Microb Ecol (2011) 61:619–625

DOI 10.1007/s00248-010-9770-y

PLANT MICROBE INTERACTIONS

Species Diversity, Distribution, and Genetic Structure

of Endophytic and Epiphytic Trichoderma Associated
with Banana Roots
Xiaomin Xia & Timothy K. Lie & Xiaoming Qian &
Zhonghui Zheng & Yaojian Huang & Yuemao Shen

Received: 24 June 2010 / Accepted: 28 October 2010 / Published online: 10 November 2010
# Springer Science+Business Media, LLC 2010

Abstract Selective isolation, molecular identification and Introduction

AFLP were used to investigate the distribution of the
various species of endophytic and epiphytic Trichoderma Trichoderma spp. are cosmopolitan soil fungi that grow
associated with banana roots and to compare and contrast rapidly, utilize diverse substrates, and resist noxious
their genetic structure. Three specific groups of Tricho- chemicals [3, 29, 30, 33]. They exhibit excellent antifungal
derma were observed in the roots of banana. Group one, action, and accordingly, some species of Trichoderma have
which made up the largest population, comprised T. been applied in the bio-control of plant diseases. Some of
asperellum, T. virens, and Hypocrea lixii, which were these, which are isolated from the soil of banana plantations
isolated from both inside and on the surface of the banana [19, 36], have proven to be effective against banana wilt
roots, while group two, made up of T. atroviride and T. caused by the soil-borne fungus Fusarium oxysporum f. sp.
koningiopsis, existed on the surface only. Group three, cubense (E.F. Sm.) W.C. Snyder & H.N. Hansen.
comprising only T. brevicompactum was isolated from the Trichoderma spp. are commonly found in soil as
inside of the roots. The AFLP analysis revealed Nei’s saprophytes, but they have also been found as endophytes
diversity indices of 0.15 and 0.26 for epiphytic T. of plants. A few publications have reported that Tricho-
asperellum and T. virens, respectively. The index values derma spp. were found in banana tissues. Early in the year
of 0.11 and 0.11 were obtained for endophytic T. 2000, Pocasangre et al. [21] surveyed the distribution of
asperellum and T. virens, respectively. The genetic diversity banana endophytic fungi in Central America and isolated a
within endophytic T. asperellum and T. virens was lower Trichoderma sp. from the central cylinder. Two years later,
than that within the epiphytes. This suggests that endo- Zum Felde [36] isolated endophytic fungi from four banana
phytic Trichoderma has a higher genetic conservation and plants of Guatemala and found that Fusarium and Tricho-
is compatible with the relatively stable microenvironments derma spp. were most commonly encountered endophytes.
inside roots. Sikora et al. [25] reported that Trichoderma atroviride
Bissett was isolated from the endorhiza of bananas and
used for the bio-control of nematodes. In spite of this, there
are few systematic studies on the species composition and
distribution of endophytic Trichoderma in banana plants
Electronic supplementary material The online version of this article [9, 20, 21, 35].
(doi:10.1007/s00248-010-9770-y) contains supplementary material, In recent years, the ecological distinctiveness of endo-
which is available to authorized users. phytes relative to other nonpathogenic plant-associated fungi
X. Xia : T. K. Lie : X. Qian : Z. Zheng : Y. Huang (*) : Y. Shen such as phyllosphere and epiphytic fungi have become one of
Key Laboratory of the Ministry of Education for Coastal the hot topics of endophyte biology [1]. Relationships
and Wetland Ecosystems, School of Life Science,
between endophytes and epiphytes have important implica-
Xiamen University,
Xiamen 361005, People’s Republic of China tions for fungal biodiversity and plant health [24]. In some
e-mail: yjh@xmu.edu.cn plants, comparative studies of endophytic and epiphytic
620 X. Xia et al.

fungi have been carried out. Legault et al. [16, 17] at 1,000×g for 30 s and 0.2 ml supernatant fluids were
investigated the endophytic and epiphytic fungi in foliage spread on Petri dishes (diameter=9 cm) containing 20 ml
of pines and showed that endophytes had higher host TSM or modified TSB medium. For each medium, five
specificity than epiphytes. Santamaría and Bayman [24] replicates were performed.
found that more morphospecies occurred as endophytic The isolation of endophytic Trichoderma was performed
fungi than as epiphytic fungi of coffee leaves. Based on by the method described by Santamaría and Bayman [24].
these studies, we speculate that the species composition, Ten pieces of the washed banana roots were surface-
distribution, and genetic structure of Trichoderma on the sterilized in 75% EtOH (3 min) and 2.6% NaClO (1 min),
surface and inside of banana roots may be different. The respectively. They were subsequently washed twice in
confirmation of the differences will help us reveal the sterilized distilled water and dried on sterile filter paper,
biodiversity, origin, and evolutionary processes of Tricho- and then five pieces of treated roots each were placed on
derma under different biological niches. the Petri dishes of TSM and modified TSB media. All
To test the speculations above, we investigated the samples were incubated at 25°C for 30 days. After several
species and distribution of endophytic and epiphytic days, fungal growth was observed on the media in the
Trichoderma associated with banana roots (Musa spp.). plates and the colonies were transferred to potato dextrose
The comparison of the genetic structure between endo- agar (PDA) medium.
phytes and epiphytes was carried out using amplified
fragment length polymorphism (AFLP) technique. Morphological Identification of Trichoderma spp.

Cultures were grown on special nutrient agar (SNA), 2%

Materials and Methods malt extract agar (MEA), and PDA at 20±1°C under
ambient daylight conditions [34]. Colony descriptions were
Selective Media for the Isolation of Trichoderma based on observations on MEA under the same light
conditions, unless otherwise specified as PDA. Growth
Root samples of banana were obtained from cultivated plants rates at 20°C, 25°C, 30°C, 35°C, and 40°C were deter-
(2.0–3.0 m in height) in Tianbao–Guotang plantations of mined after 72 h on SNA and PDA using the protocols
Guotang village, Piaoliu plantations of Jishan village, and described by Chaverri et al. [4] and Jaklitsch et al. [11].
Changtai plantations of Qidong village, Zhangzhou City, Conidiophore structure and morphology were described
Fujian Province, China. A total of 80 banana plants were from macronematous conidiophores taken from the edge of
sampled four times, in March, May, July, and October 2007. conidiogenous pustules or fascicles when conidia were
Ten pieces of roots were sampled from each plant and placed maturing, usually after 4–7 days of incubation. Morpho-
in plastic bags to prevent their drying out during transporta- logical identification was carried out using the method
tion. Trichoderma-selective medium (TSM) [7] and a described by Samuels et al. [23].
modified TSB medium (TSB medium: TSM medium
amended with benomyl) [5, 31] were used as selective media PCR Amplification of ITS of rDNA and Translation
for the isolation of Trichoderma. Modified TSB medium was Elongation Factor 1-Alpha (tef1)
composed of: 200 g of potato infusion, 20 g of glucose,
0.25 g of chloramphenicol, 0.15 g of rose bengal (Xin Zhong For DNA extraction, three mycelial plugs (diameter 5 mm)
Chemical Plant, Shanghai, China), 0.15 g of pentachloronitro- of Trichoderma were transferred from PDA to 250 ml
benzene (40% terraclor, Longruida Technology, Wuhan, Erlenmeyer flasks containing 50 ml potato dextrose broth
China), 0.05 g of streptomycin, 0.05 g of Baytan (75% and shaken at 180 rpm. After 3 days of growth at 25°C,
triadimenol, Lvye Agrochemicals, Jiangsu, China), 0.0005 g mycelia were harvested and the genomic DNA was isolated
of benomyl (50% benlate, Agro-Chemical Industry, Shanghai, using a DNA extraction kit (SBS Genetech, Shanghai,
China), and 20 g of agar (per liter of water). China). The PCR amplification of the internal transcribed
space region (ITS1, 5.8 S and ITS2) and a 0.2 kb section of
Isolation of Epiphytic and Endophytic Trichoderma the translation elongation factor (tef1) were performed
according to protocols described by Kullnig-Gradinger et
The isolation of epiphytic Trichoderma was done by a al. [15]. Sequence analysis and species identification were
modified method described by Girlanda et al. [8]. Roots carried out with the aid of the programs TrichOKey 1.0 [6]
were cut into small pieces after removing large adhering and TrichoBLAST [13] available online by the International
soil grains, and then they (4 g) were shaken in 20 ml sterile Subcommission on Trichoderma and Hypocrea Taxonomy.
distilled water for 10 min by a magnetic stirrer. After the The sequences were aligned using ClustalX 8.1 [27].
roots had been picked out, the suspension was centrifuged Phylogenetic analyses were performed using Mega 4 [26].
Endophytic and Epiphytic Trichoderma of Banana Roots 621

Strain TMC1 of Hypocrea orientalis Samuels & O. Petrini AFLP Analysis

as outgroup and the sequences from NCBI databases
reported by Hoyos-Carvajal [10] and Kubicek as reference AFLP markers were developed following the protocol
sequences. Neighbor-joining analyses were done with the described by Vos et al. [28]. DNA digestion was carried
Kimura-2-parameter model [12]. Bootstrap values were out using the restriction enzymes EcoRI and MseI. A total
calculated by 1,000 replicates. of six primer pairs, E-AG/M-CAG, E-AG/M-CAC, E-AG/

Figure 1 Phylogenetic trees showing the position of six species of (▲), FJ436162.1 (■), T. aureoviride AF194007.1 (■), AF401002.1 (■),
Trichoderma isolated from banana roots as inferred by NJ analysis of T. virens EU280090.1 (▲), T. brevicompactum EU280088.1 (▲), and T.
a ITS and b tef1. The reference ITS and tef1 sequences were obtained orientalis GQ426040.1 and EU401609.1. ▲ represents sequences
from GenBank (NCBI) and their accession numbers were T. asperellum submitted by Hoyos-Carvajal and Bissett. ■ represents sequences
EU280132.1 (▲), T. koningiopsis EU280141.1 (▲), FJ436121.1 (■), submitted by Kubicek
T. atroviride EU280133.1 (▲), AF400997.1 ■, H. lixii EU280078.1
622 X. Xia et al.

M-CTG, E-AT/M-CAG, E-AT/M-CAC, and E-AT/M-CTG, analyses, six species including T. asperellum, T. virens,
were screened during pre-experiment of AFLP. In the six Trichoderma brevicompactum G.F. Kraus, C.P. Kubicek, &
primer pairs, the primer E-AG/M-CTG produced the most W. Gams, T. atroviride, T. koningiopsis Samuels, C. Suárez,
polymorphic bands and 76 reliable bands were observed. It & H.C. Evans, and Hypocrea lixii Pat. (teleomorph of
enabled us to distinguish endophytic and epiphytic Tricho- Trichoderma harzianum Rifai) were identified (Fig. 1;
derma in AFLP analysis. Thus, this pair of primer was used Online Supplemental Table S1).
to studies the genetic diversity and structures of Tricho- Among the six species, ITS provided an unambiguous
derma. DNA fragments generated from selective amplifi- identification of T. asperellum, T. brevicompactum and T.
cations were separated on denaturing 6% polyacrylamide virens (Fig. 1a). However, phylogenetic analyses of ITS
gels containing 7.5 mol urea. The AFLP bands were scored showed that T. atroviride and T. koningiopsis formed an
for presence (1) or absence (0) by Cross linker [2]. Nei’s independent clade, which could not be separated using ITS
gene diversity index and Shannon information index was sequences. Moreover, there was some difficulty in distin-
estimated using Popgene 3.2 [32]. Phylogenic trees and guishing H. lixii from Trichoderma aureoviride Rifai using
genetic identity were analyzed using unweighted pair match ITS sequences. So H. lixii, T. aureoviride, T. atroviride, and
group analysis clustering methods performed using NTSYS T. koningiopsis were identified according to the tef1
[22]. As Trichoderma asperellum Samuels, Lieckfeldt, & sequences. The ITS clade of T. koningiopsis and T.
Nirenberg and Trichoderma virens (J.H. Mill., Giddens, & atroviride was separated into two independent clades, and
A.A. Foster) Arx are predominant species in banana roots, D19, D7, D2, D1, D22, A6, and B1 were identified as H.
we chose them to compare the genetic differences between lixii (Fig. 1b). Morphological characters were also used to
epiphytes and endophytes. supplement molecular identification. For example, the
growth rates at 30°C and 35°C in the PDA and SNA media
Nucleotide Sequence Accession Numbers were used to separate T. atroviride and T. koningiopsis
where the pigmentation of the medium separated T.
The ITS and tef1 sequences for the 72 isolations of aureoviride and H. lixii.
Trichoderma were deposited in GenBank and the sequence T. asperellum and T. virens were the dominant species of
accession numbers are from FJ610259 to FJ610307 (except Trichoderma associated with banana roots (61.1% and
FJ610291, FJ610292, FJ610286, FJ610307), FJ908713 to 18.1% of the total number of isolates, respectively).
FJ908725, FJ908727 to FJ908752 (except FJ908715, However, the proportion of H. lixii, T. brevicompactum, T.
FJ908718, FJ908723, FJ908738), FJ973431 to FJ973459, atroviride, and T. koningiopsis was relatively low with only
FJ998176 to FJ998183, and GQ131374 to GQ131399 and 8.4%, 5.6%, 4.2%, and 2.8%, respectively.
FJ969845 (Supplemental Table S1).
Distribution of Epiphytes and Endophytes

Results Four species of Trichoderma were found inside plant tissue:

T. asperellum, T. virens, T. brevicompactum, and H. lixii
Identification and Distribution of Trichoderma spp. (60.7%, 10.7%, 14.3%, and 14.3% of the total number of
endophytic isolates, respectively). Five species were isolated
One hundred and eighty-nine endophytic and epiphytic as epiphytes: T. asperellum, T. virens, T. koningiopsis, H.
Trichoderma were isolated from banana roots. Seventy-two lixii, and T. atroviride (61.3%, 22.7%, 4.5%, 4.5%, and
strains including 29 endophytes and 43 epiphytes, repre- 6.8% of the total number of epiphytic isolates, respectively).
senting the isolates collected at different times and sites, Three species, T. asperellum, H. lixii, and T. virens, were
were selected for taxonomic studies. Based on molecular found in both niches.

Table 1 Genetic diversity of epiphytic and endophytic T. asperellum and T. virens isolated from banana roots

Population No. of isolates Na Ne H I

T. asperellum Endophytes 12 1.28 1.19 0.11 0.15

Epiphytes 18 1.57 1.25 0.15 0.23
T. virens Endophytes 3 1.32 1.19 0.11 0.17
Epiphytes 10 1.84 1.43 0.26 0.40

Na observed number of alleles, Ne effective number of alleles, H Nei’s diversity index, I Shannon information index
Endophytic and Epiphytic Trichoderma of Banana Roots 623

Figure 2 Genetic similarity

among T. virens population
based on AFLP analysis
(endophytes in frame)

Genetic Diversity of Epiphytes and Endophytes endophytes that grouped into an independent cluster that
was large and separate from the epiphytes’ cluster (Fig. 3).
Nei’s diversity index (H) and Shannon information index
(I) of epiphytic T. asperellum and T. virens were higher than
those of the endophytic T. asperellum and T. virens Discussion
(Table 1). Within endophytic T. asperellum, the genetic
identify values based on the pairwise comparison of Trichoderma is a fungus with worldwide distribution. The
populations ranged from 0.91 to 1 and T. virens 0.96 to 1, results of this study showed that there were six Tricho-
while the values within epiphytic T. asperellum and T. derma species living in and on banana roots in Zhangzhou,
virens ranged from 0.75 to 1, and 0.40 to 1, respectively Fujian Province, China. To the best of our knowledge, this
(Online Supplemental Table S2, S3). is the first systematic investigation in which endophytic
Dendrograms generated with AFLP markers for T. Trichoderma were isolated from banana roots. Besides, this
asperellum and T. virens grouped epiphytic and endophytic study discovered four endophytic species, T. asperellum, T.
isolates into different clusters. Among T. virens isolates, C6, brevicompactum, T. virens, and H. lixii, inside banana roots.
C9, and C11 were endophytes which formed an independent These species were not reported as endophytes of banana
cluster separated from the epiphytic T. virens cluster (Fig. 2). by previous authors [9, 20, 21, 35].
The analysis of T. asperellum showed similar results. Out of Endophytic and epiphytic isolates of Trichoderma from
the total of 30 T. asperellum isolates analyzed, 12 were banana roots differed significantly in number and species.

Figure 3 Genetic similarity among T. asperellum population based on AFLP analysis (endophytes in frame)
624 X. Xia et al.

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Natural Sciences Foundation of China (30500632), National High biological control organisms for suppressing fusarium wilt of
Technology Research and Development Program of China banana. Plant Pathol 55:217–223
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