Diversity of Endophytic Fungi Isolated From Plant Leaves of Deciduous Dipterocarp Forest in Tak Province

Abstract

Endophytic fungi were isolated from the leaves of nine tree species from deciduous dipterocarp forest in Tak province during the wet season of 2007 and the dry season of 2008. The tree species were: Shorea obtusa, Shorea siamensis, Careya sphaerica, Morinda elliptica, Anthocephalus chinensis, Grewia eriocarpa, Terminalia chebula, Fermandoa adenophylla and Erythrina subumbrans Healthy plant leaves were prepared by a surface sterilization technique and placed on potato dextrose agar (PDA). Fungal identification was based on colony and morphological characteristics. The frequency of the fungal endophytes isolated was calculated in order to compare the distribution of fungal diversity. Two hundred and thirty one endophytic fungi were isolated from 340 leave segment samples. The diversity of endophytic fungi in the wet season was greater than in the dry season. Mycelia sterilia sp.2 was the most frequent isolate from the leaves of the different plant host species. Moreover, typical endophytic fungi genera, such as Phomopsis sp., Nigrospora sp., Fusarium sp., Pestalothiopsis sp. and Xylaria sp. were commonly found in this deciduous dipterocarp forest.

Full text

Kasetsart J. (Nat. Sci.) 43 : 182 - 188 (2009)
Diversity of Endophytic Fungi Isolated From Plant Leaves of
Deciduous Dipterocarp Forest in Tak Province
Krittapong Orachaipunlap1*, Sophon Roengsumran2 and Prakitsin Sihanonth3
ABSTRACT
Endophytic fungi were isolated from the leaves of nine tree species from deciduous dipterocarp
forest in Tak province during the wet season of 2007 and the dry season of 2008. The tree species were:
Shorea obtusa, Shorea siamensis, Careya sphaerica, Morinda elliptica, Anthocephalus chinensis, Grewia
eriocarpa, Terminalia chebula, Fermandoa adenophylla and Erythrina subumbrans Healthy plant leaves
were prepared by a surface sterilization technique and placed on potato dextrose agar (PDA). Fungal
identification was based on colony and morphological characteristics. The frequency of the fungal
endophytes isolated was calculated in order to compare the distribution of fungal diversity. Two hundred
and thirty one endophytic fungi were isolated from 340 leave segment samples. The diversity of
endophytic fungi in the wet season was greater than in the dry season. Mycelia sterilia sp.2 was the most
frequent isolate from the leaves of the different plant host species. Moreover, typical endophytic fungi
genera, such as Phomopsis sp., Nigrospora sp., Fusarium sp., Pestalothiopsis sp. and Xylaria sp. were
commonly found in this deciduous dipterocarp forest.
Key words: endophytic fungi, diversity of endphytic fungi, deciduous dipterocarp forest
1Program of Biotechnology, Faculty of science, Chulalongkorn University, Bangkok 10330, Thailand.
2Department of Chemistry, Faculty of science, Chulalongkorn University, Bangkok 10330, Thailand.
3Department of Microbiology, Faculty of science, Chulalongkorn University, Bangkok 10330, Thailand.
*Corresponding author, e-mail: toodpoy@hotmail.com
INTRODUCTION
Recently, the need for new, useful
compounds for curing human diseases has become
ever increasing because of the development and
spread of drug-resistant pathogens. Moreover,
anticancer drugs are also required due to the high
worldwide mortality rate. With the discovery of
the anticancer drug, paclitexel (Taxol), from
Pestalotiopsis microspora, a fungus that colonizes
the Himalayan yew tree Taxus wallichiana (Strobel
et al., 1996), endophytic fungi from plants have
become a new traditional source of bioactive
compounds.
Endophytes are all organisms that live
inside plant tissue for at least part of their life cycle
without causing any disease symptoms in the host
(Petrini, 1991). The symptomless nature of
endophyte occupation in plant tissue has prompted
a focus on symbiotic or mutualistic relationships
between endophytes and their hosts. The
biodiversity observed in endophytes suggests that
they can also be aggressive saprophytes or
opportunistic pathogens (Bacon and White, 2000).
Hawksworth and Rossman (1987)
estimated that there may be as many as one million
different fungal species, yet only about 100,000
have been described. As more evidence

accumulates, estimates of the actual number of
fungal species keep rising. For example, Dreyfuss
and Chapela (1994) estimated that there may be
at least one million species of endophytic fungi
alone. It seems obvious that endophytes are a rich
and reliable source of genetic diversity and novel,
undescribed species.
Currently, the study of bioactive
compounds from endophytic fungi is carried out
in association with various bioactivities, such as
immunosuppressive agents, anticancer agents and
antimicrobial agents. Moreover, study of the
biodiversity of endophytic fungi is important
because it suggests chemical diversity. There is
constant chemical innovation existing in
ecosystems as part of the evolutionary race to
survive (Redell and Gordon, 2002). It is accepted
that endophytes are ubiquitous and are an
important component of fungal diversity, even
though they are host-restricted to the tropics.
Deciduous dipterocarp forest is one such
interesting tropical site. It shows great diversity
and commonly occurs in Southeast Asian
countries, including Thailand. This type of forest
has an open canopy and is composed of small- to
medium-size xeric species. Most of the tree species
have broad leaves that are able to capture fungal
spores.
The main objective of this study was to
examine the diversity of endophytic fungi in a
deciduous dipterocarp forest in Tak province
during the wet and dry season.
MATERIALS AND METHODS
Plant samples collection
During the wet season of 2007 and the
dry season of 2008, mature, healthy plant leaves
were collected by sampling from different parts
of trees (Table 1) growing in a deciduous
dipterocarp forest in Tak province, at location north
16°44' 35.07" and east 98°48' 51.945". All plant
material was placed in clean, sterile polythene bags
and immediately transferred to the laboratory in
an icebox.
Samples were stored at 5°C and isolated
for endophytic fungi within 48 h after collection.
Isolation and identification of endophytic fungi
Leaves were carefully washed with
running tap water and then were cut into small
pieces of approximately 5×5 mm2. The specimens
were surface sterilized using the method described
by Blodett et al. (2000); the samples were
immersed in 95% ethanol for 1 min, 10% sodium
hypochlorite solution for 5 min, 95% ethanol for
Table 1 List of plant species collected for isolation of endophytic fungi.
No. Plant species Collection segment code Average leaf surface
Dry Wet (cm2)
1Shorea obtusa SOD SOW 70
2Shorea siamensis SSD SSW 67
3Careya sphaerica CSD CSW 300
4Morinda elliptica MPD MPW 70
5Anthocephalus chinensis ACD ACW 220
6Grewia eriocarpa GED GEW 80
7Fermandoa adenophylla FAD FAW 4 6
8Terminalia chebula TCD TCW 82
9Erythrina subumbrans ESD ESW 48
The first two letters in the collection segment code denote the host code of the plant species, while the third letter denotes the
season during which the fungus was isolated (W = wet, D = dry).
Kasetsart J. (Nat. Sci.) 43(5) 183

184 Kasetsart J. (Nat. Sci.) 43(5)
30 seconds and then rinsed in sterile water. The
sterile specimens were placed on potato dextrose
agar (PDA) in a Petri dish and incubated at room
temperature. Fungal colony development was
visually inspected daily. Fungal mycelia were
subcultured onto new media until pure cultures
were obtained.
The fungal isolates were examined under
a light microscope and identification to the genus
level was undertaken by referring to the key
described by Barnett and Hunter (1987) and Von
Arx (1978).
The colonization frequency (CF%) of a
single endophytic species was calculated using
Equation 1, (Gond et al., 2007):
CF% = ( Ncol / Nt ) × 100 (1)
where, Ncol = number of segments colonized by
each fungus
Nt = total number of segments studied
The similarity of endophytic fungi
between seasons was calculated and compared
according to the idex described by Jaccard (1901)
(Equation 2):
% similarity = C / (A+B+C) × 100 (2)
where, A = total number of species in season
B=total number of species in season
C=total number of species in both
seasons
RESULTS
A total of 95 isolates belonging to 7
genera and 20 species, and 136 isolates
representing 13 genera and 21 species occurred
as endophytes in the deciduous dipterocarp forest
in Tak province during the dry and wet season,
respectively. It was not possible to collect the
leaves of Erythrina subumbrans in the dry season
because leaf shed had already occurred. The
endophytic fungal colonization frequency in the
wet and dry season was 75.5 and 59.3%
respectively (Figure 1).
Mycelia sterilia sp.2 was the most
commonly found, being present on all plant hosts.
Moreover, typical endophytic fungi genera
observed were: Phomopsis sp., Nigrospora sp.,
Fusarium sp., Pestalothiopsis sp., Xylaria sp.,
Glomellera sp. and Phyllosticta sp., although 38%
percent of those were mycelia sterilia (Table 2).
The diversity of endophytic fungi between the dry
and wet seasons differed. Paecilomyces sp.2,
Phomopsis sp. 8, mycelia sterliria sp.1 and mycelia
sterliria sp.2 were the only isolates found in both
seasons.
Figure 1 Comparison of the total isolates and colonization frequency of endophytic fungi from deciduous
dipterocarp forest in Tak province during the dry and wet season.

Kasetsart J. (Nat. Sci.) 43(5) 185
Table 2 Colonization frequency (CF%) of endophytes from nine different host-tree species in deciduous dipterocarp forest in Tak province.
Endophyte SO SS CS ME AC TC GE FA ES Total
WDWDWDWDWDWDWDWDWDCF%
Collectotrichum sp. - - - - 45 - 10 - ----------55
Glomerella sp. - - -- -------5------5
mycelia sterilia sp.1 - - - - 10 5 30 - - - - - - - - - 25 - 70
mycelia sterilia sp.2 45 - 15 - - - 20 - 20 15 25 - 5 - - - 35 - 180
mycelia sterilia sp.3 - - - - - 5 - 5 - - - - - - - - - - 10
mycelia sterilia sp.4 - - - - - - - - 25 - - - - - 10 - - - 35
mycelia sterilia sp.5 - - - - - - - - 5 - - - - - - - - - 5
mycelia sterilia sp.6 - - - - - - - - - - 15 - - - 10 - - - 25
mycelia sterilia sp.7 - - - - - - - - - - - - - - - - 10 - 10
Nigrospora sp. - - - - 40 - - - 15 - - - 5 - 15 - 20 - 95
Paecilomyces sp.1 - 25 - 25 - 30 - - - 35 - 40 - - - 15 - - 170
Paecilomyces sp.2 35 - - - - 15 - 25 - - - - - 35 - 15 - - 125
Paecilomyces sp.3 - 5 - 30 - 10 - 5 - - - 5 - - - - - - 55
Pestalotiopsis sp. - - ---- ------10-----10
Phomopsis sp.1 --15 ---------------15
Phomopsis sp.2 - - 20 - - - - - - - - - - - - - - - 20
Phomopsis sp.3 - - - - - - - - - - 5 - - - - - - - 5
Phomopsis sp.4 - - - - - - - - - - 5 - - - - - - - 5
Phomopsis sp.5 - - - - - - - - - - - - 10 - 15 - - - 25
Phomopsis sp.6 - - - - - - - - - - - - 10 - - - - - 10
Phomopsis sp.7 - - - - - - - - - - - - 10 - - - - - 10
Phomopsis sp.8 - 5 - - - - - - - - - 5 5 20 - - - - 35
Phyllosticta sp. - - 15 - - - - - 10 - 25 - - - - - - - 50
Xylaria sp.1 - 10 - - - - - - - - - - - - - - - - 10
Xylaria sp.2 - 10 - - - - - - - - - - - - - - - - 10
Xylaria sp.3 - 10 - - - - - - - - - - - - - - - - 10

186 Kasetsart J. (Nat. Sci.) 43(5)
Table 2 (continued).
Endophyte SO SS CS ME AC TC GE FA ES Total
WDWDWDWDWDWDWDWDWDCF%
Xylaria sp.4 - 5 - - - - - - - - - - - - - - - - 5
Xylaria sp.5 - - - 5 - - - - - - - - - - - - - - 5
Xylaria sp.6 - - - 5 - - - - - - - - - - - - - - 5
Xylaria sp.7 - - - 5 - - - - - - - - - - - - - - 5
Xylaria sp.8 - - - - - 15 - - - - - - - - - - - - 15
Xylaria sp.9 - - - - - 5 - - - - - - - - - - - - 5
Xylaria sp.10 - - - - - 10 - - - - - - - - - - - - 10
Xylaria sp.11 - - - - - - - - - - - - - - - 10 - - 10
Xylaria sp.12 - - - - - - - - - - - - - - - 5 - - 5
Xylaria sp.13 - - - - 20 - - - - - - - - - - - - - 20
Xylaria sp.14 - - - - - - - - - - - - - - 15 - - - 15
Total CFU% 80 70 65 70 115 95 60 35 75 50 75 55 55 55 65 45 90 - 1155
Total number of 16 14 13 14 23 19 12 7 15 10 15 11 11 11 13 9 18 - 231
endophytes
Refer to Table 2 for host codes SO = Shorea obtusa, SS = Shorea siamensis, CS = Careya sphaerica, ME = Morinda elliptica, AC = Anthocephalus chinensis, GC = Grewia eriocarpa, FA =
Fermandoa adenophylla, TC = Terminalia chebula, EV = Erythrina subumbrans ( D = dry season ; W = wet season). CFU = Colony forming unit.

Kasetsart J. (Nat. Sci.) 43(5) 187
DISCUSSION
The colonization frequency was used to
compare diversity. There was a higher frequency
of endophytic fungi in the wet season than in the
dry season. Moreover, the distribution of the
endophytic fungi in the wet season was also greater
than in the dry season (Table 2). This result
suggested that moisture content was one of the
main factors that influenced diversity. The rainfall
in the wet season averaged 8.8 mm/day and there
was no rainfall in the dry season. Wet conditions
are favorable to fungal sporulation and increasing
infective fungi (Wilson, 2000).
Two hundred and thirty-one fungal
isolates were obtained and were identified to the
genus level and 40 morphotaxa were classified.
The most frequently isolated genus was
Paecilomyces sp.1 in the dry season and mycelia
sterilia sp.2 in the wet season. The highest fungal
colonization frequency and diversity of taxa was
obtained with Careya sphaerica, which has the
largest leaf area and offers much greater surface
area for inoculum capture (Mekkamol, 1998).
Moreover, coelomycetes, such as
Phomopsis sp., Xylaria sp. and Phyllosticta sp.,
were also found. The diversity of endophytic fungi
isolated in Tak province was lower than that of
endophytic fungi isolated in the Doi Suthep-Pui
area (Lumyong et al., 1998). The similarity index
showed that the endophytic fungi in both seasons
were 10.5% similar. Therefore, endophytic fungal
communities in the deciduous dipterocarp forest
in Tak province were distinctly associated with the
host plants. Factors that might cause this
phenomenon were assumed to include variations
in vegetation type and environmental parameters,
such as temperature, rainfall and humidity.
Xylariaceous fungi and mycelia sterilia
were unable to be identified in this forest type,
probably due to the lack of spore formation in the
media. Fourteen groups of xylariaceous fungi and
eight groups of mycelia sterilia were not
differentiated on potato dextrose agar (PDA),
Sabouraud dextrose agar (SDA) or malt extract
agar (MEA). Therefore, a molecular technique will
need to be used to obtain further identification of
the groups of mycelia sterilia and xylariaceous
fungi, which is expected to be more effective than
the procedures used in the present study.
CONCLUSION
In this study, the leaves from each host
were collected during the wet season of 2007 and
the dry season of 2008 to investigate any seasonal
effects. The number of isolates recovered
depended on environmental factors, such as
rainfall and humidity.
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