J Gen Plant Pathol (2015) 81:201210

DOI 10.1007/s10327-015-0587-x

FUNGAL DISEASES

Characterization of Trichoderma species isolated in Ecuador

and their antagonistic activities against phytopathogenic fungi
from Ecuador and Japan
Luis Galarza1 Yasunori Akagi2 Kazumi Takao1 Chang Sun Kim3

Nitaro Maekawa3 Akihiro Itai2 Esther Peralta4 Efren Santos4

Motoichiro Kodama1

Received: 22 September 2014 / Accepted: 9 December 2014 / Published online: 24 March 2015
The Phytopathological Society of Japan and Springer Japan 2015

Abstract Native Trichoderma spp. were isolated from here are potential biocontrol agents against important
agricultural fields in several regions of Ecuador. These pathogens of banana and cacao in Ecuador.
isolates were characterized via morphological observation
as well as molecular phylogenetic analysis based on DNA Keywords Trichoderma spp.  Biocontrol  Fusarium
sequences of the rDNA internal transcribed spacer region, oxysporum f. sp. cubense  Mycosphaerella fijiensis 
elongation factor-1a gene and RNA polymerase subunit II Moniliophthora roreri  Moniliophthora perniciosa
gene. Fifteen native Trichoderma spp. were identified as T.
harzianum, T. asperellum, T. virens and T. reesei. Some of
these strains showed strong antagonistic activities against Introduction
several important pathogens in Ecuador, such as Fusarium
oxysporum f. sp. cubense (Panama disease) and My- Trichoderma, a cosmopolitan soil-borne fungus that inter-
cosphaerella fijiensis (black Sigatoka) on banana, as well acts with root systems, soil and foliar environment (Hjel-
as Moniliophthora roreri (frosty pod rot) and Monilioph- jord and Tronsmo 1998), is an important biological agent
thora perniciosa (witches broom disease) on cacao. The for controlling plant pathogens. Trichoderma spp. have
isolates also showed inhibitory effects on in vitro colony been reported to control several phytopathogens of diverse
growth tests against Japanese isolates of Fusarium oxys- crops by various mechanisms such as the production of
porum f. sp. lycopersici, Alternaria alternata and Roselli- antifungal metabolites, competition for nutrients and space,
nia necatrix. The native Trichoderma strains characterized mycoparasitism and promoting defense mechanisms
(Hoyos-Carvajal et al. 2008; Woo and Lorito 2007). The
Electronic supplementary material The online version of this mycoparasitism of Trichoderma strains is characterized by
article (doi:10.1007/s10327-015-0587-x) contains supplementary hyphal coiling around host hyphae, haustoria and
material, which is available to authorized users. penetration into host cell walls (Abdullah et al. 2007).
& Motoichiro Kodama
Knowledge of the Trichoderma taxa is important both for
mk@muses.tottori-u.ac.jp control efficiency and environmental conservation when
Trichoderma is introduced as a biocontrol agent into the
1
The United Graduate School of Agricultural Sciences, Tottori rhizosphere of a given ecosystem.
University, 4-101 Koyama-Minami, Tottori 680-8553, Japan
To identify and characterize Trichoderma spp., mor-
2
Faculty of Agriculture, Tottori University, 4-101 Koyama- phological characteristics should be considered along with
Minami, Tottori 680-8553, Japan
molecular data from DNA sequencing (Samuels 2006). In
3
Fungus/Mushroom Resource and Research Center, Faculty of addition, a multigene approach using at least two unlinked
Agriculture, Tottori University, 4-101 Koyama-Minami,
loci is desirable for the molecular identification of closely
Tottori 680-8553, Japan
4
related Trichoderma spp. The internal transcribed spacer
Biotechnology Research Center of Ecuador, Higher
(ITS) region of the ribosomal DNA (rDNA) is one of the
Polytechnic College of the Littoral CIBE-ESPOL, Campus
Gustavo Galindo, Km 30, 5 Perimetral Road, Guayaquil, most reliable targets for identifying a strain at the species
Ecuador level (Kullnig-Gradinger et al. 2002). Genes EF-1a and

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RPB2, which encode the translation elongation factor 1-a wilt), which is caused by Fusarium oxysporum f. sp.
and the second largest subunit of RNA polymerase II, re- cubense (Ploetz 2006; Stover 1962). Cacao (Theobroma
spectively, are more variable and can be used to reflect cacao) is grown in many tropical environments (Wood and
differences within and between groups of closely related Lass 2001) and is also an economically important crop in
species (Liu and Hall 2004; Matheny 2005; Zhang et al. Ecuador and other countries. The most prominent diseases
2005). Combined use of these three genes enables the of cultivated cacao include Moniliophthora roreri, which
identification of most Trichoderma spp. at the species level causes frosty pod rot (Bowers el al. 2001; Wood and Lass
(Samuels 2006). TrichOKEY and TrichoBLAST (www. 2001), and Mo. perniciosa, which causes witches broom
isth.info) are convenient online tools for the molecular disease (Aime and Phillips-Mora 2005). These pathogens
identification of Trichoderma strains and are based on se- can cause complete yield loss. Therefore, the control of
quence comparisons of these genes (Druzhinina et al. these diseases is the most important agricultural issue in
2005; Kopchinskiy et al. 2005). However, molecular many countries of South America, Southeast Asia and
identification has also shown a high level of heterogeneity Africa.
at the species level in, e.g., T. harzianum (Chaverri et al. However, chemical controls for these diseases are un-
2003; Hermosa et al. 2000; Samuels et al. 1999). desirable in these areas for economical and environmental
Many Trichoderma species have been used for the reasons. A biocontrol method would be a preferable al-
biological control of a wide range of foliage diseases ternative for controlling these banana and cacao diseases.
(Nelson 1991). The primary species used as biocontrol The search for potential biocontrol agents against
agents are T. harzianum (Sharma et al. 2009), T. viride banana and cacao diseases in Ecuador led to the consid-
(Karthikenyan et al. 2006), T. hamatum (Harman et al. eration of Trichoderma species, which are native to various
1981), T. atroviride (Hjeljord and Tronsmo 2003), T. regions in Ecuador. The use of endogenous and domestic
asperellum (Watanabe et al. 2005) and T. virens microorganisms as biocontrol agents is the most important
(Mukherjee et al. 2006). The control efficiency for each factor in biosafety, environmental conservation and sus-
disease differs between Trichoderma strains and depends tainability in this scenario. The native Trichoderma isolates
on the target disease(s) (Harman et al. 2004). were characterized using both morphological observation
Banana [including plantain (Musa spp.)] is one of the and molecular identification. The antagonistic activity and
most important crops in the world. However, banana pro- mycoparasitism of the isolates were evaluated against
duction in tropical areas has recently faced a crisis due to banana and cacao disease pathogens common to Ecuador.
the outbreak of several diseases, such as black Sigatoka or The antagonistic activity of these isolates against the im-
black leaf streak disease, which is caused by My- portant foliar disease pathogen Alternaria alternata, as
cosphaerella fijiensis, as well as Panama disease (Fusarium well as soil-borne disease pathogens Rosellinia necatrix

Table 1 Strains of Trichoderma spp. isolated in Ecuador

Strains no. Species Location Source GenBank accession
ITS EF-1a RBP2

T1 T. harzianum Guayas Province Soil, banana LC002568 LC002569 LC002570

T2 T. asperellum Guayas Province Soil, mango LC002586 LC002587 LC002588
T3 T. harzianum Guayas Province Soil, baby bananas LC002571 LC002572 LC002573
T4 T. asperellum Guayas Province Soil, organic banana LC002589 LC002590 LC002591
T5 T. asperellum Pichincha Province Substrate, Pleurotus spp. LC002592 LC002593 LC002594
T9 T. asperellum Guayas Province Tree bark, cacao tree LC002595 LC002596 LC002597
T10 T. asperellum Guayas Province Soil LC002598 LC002599 LC002600
T13 T. asperellum Riobamba Province Soil, potatoes LC002601 LC002602 LC002603
T15 T. harzianum Riobamba Province Soil, potatoes LC002574 LC002575 LC002576
T18 T. asperellum Riobamba Province Soil, potatoes LC002604 LC002605 LC002606
T19 T. harzianum Riobamba Province Soil, potatoes LC002577 LC002578 LC002579
T20 T. harzianum Riobamba Province Soil, potatoes LC002580 LC002581 LC002582
T29 T. reesei Guayas Province Soil, rice LC002607 LC002608 LC002609
T36 T. harzianum Guayas Province Soil, rice LC002583 LC002584 LC002585
T43 T. virens Santo Domingo Province Soil, pineapple LC002610 LC002611 LC002612

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and F. oxysporum f. sp. lycopersici (from Japan) was also DNA sequencing and phylogenetic analysis
examined for comparison. of Trichoderma spp.

For the extraction of DNA, fungi were grown in 50 mL of

Materials and methods potato dextrose broth (PDB) in 100-mL Erlenmeyer flasks
at 25 C for 2 days on an orbital shaker (120 rpm). The
Isolation and identification of Trichoderma spp. resulting mycelia were ground in liquid nitrogen using a
mortar and pestle. Total genomic DNA was extracted from
Trichoderma spp. were isolated from soils of different the mycelia as described previously (Garber and Yoder
fields, cacao bark and the substrate of Pleurotus spp. in 1983).
various regions of Ecuador (Table 1). The Trichoderma PCR amplification of the ITS region of ribosomal DNA,
strains were isolated from these samples as described translation elongation factor 1-a (EF-1a) gene and RNA
previously (Elad et al. 1981). Single-spore strains were polymerase II (RPB2) gene was achieved using three sets
grown on potato dextrose agar (PDA) (Difco, Detroit, MI, of primers: ITS1 (50 -TCCGTAGGTGAACCTGCGG-30 )/
USA) at 28 C and stored at -80 C in 20 % (v/v) glyc- ITS2 (50 -GCTGCGTTCTTCATCGATGC-30 ) (White et al.
erol. Trichoderma spp. were identified via microscopic 1990), EF1-728F (50 -CATCGAGAAGTTCGAGAAGG-
observation of the morphology of conidia, conidiophores, 30 )/EF-986R (50 -TACTTGAAGGAACCCTTACC-30 ) (Sa-
phialides and chlamydospore using taxonomic keys (http:// muels 2006), and fRBP2-5F (50 -GAYGAYMGWGATC
nt.ars-grin.gov/taxadescriptions/keys/FrameKey.cfm?gen= AYTTYGG-30 )/fRPB2-7cR (50 -CCCATRGCTTGYTTRC
Trichoderma) (Samuels et al. 2014). CCAT-30 ) (Lieckfeldt et al. 1999), respectively. PCR
reactions were performed using a Thermal Cycler Dice
Pathogens TP650 (Takara Bio, Ohtsu, Japan) or a MyCycler
170-9703JA (Bio-Rad Laboratories, Hercules, CA, USA)
All pathogenic fungi used in this study are listed in with an initial step of 2 min at 95 C; followed by 30
Table 2. Four banana and cacao pathogens originating cycles of 20 s at 94 C, 20 s at 55 C, and 30 s at 72 C;
from Ecuador, My. fijiensis (Ec-01), F. oxysporum f. sp. and a final extension of 5 min at 72 C. For the molecular
cubense (Fo-01), Mo. roreri (Cp-01) and Mo. perniciosa identification of Trichoderma spp., several online tools
(MrEO-1), were obtained from stock collections at the were employed: the International Subcommission on
Biotechnology Research Center of Ecuador (CIBE- Trichoderma and Hypocrea (ISTH, www.isth.info),
ESPOL). F. oxysporum f. sp. lycopersici (Chz1-A), TrichOKEY v. 2.0 based on an oligonucleotide barcode
Rosellinia necatrix (ES-0601) and the tomato pathotype of within the ITS1 and ITS2 sequences, TrichoMARK and
A. alternata (As-27) were kindly provided by Dr. Arie TrichoBLAST (Druzhinina et al. 2005; Kopchinskiy et al.
(Tokyo University of Agriculture and Technology), Dr. 2005). A phylogenetic analysis was carried out using the
Yasuda (Tottori Prefectural Agriculture and Forest Re- MEGA 5.1 program (Tamura et al. 2011), and a neighbor-
search Institute) and Dr. D. G. Gilchrist (University of joining tree was constructed using the Kimura 2-parameter
California, Davis), respectively. The Fungus/Mushroom distance model. Confidence values were assessed using
Resource and Research Center, Tottori University 2000 bootstrap replicates of the original data.
(FMRRC) provided standard Trichoderma strains (T. har-
zianum TUFC 60692, T. atroviride TUFC 60732, T. In vitro mycoparasitism assay
koningiopsis TUFC 60205, T. citroviride TUFC 61231 and
T. longibrachiatum TUFC 60819) as references. All iso- In total, 15 Trichoderma isolates (Table 3) were selected
lates were maintained on PDA (Difco) slants or in 20 % (v/ from 30 original isolates based on growth and sporulation
v) glycerol at -80 C. abilities for further screening for their growth inhibition
Table 2 List of pathogenic Pathogen Strain Disease Origin
fungi used in this study
Mycosphaerella fijiensis Ec-01 Black sigatoka Ecuador
Fusarium oxysporum f. sp. cubense Fo-01 Panama disease Ecuador
Moniliophthora roreri Cp-01 Frosty pod rot Ecuador
Moniliophthora perniciosa MrEO-1 Witches broom disease Ecuador
Alternaria alternata tomato pathotype As-27 Alternaria stem canker of tomato USA
Rosellinia necatrix ES-0601 White root-rot Japan
Fusarium oxysporum f. sp. lycopersici Chz1-A Vascular wilt of tomato Japan

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Table 3 Inhibitory effects of Strain Species Foc Mf Mr Mp Fol Aa Rn

Trichoderma spp. against
pathogenic fungi T1 T. harzianum 67.5a b 62.2 b 63.4 b 76.5 a 70.8 a 70.0 a 73.7 a
T2 T. asperellum 70.6 a 66.9 b 60.9 b 71.5 a 70.2 a 69.7 b 74.8 a
T3 T. harzianum 72.5 a 67.8 a 66.8 b 78.7 a 73.5 a 71.7 a 82.7 a
T4 T. asperellum 73.4 a 66.7 b 77.2 a 80.9 a 70.9 a 73.0 a 73.7 a
T5 T. asperellum 71.6 a 66.7 b 75.9 a 80.9 a 71.5 a 72.2 a 73.8 a
T9 T. asperellum 72.9 a 66.7 b 76.4 a 80.6 a 69.6 b 69.7 b 78.2 a
T10 T. asperellum 72.9 a 66.7 b 80.6 a 78.2 a 69.6 b 69.7 b 76.4 a
T13 T. asperellum 72.3 a 66.7 b 74.3 a 78.1 a 71.2 a 70.9 a 73.2 a
T15 T. harzianum 65.6 b 66.7 b 64.3 b 76.8 a 68.5 b 67.2 b 75.4 a
T18 T. asperellum 72.1 a 66.7 b 64.0 b 77.2 a 73.9 a 69.9 a 78.9 a
T19 T. harzianum 65.3 b 66.7 b 65.4 b 75.9 a 69.2 b 66.0 b 78.4 a
T20 T. harzianum 71.6 a 66.7 b 73.6 a 76.9 a 73.4 a 72.2 a 84.3 a
T29 T. reesei 52.1 b 66.7 b 13.7 b 7.5 b 64.3 b 55.4 b 66.1 b
T36 T. harzianum 74.1 a 66.7 b 78.6 a 82.8 a 74.7 a 73.3 a 83.6 a
T43 T. virens 68.1 b 66.7 b 72.6 a 80.3 a 68.2 b 65.9 b 82.8 a
a
Percentage inhibition of radial growth of Fusarium oxysporum f. sp. cubense (Foc, Fo-01), My-
cosphaerella fijiensis (Mf, Ec-01), Moniliophthora roreri (Mr, Cp-01), Mo. perniciosa (Mp, MrEO-1), F.
oxysporum f. sp. lycopersici (Fol, Chz1-A), Alternaria alternata tomato pathotype (Aa, As-27), Rosellinia
necatrix (Rn, ES-0601). The experiment used a completely randomized factorial design and was repeated
three times. Analysis of variance (ANOVA) and Tukeys range test were used to test for significant
differences. Values followed by different letters differed significantly at P \ 0.05

and mycoparasitic abilities against pathogenic fungi from Results

Ecuador and Japan using the dual culture method. The
growth inhibition/antagonism test was performed in tripli- Molecular identification and phylogenetic analyses
cate on PDA by placing a mycelium disc (5 mm in di- of Trichoderma spp.
ameter) of each pathogenic fungus at one side (10 mm
from the edge) of a petri dish (90 mm in diameter); the Trichoderma spp. isolated in Ecuador were identified pre-
opposite side of each dish was inoculated with Tricho- liminarily via morphological observation in CIBE-ESPOL
derma spp. The plates were incubated at 25 C for 10 days, (Ecuador) as T. harzianum, T. viride and Trichoderma spp.
and culture diameters were measured every 24 h to monitor Further molecular identification of these Trichoderma
radial growth of each fungus. The percentage inhibition of strains was performed using sequence analyses of three
radial growth of pathogens (PIRGP) was determined with unlinked loci: the ribosomal ITS region, EF-1a gene and
the formula used by Ezziyyani et al. (2004): RPB2 gene. The strains were identified via a Blast search
PIRGP = (R1 - R2)/R1 9 100, where R1 is the colony of GenBank, DDBJ and the International Subcommission
radius (distance from the inoculation site to the edge of on Trichoderma and Hypocrea Taxonomy (ISTH), as well
colony) of the control pathogens without Trichoderma spp. as using the TrichOKEY and TrichoBLAST programs. The
inoculation and R2 is the colony radius of the pathogens identification, origin, and GenBank accession numbers of
with Trichoderma spp. inoculation. The following scale all isolates are provided in Table 1.
(Ezziyyani et al. 2004) was used to evaluate the 10-day Isolates T1, T3, T15, T19, T20, and T36 were con-
mycoparasitism against Trichoderma spp. in these dual firmed to belong to T. harzianum, section Pachybasium,
culture plates: 0: no invasion of Trichoderma on the sur- clade Harzianum. Isolates T2, T4, T5, T9, T10, T13 and
face of the pathogenic fungus, 1: 25 % invasion on the T18, which were previously identified to be T. viride via
surface of the pathogenic fungus colony, 2: 50 % invasion morphology, were identified as T. asperellum. Samuels
on the surface of the pathogenic fungus colony, 3: 100 % et al. (1999) indicated that T. asperellum could be dis-
invasion on the surface of the pathogenic fungus colony, 4: tinguished from T. viride due to its finer conidial orna-
100 % invasion on the surface of the pathogenic fungus mentation, slightly ovoid conidia, faster growth rate,
colony and sporulation on it. The dual culture assay for mostly paired branches, ampulliform phialide, and con-
each Trichoderma isolate and pathogen combination was sistent presence of chlamydospores. Two unidentified
repeated at least three times. Trichoderma spp., T43 and T29, were identified in this

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Fig. 1 Phylogenetic relations of Trichoderma spp. based on neigh- bootstrap sampled data sets supporting the clade to the right of the
bor-joining analysis of the ITS, EF-1a and RPB2 sequences. The branch and with values [50. (A) T. asperellum strains, (B) T.
number above selected branches indicates the percentage of 2000 harzianum strains, (C) T. virens strains and (D) T. reesei strains

study as T. virens (belonging to section Pachybasium (100 %) (Fig. 1). Samuels et al. (2010) described the new
clade Virens) and T. reesei (belonging to section Longi- species T. asperelloides and redescribed the closely re-
brachiatum clade Longibrachiatum) (Table 1). lated species T. yunnanense, T. asperellum and T.
The phylogenetic analysis based on ITS and the EF-1a asperelloides. These species cannot be distinguished by
and RPB2 genes indicated four distinct groups (AD) in their phenotype, biology or biogeography, and 33 % of
the isolates (Fig. 1). The first dominant group (A) was T. the T. asperellum isolates examined were identified to be
asperellum, which forms part of section Trichoderma, the recently described T. asperelloides (Samuels et al.
clade Pachybasium A or Hamatum, with high bootstrap 2010).

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T. harzianum was the second dominant group (B) show-

ing a phylogenetic branch supported with high bootstrap
values by the sequences of the three genes (Fig. 1). Druz-
hinina et al. (2010) found that the exact phylogenetic po-
sition of the majority of H. lixii/T. harzianum strains is not
clear due to a diverse network of recombining strains that
was conventionally called the pseudoharzianum matrix.
Additionally, the anamorphic tropical strain (primarily of
African origin) was called T. sp. nov. afroharzianum nom.
prov. While H. lixii and T. harzianum are evidently ge-
netically isolated, the anamorphteleomorph combination
comprising H. lixii/T. harzianum in one holomorph must be
rejected in favor of two separate species.
Two other isolates, T29 (D) and T43 (C), were identified
as T. reesei and T. virens, respectively, using phylogenetic
analysis based on the three genes (Fig. 1).

Growth inhibition and mycoparasitism

Pathogen growth inhibition on the dual culture plates was

evident at 4 days after inoculation. Mycelia of T. harzianum,
T. asperellum and T. virens but not T. reesei came into
contact with the pathogen colonies. Following contact,
green mycelia of the three species covered the pathogen
coloniesm and spores were produced, indicating strong
Trichoderma spp. mycoparasitism (Fig. 2; Supplementary Fig. 2 Dual cultures to test antagonism of Trichoderma harzianum
Figs. 1, 2, 3). strain T36 against pathogenic fungi and single cultures of each fungal
Among the isolates, T. harzianum showed the highest pathogen after 10 days at 25 C. A 5-mm-diameter mycelial disk of
PIRGP (see the Materials and Methods section) against the respective fungi was placed on plates. a Dual cultures with
pathogen on lower side of plate, Trichoderma spp. on upper side.
F. oxysporum f. sp. cubense (65.374.1 %), My. fijiensis b Single culture of each pathogen. (Foc) Fusarium oxysporum f. sp.
(62.267.8 %), Mo. roreri (63.478.6 %) and Mo. perni- cubense (Fo-01), (Mf) Mycosphaerella fijiensis (Ec-01), (Mr) Monil-
ciosa (75.982.8 %), as well as F. oxysporum f. sp. ly- iophthora roreri (Cp-01), (Mp) Mo. perniciosa (MrEO-1), (Fol) F.
copersici, (68.574.7 %), A. alternata (66.073.3 %) and oxysporum f. sp. lycopersici (Chz1-A), (Aa) Alternaria alternata
tomato pathotype (As-27), (Rn) Rosellinia necatrix (ES-0601)
R. necatrix (73.784.3 %) (Table 3). My. fijiensis showed
repeated identical radial growth when combined with dif-
ferent Trichoderma spp., resulting in identical PIRGP harzianum, T. asperellum and T. virens against all tested
values across all tests except with T3. The T. harzianum pathogens (Table 3).
T20 and T36 strains had the highest inhibitory activity On the mycoparasitism assay, the majority of T. harzia-
(above 70 %) against all fungal pathogens including both num strains had high activity (grades 34, see the Materials
the Ecuadorian and Japanese pathogens (Table 3). and Methods section), indicating 100 % coverage of the
The three isolates (T4, T5 and T13) of T. asperellum pathogen colonies as well as sporulation. Among T. har-
showed strong inhibitory activities against F. oxysporum f. zianum strains, T15, T19 and T36 exerted strong parasitism
sp. cubense, Mo. roreri, F. oxysporum f. sp. lycopersici, A. against all of the pathogens (Figs. 2, 3; Supplementary
alternata, and R. necatrix (greater than 70 % inhibition), as Fig. 1). T. asperellum strains showed slightly lower activity
well as against Mo. perniciosa (greater than 80 % inhibi- compared with T. harzianum strains (Supplementary Fig. 2).
tion) in T4, T5 and T9 (Table 3). However, these strains exerted high (grade 4) parasitism
T. virens strain T43 showed slightly lower activity (less against several pathogens, indicating that the strains may be
than 70 %) against F. oxysporum f. sp. lycopersici, A. al- useful in scenarios involving certain pathogen combina-
ternata, F. oxysporum f. sp. cubense and My. fijiensis but tions. T. virens strain T43 also showed a high degree of
high inhibition against Mo. perniciosa, R. necatrix and Mo. mycoparasitism against nearly all pathogens used in this
roreri (Table 3). However, T. reesei strain T29 appeared to study, with the exception of F. oxysporum sp. cubense
have low inhibitory activity compared with those of T. (Supplementary Fig. 3). However, T. reesei strain T29

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Fig. 3 Mycoparasitism index for strains of Trichoderma spp. against oxysporum f. sp. cubense (Fo-01), (Mf) Mycosphaerella fijiensis
seven pathogenic fungi determined using the scale described in the (Ec-01), (Mr) Moniliophthora roreri (Cp-01), (Mp) Mo. perniciosa
Materials and Methods section. Strains: T. harzianum (T1, T3, T15, (MrEO-1), (Fol) F. oxysporum f. sp. lycopersici (Chz1-A), (Aa)
T19, T20, T36), T. asperellum (T2, T4, T5, T9, T10, T13, T18), T. Alternaria alternata tomato pathotype (As-27), (Rn) Rosellinia
reesei (T29) and T. virens (T43). Pathogens: (Foc) Fusarium necatrix (ES-0601)

showed relatively low mycoparasitism against all pathogens identification of Trichoderma spp. Three different groups,
(Supplementary Fig. 3). T. harzianum, T. viride, and Trichoderma spp., were de-
termined using this method; as previously mentioned by
Lieckfeldt et al. (1999), T. viride and T. asperellum could
Discussion not be separated using this morphology-based method.
The molecular identification of Trichoderma at the
The genus Trichoderma comprises many agriculturally species level was based on a combination of several genes
useful strains that act as biological control agents through (not only a single gene sequence). Three genes [ITS, EF-1a
direct or indirect mechanisms (Lo 1998). When using and RPB2 (Kim et al. 2012)] were selected for the iden-
Trichoderma as a biocontrol agent, native and domestic tification and phylogenetic analysis of the Ecuadorian
strains are desired to prevent the disturbance of native strains. Among these genes, EF-1a has been shown to be
biodiversity and ecosystems. Thus, Trichoderma spp. better for distinguishing Trichoderma spp. because it is
should be carefully identified before application, and suit- more variable than the other genes and reflects species
able strains should be selected to fit the target fields, crops differences within and between groups of closely related
and pathogens. To facilitate this process, we identified and species (Samuels 2006). The sequence data were further
did the phylogenetic analyses of native Trichoderma strains analyzed using several online tools (www.isth.info) such as
for the present study before investigating their antagonistic TrichOKEY and TrichoBLAST (Druzhinina et al. 2005;
activities against important and intractable diseases in Kopchinskiy et al. 2005). The sequencing data of these
banana and cacao in Ecuador. three genes were useful in identifying all strains collected
Taxonomic knowledge on Trichoderma strains is im- from the agricultural soils of several crops as well as cacao
portant for further searches and identification of potential bark in various Ecuadorian provinces (Table 1). Among 15
biocontrol species and to avoid potential risk from intro- native isolates, six isolates, T1, T3, T15, T19, T20 and
ducing an unknown fungal species into the rhizosphere of a T36, were identified as T. harzianum using TrichOKEY
given ecosystem. A combination of morphological and and TrichoBLAST based on sequence homology with
molecular methods is desirable for the reliable and accurate greater than 99 % of the genes tested.
identification of Trichoderma spp. The few morphological T. viride is a paraphyletic group, and an integrated
characteristics with limited variation in Trichoderma spp. morphological/molecular approach has been used to con-
may lead to an overlap and misidentification of the species firm the reclassification of types I and II of T. viride into
(Kullnig-Gradinger et al. 2001). In this study, web-based two species (Samuels et al. 2010). Type I is the true T.
taxonomic keys (http://nt.ars-grin.gov/taxadescriptions/ viride species, which also includes the anamorph of H. rufa
keys/FrameKey.cfm?gen=Trichoderma) (Samuels et al. and is grouped together with the strains of T. atroviride and
2014) were used for the preliminary morphological T. koningii. Type II represents the new species T.

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asperellum (Lieckfeldt et al. 1999; Samuels et al. 1999), inhibition and mycoparasitism against some pathogens. T.
which conidiates more rapidly and produces darker, ovoid virens was reported to inhibit mycelial growth of several
conidia rather than globose conidia. Several isolates in this pathogens such as Rhizoctonia solani and Pythium ultimum
study were initially identified as T. viride via morpho- (Hjeljord and Tronsmo 1998). T. virens T43 showed a high
logical key; these strains were subsequently identified to be PIRGP with mycoparasitism against nearly all pathogens
T. asperellum using molecular analysis. Samuels et al. used in this study. These T. asperellum and T. virens strains
(2010) re-evaluated the taxonomy of T. asperellum using are also useful as candidate strains for field tests. T. reesei
different methods, including the sequence analysis of the T29 exerted only weak antagonistic activities compared
EF-1a and RPB2 genes, and identified several strains as with the other species.
belonging to the new species T. asperelloides. In our study, The antagonism and mycoparasitism of Trichoderma are
we found a correlation of one isolate (T4) with T. not properties belonging to a single species, and different
asperelloides via EF-1a gene analysis. However, all other strains of the same species can vary in their biocontrol
data indicated the isolate would belong to T. asperellum. potential. These biocontrol activities can involve produc-
Further molecular taxonomical analysis would be neces- tion of cell-wall-degrading enzymes such as b-1,3-glu-
sary using additional sequences to identify the isolate. canase, N-acetyl-glucosaminidases (NAGAse), chitinase,
Another isolate was identified as T. reesei (T29) with the acid phosphatase, acid proteases and alginate lyase
sexual state Hypocrea jecorina, section Longibrachiatum (Qualhato et al. 2013). The antagonistic activities can also
clade Longibrachiatum. This species is useful in industry, vary according to the target pathogens, as indicated in this
such as textile and paper manufacturing, due to its high study. Therefore, it is important to select the most effective
cellulose production (Seidl et al. 2008). As a biocontrol and suitable strains in accordance with the target diseases.
agent, T. reesei was reported to exert antifungal activity via In this study, several Ecuadorian strains of T. harzianum,
the production of degrading enzymes, e.g., 32-kDa endo- e.g., T15 and T. asperellum, e.g., T4 showed high an-
chitinase (Harjono and Widyastuti 2001). Another isolate tagonistic activities against important banana and cacao
was identified as T. virens (T43) section Pachybasium pathogens in the country, indicating that they are potential
clade Virens. This species has also been used as a model biocontrol candidates. In addition, these candidate strains
Trichoderma strain for research on biocontrol mechanisms, were isolated from diverse areas and sources.
and the draft genome sequence has recently been identified A thorough understanding of the molecular mechanisms
(http://genome.jgi-psf.org/Trive1). Most isolates were of mycoparasitism as well as the development of more
placed in their correct clade using phylogenetic analysis. effective biocontrol methods with rapid and easy screen-
Members of Trichoderma spp., such as T. martiale, have ings are important for the future application of candidate
been reported to be potential biocontrol agents against strains. Using a subtraction hybridization approach,
cacao black pod disease (Hanada et al. 2009). T. oval- Scherm et al. (2009) identified potential marker genes that
isporum is also used for the biocontrol of frosty pod rot of could be used to rapidly screen and assess the biocontrol
cacao in an integral pest management program (Krauss potential of T. harzianum strains. The involvement of cell-
et al. 2010). T. harzianum is a typical species used for the wall-degrading enzymes in the mycoparasitism of T. har-
biocontrol of many diseases including a cacao disease zianum was also studied via the functional analysis of ge-
(Garcia et al. 2012). In banana production, Trichoderma nes that encode enzymes, e.g., ech-42 (Carsolio et al.
spp. have been used for integrated pest management pro- 1994), qid74 (Rosado et al. 2007) and Thctf1 (Rubio et al.
grams in which the fungus is applied some days before 2008).
planting (Perez et al. 2009). T. harzianum and T. asperel- In the present study, several candidate strains were
lum were also used for the biocontrol of banana fruit rot identified to act against important and intractable diseases
pathogens (Adebesin et al. 2009). of banana and cacao in Ecuador. Field tests of the candi-
Among the four Trichoderma species identified in this date strains against F. oxysporum f. sp. cubense (Panama
study, T. harzianum, T. asperellum and T. virens have been disease) and My. fijiensis (black Sigatoka) on banana as
reported to be the most potent biocontrol agents against a well as Mo. roreri (frosty pod rot) and Mo. perniciosa
variety of pathogens (Hjeljord and Tronsmo 1998; Jeger (witches broom disease) on cacao are now underway in
et al. 2009). Similar to the previous studies, several banana and cacao fields in Ecuador.
Ecuadorian T. harzianum isolates showed high antagonistic
activities in growth inhibition and mycoparasitism tests. T. Acknowledgments We thank T. Arie, T. Yasuda and D.
G. Gilchrist for providing the fungal strains. This work was supported
harzianum T15, T19 and T36 showed exceptional activities by the Global COE Program Advanced Utilization of Fungus/
in both criteria, and related isolates could be good candi- Mushroom Resources for Sustainable Society in Harmony with Na-
date strains for further field tests. Several strains of T. ture, MEXT, Japan. We thank the National Secretary of Higher
asperellum, e.g., T4, T5 and T13, also showed high growth Education, Science, Technology and Education of Ecuador and the

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