Colletotrichum sp.

AJ301939 100%

Colletotrichum sp.

1

1 Tm6-1 AY423480 C. gloeosporioides

AJ301974 98%

C. gloeosporioides

10

6 Tm4-1 AY423474 C. gloeosporioides

AJ301907 99%

C. gloeosporioides

6

4 Tm5-1

1

AY423475 C. gloeosporioides

AJ301986 100%

Fusarium solani

9

6 Tm1-5

2

AY433806 Fusarium sp.

AM412637 99%

Aspergillus nidulans

3

1 Tm6 AY452983 Aspergillus nidulans AF455505 98%

Phanerochaete sp.

1

1 Tm1-1 AY433811 Phanerochaete sordida AB210078 97%

Rhizoctonia solani

1

1 Tm1-4 AY433813 Rhizoctonia solani

AF153780 97%

Fungal endophyte sp.1 3

3 Tm4-6 AY433808 Fungal endophyte

EF420019 96%

Fungal endophyte sp.2 3

1 Tm3-5

3

AY456192 Aphyllophorales

EF060457 98%

Fungal endophyte sp.3 2

2 Tm3-3 AY433812 Basidiomycete

AY730555 99%

1

Represented 5 isolates, rDNA ITS sequences AY423475 to AY423479.

2

Represented isolate Tm6-3-2, AY433805.

3

Represented isolate Tm8-1, AY433810.

WANG et al. ¡X Endophytic fungi in

Taxus mairei

41

to identify the endophytes, and then confirmed the

identification by ITS rDNA sequences analysis. Because

many fungi of the class Ascomycetes and Basidiomycetes

do not produce sexual structures in artificial media,

it is difficult to identify these fungi using traditional

microscopic methods only.

We limited the present investigation to culturable fungi.

In other words, we neglected obligate biotrophs. The

biodiversity could thus be underestimated.

Colletotrichum species were the endophytes most

frequently isolated from T. mairei in Fu-Shan, and these

have not yet been reported as endophytes of Taxus though

they have been reported as common endophytes from

other plants (Frohlich et al., 2000; Larran et al., 2001;

Photita et al., 2001; Cannon and Simmons, 2002; Arnold

et al., 2003).

It is interesting that anthracnose caused by C .

gloeosporioides on T. mairei has been reported in Taiwan

(Fu et al., 2003). This pathogen has appeared on cuttings

and seedlings in nurseries and on larger plants grown in

plantations. The vigor of both the host and the fungus

depends on temperature. Higher temperature places stress

on the host plants. Under lower temperature, the host

plants are more vigorous and less susceptible to attack.

Symptoms appeared within 7 days when the temperature

was over 32¢XC, and symptoms were delayed when it was

below 24¢XC. The average yearly air temperature of Fu-

Shan Nature Reserve is 18.3¢XC (Taiwan Forestry Research

Institute Website, http://www.tfri.gov.tw/tfe/2fnew/

10.htm). Thus, perhaps due to temperature limitation, C.

gloeosporioides infects T. mairei as a common endophyte

without disease expression in Fu-Shan. However, this may

merely indicate that some isolates are well adapted for an

endophytic mode of life and might be potential pathogens.

The ecological roles of endophytes are diverse and

varied. Colletotrichum gloeosporioides is a worldwide

plant pathogen that infects many plant species (Brown et

al., 1998; Jeger and Bailey, 1992). Colletotrichum musae

and C. gloeosporioides have been found as endophytes

in banana, but these fungi also cause anthracnose of

banana fruits (Photita et al., 2001), supporting the idea that

pathogens may spend part of their lives in an endophytic

stage (Brown et al., 1998).

Xylariaceous fungi and Alternaria were reported as

the most commonly isolated endophytes. Other genera

commonly isolated include Idriella, Phoma, Phomopsis,

and Phyllosticta (Rodrigues and Petrini, 1997; Caruso et

al., 2000). In this study, Colletotrichum species were the

most common endophyte in T. mairei, but none of the

other fungi mentioned above were found.

Fusarium solani endophytic isolates were taken

from six trees. ITS sequence comparisons revealed their

identities to be: Cycas taiwaniana, Pinus morrisonicola,

Pinus taiwanensis, Pseudotsuga wilsoniana, Keteleeria

davidiana, Taiwania cryptomerioides, Chamaecyparis

formosensis, Taxus baccata, Taxus floridana, and

Calocedrus formosana from Fu-Shan (data not shown).

Thus, Fusarium solani was the second most frequently

isolated species in our samples.

In previous studies, the genus Fusarium is a frequent

endophyte in other plants (Bills and Polishook, 1992;

Suryanaryanan and Kumaresan, 2000; Photita et al., 2001;

Cao et al., 2002; Larran et al., 2002). However, these

studies generally do not supply the species name. GenBank

contains the ITS sequences of only two Fusarium

endophytes, Fusarium arthrosporioides AY575714 and

Fusarium oxysporum strain F35 AY555719, and they

differ from our ITS sequence of Fusarium solani. Our ITS

sequence showed 99% similarity (533 bp/536 bp) to the

reference sequence AM412637, Fusarium solani (Azor et

al., 2007).

The other endophytic species Aspergillus nidulans

and Rhizoctonia solani have been described in previous

studies (Bills and Polishook, 1992; Rodrigues and Petrini,

1997; Suryanaryanan and Kumaresan, 2000; Photita et al.,

2001). The results obtained in this work are in agreement

with Wang et al. (2002), who reported Aspergillus sp. as

a common endophytic fungus in their investigation of T.

mairei.

Phanerochaete and Rhizoctonia are both common

saprobic fungi. Phanerochaete causes white rot of conifer

logs and stumps. When the host is weakened, it is the

dominant fungus of the decaying process (Eriksson et al.,

1990). Petrini (1991) mentioned the symptomless tissues

may be in ecological species equilibrium, and when

senescence process starts, this tissue gradually allows the

establishment of new, mainly saprobic fungal species.

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