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Abstract Endophytic isolates of Trichoderma species
are being considered as biocontrol agents for diseases
of Theobroma cacao (cacao). Gene expression was
studied during the interaction between cacao seedlings
and four endophytic Trichoderma isolates, T. ovali-
sporum-DIS 70a, T. hamatum-DIS 219b, T. harzianum-
DIS 219f, and Trichoderma sp.-DIS 172ai. Isolates DIS
70a, DIS 219b, and DIS 219f were mycoparasitic on the
pathogen Moniliophthora roreri, and DIS 172ai pro-
duced metabolites that inhibited growth of M. roreri in
culture. ESTs (116) responsive to endophytic coloni-
zation of cacao were identified using differential display
and their expression analyzed using macroarrays.
Nineteen cacao ESTs and 17 Trichoderma ESTs were
chosen for real-time quantitative PCR analysis. Seven
cacao ESTs were induced during colonization by the
Trichoderma isolates. These included putative genes for
ornithine decarboxylase (P1), GST-like proteins (P4),
zinc finger protein (P13), wound-induced protein (P26),
EF-calcium-binding protein (P29), carbohydrate oxi-
dase (P59), and an unknown protein (U4). Two plant
ESTs, extensin-like protein (P12) and major intrinsic
protein (P31), were repressed due to colonization. The
plant gene expression profile was dependent on the
Trichoderma isolate colonizing the cacao seedling. The
fungal ESTs induced in colonized cacao seedlings also
varied with the Trichoderma isolate used. The most
highly induced fungal ESTs were putative glucosyl
hydrolase family 2 (F3), glucosyl hydrolase family 7
(F7), serine protease (F11), and alcohol oxidase (F19).
The pattern of altered gene expression suggests a
complex system of genetic cross talk occurs between the
cacao tree and Trichoderma isolates during the estab-
lishment of the endophytic association.
Keywords Antibiosis ÆEndophyte ÆMycoparasitisim Æ
Theobroma cacao ÆTrichoderma
Introduction
Theobroma cacao (cacao), the source of chocolate, is
grown in many tropical countries (Wood and Lass
2001). Cacao is grown in a range of cropping systems
such as full sun, or more traditionally under shade.
Cacao diseases cause severe yield losses in many areas
where cacao is grown (Bowers et al. 2001; Wood and
Lass 2001). The major diseases of cacao include Black
Pod (Phytophthora species), Witches’ Broom (Crini-
pellis perniciosa), and Frosty Pod Rot (Moniliophthora
Electronic Supplementary Material Supplementary material is
available to authorised users in the online version of this article
at http://dx.doi.org/10.1007/s00425-006-0314-0.
B. A. Bailey (&)ÆH. Bae ÆM. D. Strem
Sustainable Perennial Crops Laboratory,
BARC-West, Beltsville, MD 20705, USA
e-mail: baileyb@ba.ars.usda.gov
D. P. Roberts
Sustainable Agricultural Systems Laboratory,
BARC-West, Beltsville, MD 20705, USA
S. E. Thomas ÆJ. Crozier ÆK. A. Holmes
CABI, UK Centre (Ascot), Silwood Park, Berks, UK
G. J. Samuels
Systematic Botany and Mycology Laboratory,
BARC-West, Beltsville, MD 20705, USA
I.-Y. Choi
Soybean Genomics and Improvement Laboratory,
BARC-West, Beltsville, MD 20705, USA
Planta
DOI 10.1007/s00425-006-0314-0
123
ORIGINAL ARTICLE
Fungal and plant gene expression during the colonization
of cacao seedlings by endophytic isolates of four
Trichoderma species
B. A. Bailey ÆH. Bae ÆM. D. Strem Æ
D. P. Roberts ÆS. E. Thomas ÆJ. Crozier Æ
G. J. Samuels ÆIk-Young Choi ÆK. A. Holmes
Received: 9 February 2006 / Accepted: 18 April 2006
Springer-Verlag 2006
roreri). Although the distributions of the pathogens
vary, all three diseases occur in South and Central
America (Bowers et al. 2001; Wood and Lass 2001).
Fungicides are used to control cacao diseases with
varying success and at significant cost to smallholder
farmers (Purdy and Schmidt 1996; Adejumo 2005). We
are considering biocontrol of cacao diseases as an
alternative strategy to fungicide use and as a compo-
nent of integrated pest management.
In their native habitats, Theobroma species, includ-
ing cacao, are understory forest trees found in tropical
regions of Central and South America (Wood and Lass
2001). As such, Theobroma species exist in some of the
most diverse ecosystems in the world (Wood and Lass
2001). With more extensive study has come the reali-
zation that Theobroma species carry a portion of this
regions diverse microbial community in unique endo-
phytic associations (Arnold et al. 2003; Rubini et al.
2005). Among the endophytic fungi associated with
cacao and other forest inhabitants are many species of
Trichoderma (Samuels et al. 2000; Holmes et al. 2004).
These Trichoderma species inhabit different plant tis-
sues including roots, trunks, stems, leaves and fruit.
Trichoderma species have recently been described
as ‘‘opportunistic, avirulent plant symbionts with po-
tential to control plant diseases’’ (Harman et al. 2004).
Much of the research concerning Trichoderma species/
plant interactions centers on the concept of Tricho-
derma species as common soil inhabitants (Harman
et al. 2004). Increasing evidence indicates certain
Trichoderma species have significant potential for
biocontrol of plant diseases in the plant canopy where
environmental conditions, microbial communities, and
plant tissues differ greatly from those encountered in
the root zone (Wilson 1997). For example, T. strom-
aticum, an epiphytic mycoparasite, has already proven
to be beneficial for control of C. perniciosa, the causal
agent of witches’ broom of cacao (Bastos 1996;Sam-
uels et al. 2000).
We have isolated a collection of endophytes from
the above ground portions of cacao trees, related
Theobroma species, and other plant species found
associated with Theobroma species in tropical forest.
This collection of endophytic fungi represents a po-
tential source of biocontrol agents for cacao diseases.
More than 100 isolates from diverse Trichoderma
species are included in the collection (Evans et al.
2003). The objectives of the research presented here
include the characterization of the responses of both
the endophytic Trichoderma species and the cacao
seedlings following establishment of an endophytic
relationship. As part of this research we used a seedling
assay and differential display, macroarray, and real-
time quantitative PCR (Q-PCR) to characterize the
interactions between four Trichoderma species and
cacao at the molecular level.
Materials and methods
Trichoderma isolates and their culture
The isolation of Trichoderma endophytes was de-
scribed by Evans et al. (2003). Four isolates, repre-
senting four phylogenetically distinct species of
Trichoderma, were chosen for study (Holmes et al.
2004). DIS 70a (T. ovalisporum) was isolated from high
tropical forest along the Pan
˜acocha-Rı
´o Yanayacu,
Napo River, Sucumbios Province, Ecuador in 1999
(Holmes et al. 2004). The tissue was a witches’ broom
on a liana, identified by a local Quechua guide as aya-
huasca (Banisteriopsis caapi, Malpighiaceae). DIS 219b
(T. hamatum) and DIS 219f (T. harzianum) were iso-
lated from a pod of Theobroma gileri found in Gua-
dual, Lita, Esmeraldas Province, Ecuador (Evans et al.
2003). DIS 172ai (‘Tkon 21’ in Holmes et al. 2004)
represents an undescribed Trichoderma species that is
widely distributed in tropical American and African
soils. DIS 172ai was isolated from the stem of a 50–
60 year old Theobroma grandiflorum (cupuac¸u) tree
located in Brazil (Embrapa, Belem, Para).
Soluble inhibitory metabolite production
Conidia from Trichoderma isolates were harvested
from 2-week-old cultures grown on 20% PDA at 25C
and subsequently filtered through glass wool to remove
mycelia. Three flasks containing 150 ml of minimal
salts broth (MIN; Srinivasan et al. 1992) were each
inoculated with 1 ml of a 1·10
6
conidial suspension of
the endophyte and incubated in an orbital incubator at
25C and 110 rpm. After 7 days growth, mycelia were
collected in cheese cloth and the liquid sterilized by
filtration through a 0.22 lm filter (Millipore). Sterile
filtrate was placed in a 90C water bath for 2 h to
inactivate enzymes, then added to an equal volume of
strengthened agar, 3% MIN [3% agar no. 3], and
poured into Petri dishes. A 4-mm-diam. plug of
M. roreri, from the growing edge of a 7-day-old colony,
was placed in the center of the Petri dish. Controls
were prepared by replacing the fungal filtrate with
uninoculated MIN broth. Three replicate plates were
used for each test and all plates were incubated at
25C. Inhibition of mycelial growth of M. roreri was
recorded as the difference between mean radial growth
in the presence and absence of the fungal filtrate.
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123
Mycoparasite screening
The Trichoderma isolates were screened for mycopar-
asitic ability using a pre-colonized plate method as
previously described (Evans et al. 2003; Holmes et al.
2004). A strip of inoculum, 2.5·0.5 cm, excised from a
freshly sporulating colony of the Trichoderma isolate,
was placed at one edge of a 9-cm-diam. PDA plate
wholly pre-colonized by an isolate of M. roreri. Five
replicate plates were prepared. The plates were main-
tained at 25C in the dark, and on a weekly basis, a
total of 15 samples from each replicate were removed
with a 5-mm-diam. cork borer starting at the M. roreri
inoculum. These samples were plated onto 20% PDA
and incubated at 25C under black light (near UV) and
observed over 14 days for the growth of the Tricho-
derma sp. or M. roreri. The percentage colonization
was determined. This was carried out for 5 weeks or
until complete colonization by the Trichoderma isolate
had occurred.
Enzyme assays for T. ovalisporum isolate DIS 70a
Isolate DIS 70a was grown 8 days at room temperature
and 75 rpm in 250 ml Erlenmeyer flasks containing
25 ml Wiendling’s minimal salts (Jones and Hancock
1987) plus 0.2% carboxymethylcellulose, crystalline
cellulose, glycerol, pectin, or cacao seedling extract.
Cacao seedling extract was prepared by grinding 7-day-
old cacao seedlings minus the cotyledons in liquid
nitrogen and lyophilizing the resulting powder. The
lyophilized powder was used directly in culture med-
ium. The remaining chemicals were reagent grade. DIS
70a was also grown as above in V8 broth. Three rep-
licate cultures of each isolate were prepared from each
medium. Culture filtrate was prepared by passing the
culture through a sterile 0.2 lbottle filter unit. Dry
weight of mycelia collected on the filter units from
each replicate culture was determined. Sterile culture
filtrate was stored at –80C until used.
Culture filtrate was assayed for carboxymethylcel-
lulase (CMC’ase), crystalline cellulase, chitinase, pol-
ygalacturonase, pectate lyase, and protease activities.
For CMC’ase activity, culture filtrate was mixed with
50 mM MES buffer, pH 5.0, and 0.2% carboxymeth-
ylcellulose and incubated at 37C. For crystalline cel-
lulase activity, culture filtrate was mixed with 100 mM
MES buffer, pH 5.0, and 0.2% crystalline cellulose
(Sigmacell, Sigma Chemical Company, St. Louis, MO,
USA) and incubated at 37C. For polygalacturonase
activity, culture filtrate was mixed with 100 mM suc-
cinate, pH 5.0, 10 mM EDTA, and 0.2% polygalactu-
ronic acid and incubated at 37C. Reducing sugars
liberated in these reactions were determined by the
method of Nelson (1944) with glucose as standard for
carboxymethylcellulase and crystalline cellulase activ-
ity and galacturonic acid as standard for polygalactu-
ronase activity. One unit of CMC’ase or crystalline
cellulase activity was defined as the amount of enzyme
that released 1 lg glucose reducing equivalent per min
per lg dry weight mycelium. One unit of polygalactu-
ronase activity was defined as the amount of enzyme
that released 1 lg galacturonic acid reducing equiva-
lent per min per lg dry weight mycelium. For pectate
lyase activity, culture filtrate was mixed with 50 mM
Tris, pH 8.5, 1.5 mM CaCl
2
, and 0.2% polygalacturonic
acid and incubated at 37C. One unit of pectate lyase
activity was the amount of enzyme that increased
absorbance at 232 nm one unit per hour per lg dry
weight mycelium. Chitinase activity was determined by
incubating culture filtrate with 50 mM Tris, pH 7.0 and
200 lg chitin azure (Sigma Chemical Co.) at 37C.
Chitinase activity was also determined in 50 mM Tris,
pH 8.0 and in 50 mM succinate buffer, pH 5.0. One
unit of chitinase activity was defined as the amount of
enzyme that increased absorbance at 575 nm one unit
per hour per lg dry weight mycelium. Protease activity
was determined by incubating culture filtrate with
50 mM Tris, pH 7.0 and 200 lg azocoll (Sigma
Chemical Co.) at 37C (Chavira et al. 1984). One unit
of protease activity was the amount of enzyme that
increased absorbance at 520 nm one unit per hour per
lg dry weight mycelium. Autoclaved culture filtrates
were used as negative controls in determinations of all
enzyme activities. Lower limits of detection were
0.198 U for CMC’ase and crystalline cellulase, 0.386 U
for polygalacturonase, 0.1 U for pectate lyase, 0.01 U
for chitinase, and 0.01 U for protease activities.
Seedling production
Open pollinated seeds of T. cacao variety Comum
(Lower Amazon Amelonado type) were collected by
Alan Pomella from established plantings at the Al-
mirante Cacau, Inc. farm (Itabuna, Bahia, Brazil). The
seed coat was removed and the seed surface sterilized
by incubation in 14% sodium hypochlorite for 3 min
followed by three washes in sterile distilled water.
Three sterile seeds were placed side-by-side with the
radical tips oriented in the same direction on 1.5%
water agar in 10-cm-diam Petri dishes and sealed with
parafilm. Seeds were pre-germinated under fluorescent
lights at 23C. After 3 days, two 0.6-cm agar plugs of
one of the four Trichoderma isolates were placed on
the water agar surface below the emerging roots. The
Trichoderma isolate was allowed to grow out of the
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123
agar plug through the water agar and onto the cacao
seedlings. The seedlings were rated for root discolor-
ation after 6 days of colonization using a scale from 0
(no discoloration) to 4 (severe browning).
Seedlings were subsequently processed in one of
three ways: (1) Dissection into 1-cm sections of roots,
stems, cotyledons, and plumules, surface sterilized as
previously described and plated on CDA. (2) Cotyle-
dons were removed and the seedlings frozen in liquid
nitrogen and stored until used for RNA isolation. (3)
After being colonized by Trichoderma, the germinating
seeds were planted in sterile soilless mix (80 g of 2:2:1,
sand:perlite:promix) in double magenta boxes (20-cm
high). Sterile distilled water (20 ml) was added to dry
soilless mix after planting and the seedlings grown on
fluorescent light benches at 23C for 2 weeks. The
2-week-old cacao seedlings were dissected into 1-cm
sections of roots, stems, cotyledons, plumules, and
leaves, surface sterilized as previously described, and
the sections incubated on cornmeal agar plates. All
plates were incubated at room temperature on the lab
bench (23C) for 5–7 days until the endophytic Trich-
oderma isolates grew out of the cacao tissue sections.
At least four replicate plates of three seedlings each
were prepared for each Trichoderma isolate and the
control (uninoculated seedlings) for each of the three
seedling processes. Replicate seedling plates were ini-
tiated two at a time using seeds from separate ship-
ments. Four seedlings from independent replicate
seedling plates were dissected for each Trichoderma
isolate and the control for seedling processes 1 and 2 in
order to verify endophytic colonization of the cacao
seedlings.
Statistical analysis
The data were expressed as percent growth inhibition
for the antibiosis study and percent colonization for
the mycoparasitism and seedling colonization studies.
The data were analyzed by analysis of variance using
the SAS general linear models procedure (SAS
Institute, Cary, NC, USA). The least significant dif-
ference (P< 0.05) is provided for means comparisons
for each tissue in each experiment.
RNA isolation and differential display
Fungal isolates were grown on CDA plates, [1.7%
Difco corn meal agar (DIFCO laboratories, Detroit,
MI, USA) plus 20% dextrose] in an incubator at 23C
for 5 days before use. Two agar plugs were added to
50 ml of clarified V8 broth in a 250 ml flask, three
flasks for each isolate. Mycelia of each isolate were
produced by growth at 23C in stationary cultures for
7 days. Mycelia were collected by filtration onto #2
Whatman paper, frozen in liquid nitrogen, and stored
in liquid nitrogen before use in RNA extractions.
Total RNA was isolated from cacao seedlings and
treated with DNase I as previously described (Bailey
et al. 2005a). Total RNA of fungal mycelia was ex-
tracted using RNeasy mini kit (Qiagen, Valencia, CA,
USA) according to the manufacturer’s recommenda-
tion, with an extra DNase I treatment. Using the
GenHunter Corporation (Nashville, TN, USA) RNA-
spectra Red Fluorescent mRNA Differential Display
System, the RNA was reverse-transcribed with three
unlabeled anchor primers following manufacturers
instructions. Subsequent PCR reactions used all 8
random primers included in RNAspectra
TM
Red Kit 1,
with the incorporation of the fluorescent label using
labeled anchor primers. The samples were electro-
phoresed on a 4-mm-thick denaturating polyacryl-
amide (6% acrylamide, 8 M urea) sequencing gel. The
GenHunter procedure was followed for loading and
electrophoresis. Gel images were captured using an
Amersham Biosciences Typhoon 8600 Variable Mode
Imager (GE Healthcare, Piscataway, NJ, USA) with a
532 green laser, and Cy3, 555, BP20 emission filter.
Bands of interest were selected, excised and eluted
from the gel, and reamplified following the procedure
described in the GenHunter protocol. The reamplified
cDNA fragments were cloned using the PCR-TRAP
Cloning System (GenHunter). Plasmids containing
DNA from bands of interest from the differential dis-
play were isolated using a Wizard Plus SV Minipreps
DNA kit (Promega, Madison, WI, USA) following kit
directions. The mini-prepped plasmids were sequenced
and then identified using BLAST.
Macroarray procedure
Plasmid DNA (2 lg) was diluted with sterile distilled
water and transferred into each well of a 96-well
microtiter plate to give 16 ll. Denaturing solution
(40 ml 2 M NaOH plus 2 ml 1% bromophenyl blue)
was added at a rate of 4 ll per well. Empty wells were
loaded with 16 ll water plus 4 ll NaOH/dye. The
plates were incubated at room temperature for 20–
30 min before printing.
Two different plates were set up for each printing.
Using a 9-hole grid template, each plate was stamped
on each membrane (Zeta-Probe GT, BioRad, Hercu-
les, CA, USA) four times in either a 4-corner pattern
or a compass-point (diamond) pattern, leaving the
center of the 9-hole grid empty, giving four identical
replications per blot. This process resulted in the
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123
transfer of approximately 25 ng of plasmid DNA for
each of four spots for each plasmid. Immediately after
printing, the membranes were crosslinked using a
Stratagene (La Jolla, CA, USA) UV Stratalinker 1800,
air-dried and then stored at –20C.
Radioactive probes were made from 5 lg RNA
using Superscript III First Strand Synthesis System
(Invitrogen, Carlsbad, CA, USA) following the
instructions supplied with the kit, except volumes were
doubled. dCTP-aP
32
was supplied by Perkin Elmer
Life and Analytical Sciences (Boston, MA, USA).
Unincorporated nucleotides were removed with an
Edge Biosystems (Gaithersburg, MD, USA) Performa
DTR-RT gel filtration cartridge spin column. Three
independent biological samples were used to create
probes for each Trichoderma isolate/T. cacao combi-
nation and controls.
Membranes were pre-wetted in 1·SSC and incu-
bated twice in BD Biosciences-Clontech (Palo Alto,
CA, USA) ExpressHyb buffer for 30 min at 65C.
Fresh, pre-warmed ExpressHyb buffer was placed in
each bottle and the cleaned probe added to the buffer
and gently mixed before returning the bottle to the
incubation oven. Membranes were hybridized 2 h at
65C with constant rotation. Blots were washed fol-
lowing the ExpressHyb protocol, exposed to a Molec-
ular Dynamics (Amersham Biosciences, part of GE
Healthcare) Storage Phosphor screen, and visualized
using an Amersham Biosciences Typhoon 8600 Vari-
able Mode Imager.
Real-time quantitative PCR
Four micrograms of each RNA sample isolated as
described above were used to generate first strand
cDNA using SuperScript III RNase H
–
Reverse
Transcriptase (Invitrogen) with Oligo (dT)
20
primer.
The synthesized first strand cDNA was diluted tenfold
and used as template for Q-PCR. The Q-PCR analysis
of gene expression was performed using Mx3005P
Q-
PCR System and Brilliant
SYBR
Green Q-PCR
Master Mix (Stratagene, La Jolla, CA, USA). Primers
(Supplementary File S1), 23–27 oligomers, for selected
genes were designed to generate a product of 200–
250 bp, and to have a T
m
(melting temperature) of
60±3C using the Primer 3 program of Biology Work-
Bench (http://www.workbench.sdsc.edu/). A dissocia-
tion (melting) curve was run for each sample at the end
of the amplification reaction to verify a single product
was amplified in the PCR reaction (95C for 1 min,
55C for 30 s and 95C).
Reactions contained 12.5 llof2·Brilliant SYBR
Green Q-PCR Master Mix, 5 ll of tenfold diluted
cDNA, 2,500 nM of each gene-specific primer and di-
luted reference dye (final concentration = 300 nM) in
a final volume of 25 ll. A master mix of cDNA, 2·
SYBR
Green QPCR Master Mix, and reference dye
was prepared to reduce pipetting errors and to ensure
the same amount of reagent in each well. A threshold
of 0.1 was manually defined to obtain a threshold cycle
(C
T
) value, which is the cycle number that is required
for the SYBR
Green fluorescent signal (DRn) to cross
the threshold value. Averages and standard errors
(SEs) for C
T
values were calculated for each gene of
interest based on three replications with three different
biological samples. The following default thermal
profile was used: 95C for 10 min, 40 cycles of 95C for
30 s, 55C for 1 min, and 72C for 30 s.
The constitutively expressed cacao ACTIN gene
(P55) and Trichoderma ACTIN gene, (AY376676)
were used as expression references. PCR efficiencies
(E) of all primers were calculated using dilution curves
with five dilution points, threefold dilution, and the
equation E= [10
(–1/slope)
] – 1 as described previously
by Pfaffl (2001). To compare data from different PCR
reactions and cDNA templates, C
T
values for all genes
of interest (GOI) (C
TGOI
) were normalized to the C
T
values of ACT (C
TACT
) for each replication. The rel-
ative expression ratios of the target plant genes
(treatment/control) were computed using the following
equation: ðEGOIÞDCTGOI =ðEACT ÞDCTACT
hi
;where DC
T
=
C
TC
–C
TT
,C
TT
is the C
T
for treatment, and C
TC
is the
C
T
for the corresponding control. The data are
presented as Log
10
values of the fold induction/
repression ± standard error. The data for plant genes is
expressed as Log
10
of the induction/repression ± stan-
dard error for each Trichoderma isolate/cacao seedling
combination. Mean fold induction was calculated
after determination of individual replication fold
induction.
Since some target genes failed to amplify in specific
Trichoderma isolate/cacao seedling combinations or in
fungal mycelia controls, the data for fungal ESTs are
presented as Log
10
of expression levels (EXP
GOI
)
normalized against Trichoderma ACTIN (Funact)
using the equation EXP
GOI
=E
GOI
DC
where DC=C
TACT
–
C
TGOI
,C
TACT
is the C
T
for Funact in the sample, and
C
TGOI
is the C
T
for the corresponding gene of interest
in the sample. The ratios of Fungal EST expression in
treatment/Fungal EST expression in mycelia were
calculated as described above for fungal ESTs where
appropriate controls were available.
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123
Results
Soluble inhibitory metabolite production
and mycoparasitism
The Trichoderma isolates varied in their abilities to
produce metabolites that inhibited growth of M. ror-
eri, the Frosty Pod pathogen (Table 1). Culture fil-
trates from DIS 172ai grown in MIN medium
completely inhibited growth of M. roreri when
incorporated into MIN agar while the DIS 219f fil-
trate inhibited growth of M. roreri 46%. Culture fil-
trates from the other two isolates were not inhibitory.
The isolates studied also varied in their ability to
colonize M. roreri (Table 1). DIS 70a, DIS 219b, and
DIS 219f completely colonized the psuedostroma of
M. roreri on precolonized plates after 5 weeks. DIS
172ai was a poor mycoparasite of M. roreri eliminat-
ing the pathogen on only 12% of the agar plugs from
precolonized plates after 5 weeks.
Enzyme assays for T. ovalisporum isolate DIS 70a
b-Glucanase, cellulase, polygalacturonase, and prote-
ase activities were detected in filtrates of DIS 70a li-
quid cultures grown on cacao extract or V8 Juice as
substrate (Table 2). The b-glucanase, cellulase, poly-
galacturonase, and protease activities were also de-
tected when carboxymethyl cellulose, cellulose,
polygalacturonic acid, and gelatin were used as sub-
strates, respectively. The b-glucanase, cellulase, and
protease enzyme activities detected showed a depen-
dence on the substrate used with cacao extract and V8
juice serving as complex substrates inducing multiple
enzyme activities. Polygalacturonase activity was par-
ticularly high among the enzymes assayed.
Colonization of cacao seedlings
DIS 219b and DIS 219f caused intense discoloration of
the root surface of cacao seedlings within 6 days of
Table 1 Colonization of cacao seedlings and antibiosis and mycoparasitism of Moniliophthora roreri by endophytic Trichoderma
isolates
Control Trichoderma isolate
DIS 70a DIS 219b DIS 219f DIS 172ai LSD
0.05
Antibiosis
a
–2 11 46 100 36
Mycoparasitism
b
100 100 100 12 4
Discoloration rating
c
0.1 1.7 3.9 3.0 2.8 0.9
Seedling colonization (plate culture)
d
Root 0 75 87 100 100 26
Stem 0 100 100 87 63 36
Cotyledon 0 87 100 100 100 17
Plumule 0 50 100 50 50 67
Seedling colonization (magenta box culture)
e
Root 0 100 100 100 100 0
Stem 0 100 100 100 100 0
Bark 0 100 100 100 100 0
Xylem 0 75 100 100 67 37
Cotyledon 0 100 100 100 100 0
Plumule 0 80 100 100 100 26
Leaf 0 60 100 100 87 35
Colonization studies included pregerminated cacao seeds inoculated with agar plugs of the Trichoderma isolate. The Trichoderma
isolates were allowed to colonize the seedlings. Ratings for root discoloration were made and seedling colonization determined.
Alternatively, the seedlings were planted in sterile soil and grown 2 weeks prior to determining seedling colonization. Studies of
antibiosis and mycoparasitism were carried out as described in the Materials and methods
a
Means represent percent inhibition of growth of M. roreri on plates including filtrates from Trichoderma isolates grown on MIN
medium
b
Means represent percentage of plate precolonized with M. roreri where Trichoderma isolate was re-isolated and M. roreri eliminated
c
Rating for discoloration of the root zone was determined using a scale of 0 (no discoloration) to 4 (dark brown discoloration) prior to
harvest
d
Seven days after inoculation, seedlings were harvested, cut into sections, surface sterilized, and plated on cornmeal agar. Data
represents mean percentage of tissue pieces colonized by the test Trichoderma isolate
e
Seedlings were planted in sterile soil in magenta box culture. Magenta-box-culture seedlings were sampled similar to plate culture
seedlings. Data represents mean percentage of tissue pieces colonized by the test Trichoderma isolate
Planta
123
inoculation while DIS 172ai and DIS 70a caused less
intense root responses but did discolor cacao roots
(Table 1). The roots of uninoculated seedlings re-
mained white. Using 10·to 50·magnification with a
stereoscopic microscope, all four fungi were observed
growing on the surface of inoculated cacao seedlings.
When seedling tissues were surface sterilized and pla-
ted on cornmeal dextrose agar (CDA) plates, the
Trichoderma isolates were observed growing out of all
cacao tissues. All four isolates could be re-isolated
from all parts of colonized seedlings (Table 1). When
considered across the Trichoderma isolates tested,
plumules were least colonized at 62.5% and cotyledons
were most heavily colonized at 96.8%. DIS 219b ten-
ded to be the most aggressive in colonizing cacao
seedlings under the conditions used. When colonized,
germinating seeds were planted in sterile soil in ma-
genta box culture, the seedlings emerged and grew
normally. The Trichoderma isolates were observed
growing out of surface sterilized cacao tissue isolated
from magenta box grown seedlings when plated on
CM-dextrose agar (Table 1). Under the conditions
used, Trichoderma isolates maintaining the colony
morphologies for the isolates used in the inoculations
were the only microbes observed growing out of the
cacao tissues. DIS 219b and DIS 219f completely col-
onized all tissues sampled and, roots, stems, bark, and
cotyledons were completely colonized by all four iso-
lates. Among the additional tissues sampled from
emerged seedlings in the magenta box culture, leaf and
xylem tended to be less colonized although all four
isolates were isolated from these tissues more than
once.
Differential display analysis
Differential display was carried out using total RNA
from cacao seedlings individually colonized by DIS
70a, DIS 219b, DIS 219f, and DIS 172ai plus several
Trichoderma isolates not further studied. 164 EST
clones were sequenced and analyzed for putative pro-
tein function. A total of 116 independent ESTs were
identified and a putative function or conserved domain
identified for 59 ESTs (Supplementary File S2). Thirty-
nine ESTs were most closely aligned with plant genes
and 16 ESTs were most closely aligned with fungal
genes. The majority (11 of 16) of ESTs aligning with
fungal genes originated from DIS 219b-colonized
seedlings.
Macroarray analysis of cacao gene expression
in response to endophytic colonization by
Trichoderma isolates
An additional 21 ESTs (Supplementary File S2) iden-
tified from previous studies of cacao stress responses
were included in the macroarray (Bailey et al. 2005a,b).
Fifty-nine ESTs of cacao origin and 57 ESTs of uncer-
tain origin (Trichoderma species or cacao) were in-
cluded in the macroarray analysis (Supplementary File
S2). The fungal ESTs were included on the macroarray
but were generally not detectable by macroarray
Table 2 Enzyme assays for T. ovalisporum isolate DIS 70a
Substrate Mycelium
a
g dry wt b-Glucanase
b
Cellulase
b
Pgase
c
Protease
d
Glycerol 0.010(0.001)
e
BDT BDT 8.10(0.71) 0.005(0.002)
CMC 0.007(0.001) 2.58(0.03) – – –
Cellulose 0.042(0.001) – 0.08(0.04) – –
PGA 0.011(0.001) – – – 0.026(0.004)
Cacao 0.026(0.004) 1.01(0) 0.64(0.03) 3.89(0.13) 0.013(0.003)
V8 Juice 0.110(0.040) 0.21(0.01) 0.10(0.02) 0.27(0.06) 0.009(0.003)
Isolate DIS 70a was grown 8 days in Wiendling’s minimal salts plus 0.2% carboxymethylcellulose, crystalline cellulose, glycerol, pectin,
or powdered lyophilized cacao seedling. DIS 70a was also grown in V8 broth. Three replicate cultures of each isolate were prepared
from each medium. The culture filtrates were assayed for b-glucanase, cellulase, chitinase, polygalacturonase, pectate lyase, and
protease activities. Autoclaved filtrate was used as negative controls in determinations of all enzyme activities. Pectate lyase and
chitinase activities were below the level of detection
BDT Below the level of detection, – not done
a
Dry weight of mycelium in 25 ml culture filtrate
b
One unit of b-glucanase or cellulase activity is the amount of enzyme that released 1 lg glucose reducing equivalent per minute per g
dry weight mycelium
c
One unit of polygalacturonase (Pgase) activity is the amount of enzyme that released 1 lg galacturonic acid reducing equivalent per
min per g dry weight mycelium
d
One unit of protease activity was the amount of enzyme that increased absorbance at 520 nm one unit per hour per g dry weight
mycelium
e
Numbers in parenthesis represent standard errors
Planta
123
analysis and these results are not further discussed
(Supplementary File S2). Sixty ESTs could be detected
on the macroarrays in at least one of the treatment
combinations, 32 ESTs of plant origin related to pub-
lished sequences and 28 ESTs of uncertain origin
(Trichoderma species or cacao) unrelated to reported
sequences. Many of the EST clones included on the
macroarray were not detected using probes derived
from total RNA isolated from either fungal-colonized
or control cacao tissues suggesting their expression
levels were low in planta. Data on a subset of the ESTs
carried forward to QPCR are presented in Fig. 1. Ca-
cao-derived ESTs P3, P4, P13, P26, P59, and U4 were
induced due to colonization by at least three of the four
isolates studied with the greatest induction being 18.5-
fold for EST P59 when cacao seedlings were colonized
by DIS 219b. Expression of EST P12 was repressed in
cacao seedling due to colonization by isolates DIS 172ai
and DIS 219b. Colonization by Trichoderma isolates
DIS 219b and DIS 172ai resulted in very similar pat-
terns of both induction and repression for the cacao-
derived ESTs being studied.
Real-time quantitative-PCR analysis
A subset of 19 cacao-derived ESTs (Table 3) was se-
lected for further study using Q-PCR based partially
on the macro-array results (induced or repressed) and
in some cases on the tentative ID of the gene itself.
This allowed analysis of some genes that were not
detected on the macro-array but were of particular
interest based on their putative function. In addition,
17 ESTs (Table 3) characterized as fungal in origin
based on sequence analysis were used for Q-PCR
analysis. For the ESTs showing similarity to plant gene
sequences, all were detectible in the non-colonized
cacao seedling at some level. In some cases, depending
upon the isolate being studied, primer sets for fungal
genes failed to amplify the target gene transcript in
colonized plant tissue or in the control fungal biomass.
In eight fungal EST/fungal isolate combinations, seven
of which occurred with DIS 70a, the primer sets for
fungal genes amplified target gene transcript in colo-
nized plant tissue but not in the control fungal biomass.
In all cases the targeted gene could be amplified using
fungal genomic DNA as a template. In three fungal
EST/fungal isolate combinations, the target transcript
could be amplified in the control fungal biomass and
not in the colonized plant.
T. ovalisporum-isolate DIS 70a interaction
with cacao
Based on real time Q-PCR analysis of colonization of
cacao seedlings, isolate DIS 70a induced accumulation
of transcripts for five plant ESTs more than twofold
(P1, P4, P13, P59, and U4) (Fig. 2a). Transcript levels
for ESTs P1, P4, P59, and U4 accumulated in DIS 70a-
colonized tissues to levels fivefold more than these
found in the controls. EST P59 was most highly in-
duced, accumulating in DIS 70a-colonized tissues to
levels 60-fold more than levels found in non-colonized
seedlings. Transcripts for plant ESTs P12, P31, and P41
decreased in DIS 70a-colonized tissues more than 50%
with P12 being decreased more than fourfold.
Transcripts for 12 fungal ESTs including FunTef1
and FunAct (Fig. 2b) were detected in cacao seedlings
colonized by DIS 70a. FunTef1 was not induced during
colonization, showing a constitutive expression level in
planta and in pure fungal mycelia relative to FunAct.
ESTs F3 and F9 were most strongly induced at 6.4-fold
and 2.4-fold, respectively, of the expression levels ob-
served in liquid-culture-grown DIS 70a mycelia.
Transcripts of seven fungal ESTs (F2, F5, F7, F11, F12,
F14, and F19) were detected in colonized seedlings but
were not detected in liquid-culture-grown mycelia.
T. hamatum isolate DIS 219b interaction with cacao
Transcript levels for eight plant derived ESTs (P1,
P4, P13, P26, P29, P57, P59, and U4) accumulated
in seedlings colonized by DIS 219b to more than
Fig. 1 Expression levels and fold induction/repression values for
11 cacao ESTs on macroarrays probed with cDNA derived from
cacao seedlings colonized with four Trichoderma isolates. Six
days were allowed for colonization of cacao seedlings on water
agar before harvest. Sterile cacao seedlings were maintained as
controls. The data are presented as the Log
10
value of the gene of
interest (GOI) expression level normalized against cacao ACTIN
(P55). The fold induction/repression in response to colonization
is presented numerically over expression level bars. Fold changes
in expression less than ±2 are indicated by ns (nonsignificant)
Planta
123
threefold the levels observed in non-colonized seed-
lings (Fig. 3a). Transcript levels for plant ESTs P1, P4,
P59, and U4 were induced more than tenfold in seed-
lings colonized by DIS 219b. Transcript levels for plant
EST P44 decreased fourfold and transcripts for plant
ESTs P12 and P31 decreased more than 100-fold in
these same seedlings.
Fifteen fungal genes were detected in cacao seed-
lings colonized by DIS 219b including FunTef1 and
FunAct (Fig. 3b). FunTef1 transcript was induced 4.5-
fold in the DIS 219b-colonized cacao seedlings com-
pared to transcript levels observed in liquid-culture-
grown DIS 219b mycelia. Transcript levels for six
fungal ESTs (F4, F6, F12, F13, and F15) were induced
less than FunTef1. Of the remaining fungal ESTs
whose transcripts were detected in DIS 219b-colonized
cacao seedlings, five were induced more than 20-fold
with F3, F7, and F11 being most highly induced at 125-
fold, 2514-fold, and 116,653-fold, respectively. Tran-
scripts for EST F21 were detected in liquid-culture-
grown mycelia but not in DIS 219b-colonized cacao
seedlings.
DIS 219f-T. harzianum interaction with cacao
Plant ESTs P1, P4, P13, and P57 were induced in DIS
219f-colonized seedlings by just over twofold compared
to non-colonized, seedlings, respectively. Transcripts
Table 3 Summary of ESTs studied in cacao seedlings colonized by Trichoderma species using macro-array and Q-PCR analysis
Clone
no.
Accession
no.
Size
(bp)
Source
a
Clone ID Putative ID Identity(%)/
expected ratios
P1 DW246134 >389 DIS 219b AF029349 Lycopersicon esculentum/ornithine decarboxylase 69/1E-56
P3 DW246135 646 DIS 219b CNS0A0ZN Arabidopsis thaliana/unknown protein 81/4E-95
P4 DW246136 321 DIS 219b AB087837
NM_120357
Pisum sativum /glutathione S-transferase
Arabidopsis thaliana /In2-1 protein, putative
65/6E-9
63/6.0E-8
P12 DW246137 407 DIS 70a AM117766 Theobroma cacao/unknown protein/extensin-like protein
Protease inhibitor/seed storage/LTP family
97/5E-80
P13 DW246138 215 DIS 219b CA992708 Gossypium hirsutum /EST-Zinc-Finger protein, putative
Cys2-His2 type
87/7.0E-12
P18 DW246140 415 DIS 219b NM_148885 Arabidopsis thaliana/Zinc-Finger protein 74/2E-25
P20 DW246141 354 DIS 219b AAM62748 Arabidopsis thaliana/B-cell receptor-associated protein 67/4E-9
P25 DW246142 313 DIS 219b NM_122210 Arabidopsis thaliana/expressed protein 86/7E-55
P26 DW246143 190 DIS 219b CA798633 Theobroma cacao/Putative wound protein 100/4.0 E-30
P29 DW246144 440 DIS 70a AF531362 Gossypium barbadense/EF-hand, calcium binding motif 92/6E-49
P30 DW246145 363 DIS 70a CK144295 Theobroma cacao/ORFX/fw2.2-like 99/3E-78
P31 DW246146 662 DIS 219f BT012976 Lycopersicon esculentum/tonoplast intrinsic protein 89/2E-75
P40 572 Published CK144296 Theobroma cacao/Apoplastic quiacol peroxidase-like
P41 303 Published CK144297 Theobroma cacao/Photosystem I 24 kDa protein-like
P44 557 Published CF973685 Theobroma cacao/Chitinase-TcChiB
P55 477 Published CA797197 Theobroma cacao/Actin
P57 423 Published CF974274 Theobroma cacao/chloroplast elongation factor
P59 DW246148 517 DIS 219b AF503442 Nicotiana spp./Nectrin 5-glucose oxidase activity 73/3E-28
U4 DW246147 356 DIS 219b No homology
F2 DW246118 273 DIS 219b XP_369811 Magnaporthe grisea/Vacuolar ATP synthase subunit 89/4.0 E-07
F3 DW246119 621 DIS 219b XM_654907 Aspergillus nidulans /Glycosyl hydrolases family 2 69/4.0 E-73
F4 DW246120 461 DIS 219b AY850350 Magnaporthe grisea /eukaryotic trans. initiation factor 3 70/4.0 E-10
F5 DW246121 377 DIS 219b XM_385607 Gibberella zeae/Phenylalanyl-tRNA synthetase 86/8.0 E-23
F6 DW246122 300 DIS 219b XM_369855 Magnaporthe grisea/ATP dependent DNA ligase 93/2.0 E-10
F7 DW246123 243 DIS 219b XM_745951 Aspergillus fumigatus /Glycosyl hydrolase family 7 78/4.0 E-15
F9 DW246124 216 DIS 219b XM_327884 Neurospora crassa/Uracil phosphoribosyltransferase 72/4.0 E-18
F11 DW246125 247 DIS 219b AY258899 Trichoderma hamatum/alkaline proteinase 100/1.0 E-53
F12 DW246126 536 DIS 219b XM_322385 Neurospora crassa/Enoyl-CoA hydratase/isomerase 71/6.0 E-55
F13 DW246127 518 DIS 219b XM_390361 Gibberella zeae/DnaJ molecular chaperone 62/2.0 E-55
F14 DW246128 263 DIS 219b CF872154 Trichoderma reesei/nuclear pore membrane glycoprotein 64/2.0 E-04
F15 DW246129 514 DIS 70a XM_386421 Gibberella zeae/actin depolyem. factor/cofilin-like 73/5.0 E-28
F19 DW246131 695 DIS 219f AF232903 Cochliobolus victoriae/alcohol oxidase 76/1.0 E-103
F20 DW246132 363 DIS 219f XM_389986 Gibberella zeae/scavenger mRNA decapping enzyme 81/8.0 E-44
F21 DW246133 264 DIS 219f CK434057 Trichoderma harzianum/unknown 65/2.0 E-28
FunTef1 230 Published AF348101 Trichoderma harzianum/TEF1
Funact 283 Published AY376676 Trichoderma sp./actin
a
Source for P#, U#, and F# ESTs represents the cacao/Trichoderma isolate combination from which the EST was isolated using
Differential Display
Planta
123
for plant ESTs P26, P59, and U4 increased 5.4-fold,
19.2-fold, and 8.5-fold, in seedlings colonized by DIS
219f as compared to non-colonized seedlings (Fig. 4a).
Transcript levels for plant EST P12 decreased by more
than twofold in DIS 219f-colonized seedlings when
compared to non-colonized seedlings.
Transcripts for five fungal ESTs were detected in
cacao seedlings colonized by DIS 219f including
FunTef1 and FunAct (Fig. 4b). FunTef1 was not in-
duced during colonization, showing a consistent
expression level in seedlings colonized by DIS 219f
and in pure fungal mycelia when compared to FunAct
expression levels. ESTs F9 and F21 accumulated in
cacao seedlings colonized by DIS 219f to levels 99.5
and 6.7-fold levels detected in mycelia grown in liquid
culture, and EST F19 was induced more than
1,000,000-fold in DIS 219f-colonized seedlings. Tran-
scripts for ESTs F4, F15 and F20 were detected in
liquid-culture-grown DIS 219f mycelia but not in DIS
219f-colonized seedlings.
DIS 172ai-T. species interaction with cacao
Transcript levels for 7 of 20 plant ESTs (P1, P4, P13,
P26, P29, P59, and U4) accumulated in seedlings col-
onized by DIS 172ai to more than threefold the levels
observed in non-colonized seedlings (Fig. 5a). Tran-
scripts for plant ESTs P1, P4, P59, and U4 accumulated
in seedlings colonized by DIS 172ai to levels that were
tenfold higher than the levels observed in non-colo-
nized seedlings. Transcripts for plant ESTs P12 and
P31 decreased by more than tenfold in these same
colonized seedlings when compared to transcript levels
observed in non-colonized seedlings.
Transcripts for eight fungal ESTs were detected in
the DIS 172ai-colonized cacao seedlings including
Fig. 2 Fold induction and expression levels for cacao and fungal
ESTs in cacao seedlings colonized by Trichoderma ovalisporum
DIS 70a. aData for plant ESTs is presented as Log
10
of the fold
induction/repression. bData for fungal ESTs is presented as
Log
10
EXP
GOI
for expression level of each EST relative to
Fungal ACTIN (Funact). Bars represent Fungal EST expression
in colonized plant tissue (Plant) or pure fungal mycelia
(Mycelia). The fold induction/repression in response to coloni-
zation is presented numerically over expression level bars where
ESTs were amplified in both treatment and control samples
Fig. 3 Fold induction and expression levels for cacao and fungal
ESTs in cacao seedlings colonized by Trichoderma hamatum DIS
219b. aData for plant ESTs is presented as Log
10
of the fold
induction/repression. bData for fungal ESTs is presented as
Log
10
EXP
GOI
for expression of each EST relative to Fungal
ACTIN (Funact). Bars represent Fungal EST expression in
colonized plant tissue (Plant) or pure fungal mycelia (Mycelia).
The fold induction/repression in response to colonization is
presented numerically over expression level bars where ESTs
were amplified in both treatment and control samples
Planta
123
ESTs FunTef1 and FunAct (Fig. 5b). EST F3 was most
strongly induced in planta at 15.2-fold the level found
in liquid-culture-grown mycelia. Expression levels for
the remaining seven ESTs were not altered during
colonization. Transcript for F19 was not detected in
liquid-culture-grown DIS 172ai mycelia but was de-
tected in colonized cacao seedlings.
Discussion
Trichoderma isolates with biocontrol potential are
endophytic on cacao
Trichoderma ovalisporum DIS 70a has been more
intensively studied than the other three isolates tested
here (Holmes et al. 2004). In studies by Holmes et al.
(2004), and verified here, DIS 70a completely colo-
nized the pseudostroma of the Frosty Pod pathogen
M. roreri in plate culture screens. DIS 70a established
an endophytic association with cacao seedlings in
greenhouse studies and could be re-isolated from the
apical meristem and the younger tissues (Holmes et al.
2004). DIS 70a was also observed overgrowing the
pseudostroma of M. roreri on pods in the field and
could be re-isolated from surface-sterilized pods
10 weeks after the inoculation (Holmes et al. 2004).
Less is known about the biocontrol abilities of the
other three isolates tested. However, as shown here
(Table 1), T. harzianum isolate DIS 219f and T.
hamatum isolate DIS 219b actively colonized colonies
of M. roreri grown on 20% PDA. Trichoderma sp.
Isolate DIS 172ai was a poor mycoparasite of M. roreri
but produced metabolites capable of completely
inhibiting growth of M. roreri in culture. The culture
filtrates of the other three isolates were significantly
Fig. 4 Fold induction and expression levels for cacao and fungal
ESTs in cacao seedlings colonized by Trichoderma harzianum
DIS 219f. aData for plant ESTs is presented as Log
10
of the fold
induction/repression. bData for fungal ESTs is presented as
Log
10
EXP
GOI
for expression of each EST relative to Fungal
ACTIN (Funact). Bars represent Fungal EST expression in
colonized plant tissue (Plant) or pure fungal mycelia (Mycelia).
The fold induction/repression in response to colonization is
presented numerically over expression level bars where ESTs
were amplified in both treatment and control samples
Fig. 5 Fold induction and expression levels for cacao and fungal
ESTs in cacao seedlings colonized by Trichoderma species DIS
172ai. aData for plant ESTs is presented as Log
10
of the fold
induction/repression. bData for fungal ESTs is presented as
Log
10
EXP
GOI
for expression of each EST relative to Fungal
ACTIN (Funact). Bars represent Fungal EST expression in
colonized plant tissue (Plant) or pure fungal mycelia (Mycelia).
The fold induction/repression in response to colonization is
presented numerically over expression level bars where ESTs
were amplified in both treatment and control samples
Planta
123
less inhibitory toward M. roreri. Antibiosis, the pro-
duction of antimicrobial compounds, and mycopara-
sitism, the feeding on a fungus by another organism,
are major mechanisms whereby Trichoderma species
provide protection to plants against plant pathogens
(Chet et al. 1998; Howell 2003; Harman et al. 2004).
Data presented here (Table 1) verify that all four
isolates studied are capable of establishing endophytic
associations with cacao. They differ in their abilities to
cause root discoloration but all four isolates were able
to colonize all plant parts. Root discoloration was
superficial and cacao seedlings grew normally when
planted. The root discoloration may be related to the
synthesis of antimicrobial compounds. The induction
of terpenoid biosynthesis was closely correlated with
the ability of Trichoderma virens isolates to protect
cotton seedlings against Rhizoctonia solani (Howell
et al. 2000). Our results indicate DIS 219f and DIS
219b are slightly more aggressive in colonizing cacao
seedlings than DIS 70a and DIS 172ai, based on the
root discoloration and colonization data.
Additional data on enzyme production in response
to growth on cacao extract indicates T. ovalisporum
isolate DIS 70a has many of the enzyme activities re-
quired to breakdown plant cell wall components
including b-glucanase, cellulase, polygalacturonase,
and protease activities (Table 2). Particularly, polyga-
lacturonase activity, required for degradation of the
middle lamella, may be essential for movement of
Trichoderma isolates between cacao cells. b-Glucan-
ase, cellulase, and protease activities have also been
associated with the ability of Trichoderma isolates to
parasitize plant pathogens (Chet et al. 1998).
Gene expression in cacao shows endophyte-
isolate-specific induction patterns
All the plant ESTs studied that showed altered
expression responded to colonization by more than one
Trichoderma isolate suggesting some similarity of re-
sponse across isolates (Table 4). The expression pro-
files of cacao ESTs were not identical when
comparisons were made between Trichoderma isolate/
cacao interactions ESTs indicating Trichoderma isolate
dependent differential regulation.
Careful study of the plant responses to the four
endophytes suggests these four groupings for the plant
genes showing altered gene expression (Table 4): ESTs
P1, P4, P12, P13, and P31; EST P26; EST P29; and
ESTs P59 and U4. Where grouped with other ESTs,
the ESTs showed coordinated expression patterns
being either induced or repressed together. Based on
the QPCR results, cacao ESTs P1, P4, and P13 were
induced in response to colonization by all four Trich-
oderma isolates but only twofold by DIS 219f. EST P12
was repressed in cacao seedlings by colonization with
all four Trichoderma isolates, only 60% with DIS 219f
and, EST P31 was repressed in cacao seedlings colo-
nized by DIS 70a, DIS 219b, and DIS 172ai. EST P26
was induced in response to colonization with DIS 219f,
DIS 219b, and DIS 172ai. EST P29 was induced in
Table 4 Summary table for Q-PCR results indicating induction/
repression of ESTs identified in Trichoderma spp./Theobroma
cacao interactions
Clone Trichoderma isolate
DIS 70a DIS 219b DIS 219f DIS 172ai
P1 +
a
2+ + 2+
P3 d
b
ddd
P4 + 2+ + 2+
P12 –
c
2– – 2–
P13 + 2+ + +
P18 – d – d
P20 d d d d
P25 d d d d
P26 d + + +
P29 d + d +
P30 d d d d
P31 – 2– d 2–
P40 d d d d
P41 – d d d
P44 d d d –
P55 d d d d
P57 d + + d
P59 2+ 2+ 2+ 2+
U4 2+ 2+ + 2+
F2 ¥
d
+ndnd
F3 + 3+ nd ++
F4 d + nd d
F5 ¥+ndnd
F6 nd
e
+ndnd
F7 ¥4+ nd nd
F9 + 2+ 2+ d
F11 ¥6+ nd nd
F12 ¥+ndnd
F13 nd + nd nd
F14 ¥+nd–
F15 d – nd d
F19 ¥2+ 7+ ¥
F20 nd nd nd nd
F21 nd nd + nd
FunTef1 – + + –
FunAct d d d d
a
+, 2+, 3+, 4+, 5+, 6+, and 7+ represent 2, 10
1
,10
2
,10
3
,10
4
,10
5
,
10
6
-fold induction of gene expression
b
d, detected but not induced
c
– and 2– represent 2 and 10
1
-fold repression of gene expression
d
¥, combinations were fungal gene expression was detected in
planta but not in the control mycelia. At least a 16-fold induction
is predicted for fungal transcripts detected in planta but not in
control mycelia
e
nd, expression was not detected in planta
Planta
123
cacao seedlings by DIS 219b and DIS 172ai. ESTs P59
and U4 were induced more than eightfold in cacao
seedlings by colonization with each of the four Trich-
oderma isolates studied.
Several of the cacao ESTs induced due to coloni-
zation by these Trichoderma isolates share homology
with genes reported to function in plant responses to
environmental stresses. EST P1 shows close homology
to the gene for ornithine decarboxylase (Table 3), a
primary control point in polyamine biosynthesis.
Ornithine decarboxylase and polyamines have been
associated with developmental changes (Walden et. al.
1997), abiotic stresses such as drought (Capell et al.
2004), and resistance to biotic stresses including plant
disease (Yoo et al. 2004; Walters 2000). EST P13 is
related to zinc finger proteins (Table 3) commonly
associated with responses to biotic and abiotic stresses
including plant symbiont and plant pathogen interac-
tions (Kim et al. 2004). EST P4 is related to a family of
glutathione-S-transferase (GST)-like proteins (Ta-
ble 3). Among their many activities GSTs have a broad
role in protecting cells from oxidative injury by
detoxifying compounds that would otherwise damage
plant cells (Dean et al. 2005). The activity of GSTs
contributes to resistance of plants to both biotic and
abiotic stress (Dixon et al. 2002; Perl-Treves et al.
2004). Included within the GST family are the In2-
related genes. Although the function of In2-related
genes is unknown, they have been shown to carry
GSH-dependent thiol transferase activity that could
dethiolate S-glutathionylated proteins that accumulate
during oxidative stress (Dixon et al. 2002; Dixon et al.
2005). EST P26 is nearly identical to a previously
published cacao EST CA798633 (Table 3) that is re-
lated to a Medicago sativa gene (MSA248337) encod-
ing a putative wound-induced protein (BLASTX,
Identity = 61%, Expect = 5 E-15) with unknown
function and associated with alfalfa nodule develop-
ment (Jimenez-Zurdo et al. 2000) and general wound
responses (Parsons and Mattoo 1991). EST P29 carries
an EF-hand, calcium-binding motif (Table 3). EF-
hand, calcium-binding motifs are found in a diverse
super-family of proteins involved in calcium signaling
leading to the targeted activation or inactivation of
proteins. Proteins carrying EF-hand, calcium-binding
motifs are often characterized as calmodulin-like pro-
teins and are involved in various developmental pro-
cesses and responses to stress (McCormack et al. 2005).
P59 shares homology to a recently characterized family
of carbohydrate oxidase encoding genes in plants that
also share homology with berberine bridge-forming
enzymes (Carter and Thornburg 2004;Huetal.2003).
These carbohydrate oxidases produce hydrogen
peroxide as a product and have been shown to be
important in plant defense against microbes (Carter
and Thornburg 2004; Hu et al. 2003). EST U4 has no
known homology to established sequences.
Expression of plant ESTs P12 and P31 was repressed
in cacao by colonization with the Trichoderma isolates
studied although to a much lesser degree in seedlings
colonized by DIS 70a and DIS 219f. EST P12 is nearly
identical to T. cacao EST AM117766 where they
overlap (BlastN, Identity = 97%, Expect = 5 E-80)
and, EST CA796489 is closely related to a group of
extensin-like proteins (Table 3). Extensins are typically
associated with cell walls but the exact function of this
group of extensin-like proteins is unknown (Hotze et al.
1994). P31 is closely related to the major intrinsic pro-
tein (MIP) superfamily (Table 3). Members of the MIP
superfamily in plants, also called aquaporins, function
as membrane channels that selectively transport water,
small neutral molecules, and ions out of and between
cells. A possible correlation comes from observations in
tobacco (Smart et al. 2001) and Arabidopsis (Jang et al.
2004) that transcript levels for some aquaporins decline
in response to drought and drought is known to induce
polyamines biosynthesis in plants (Capell et al. 2004).
The repression of MIP gene expression may reduce
membrane water permeability and encourage water
conservation during periods of drought (Smart et al.
2001). Enhanced drought tolerance is commonly asso-
ciated with endophyte-colonized grasses (Schardl et al.
2004) and has been demonstrated with root-colonizing
T. harzianum isolate T22 (Harman 2000). Enhanced
drought tolerance would be a valuable trait in cacao
since the crop is very sensitive to prolonged drought, a
problem exacerbated by the cultivation of cacao in full
sun (Belsky and Siebert 2003).
Fungal gene expression is altered during
colonization of cacao seedlings
The pattern of detectable fungal gene expression var-
ied with the Trichoderma isolate being studied (Ta-
ble 4). The ESTs showing the greatest level of
induction during colonization of cacao seedlings were
EST F7 for DIS 70a, EST F11 for DIS 219b, EST F19
for DIS 219f, and EST F3 for DIS 172ai. It is notable
that some fungal transcripts were detected in planta
but not in liquid-culture-grown fungal mycelia, this
occurring most often with DIS 70a. Several transcripts
were detected in liquid-culture-grown mycelia but not
in planta. At least 16-fold induction can be predicted
for those fungal transcripts detected in planta and not
detected in mycelia grown in clarified V8 broth. The
primer sets used for amplifying fungal cDNAs for
Planta
123
Q-PCR were effective in most cases in amplifying the
target cDNAs for DIS 219b (18) and DIS 70a (13) and
less so for DIS 219f (8) and DIS 172ai (7). The majority
of the fungal EST clones originated from DIS 219b-
colonized cacao seedlings and, DIS 172ai was not used
in the differential display from which the ESTs were
derived. Some of the differences are likely due to
sequence differences for the gene in question between
the Trichoderma isolates used in combination with the
specific primer sets used for Q-PCR. The primer sets
used for each fungal-derived EST were developed from
a single Trichoderma isolate (Table 3). The diversity of
ESTs studied allowed identification of functional
primer sets for all four Trichoderma isolates (species)
studied.
The fungal ESTs identified can be loosely grouped
into three groups: ESTs for enzymes potentially in-
volved in nutritional support of the fungus and direct
interaction with cacao tissue (F3, F7, F11, F12, and
F19); ESTs for enzymes involved in basic cell functions
(F2, F4, F5, F6, F12, F13, F15); and ESTs with un-
known function (F21). Included in the first group are
fungal genes associated with carbohydrate hydrolysis
(F3, F7, F19), protein digestion (F11), and lipid
metabolism (F12). These enzymes are of obvious
importance in the establishment of endophytic associ-
ations, potentially functioning in acquisition of nutri-
ents from the cacao seedling and aiding in the
colonization process itself by Trichoderma and in the
mycoparasitic interaction. Hydrolytic enzymes are
important in the parasitism of other fungi by Tricho-
derma species facilitating the digestion of cell walls
(Chet et al. 1998; Harman et al. 2004). Fungal ESTs F3
and F7 have homology with hydrolases with unspeci-
fied activities (Table 3). F3 is expressed by DIS 70a,
DIS 219b, and DIS 172ai in liquid culture and induced
during colonization of cacao by these isolates from 6 to
124-fold. EST F3 is related to genes encoding glucosyl
hydrolase family 2 proteins that have diverse activities
including b-galactosidase, b-mannosidase and b-glucu-
ronidase activities. EST F7 was detected in cacao tis-
sues colonized by DIS 70a and DIS 219b and was
highly induced in both Trichoderma species/cacao
interactions. EST F7 is most closely related to genes
encoding a newly characterized xyloglucan hydrolase
family that is thought to function in the digestion of
cell wall carbohydrates (Gielkens et al. 1999; Grishutin
et al. 2004). EST F11 encodes a serine protease
(Table 3). Serine proteases have been shown to func-
tion both in mycoparasitism by T. hamatum (Steyaert
et al. 2004) and T. harzianum (Geremia et al. 1993) and
in the endophytic association between Acremonium
typhinum and the grass, Poa ampla (Lindstrom et al.
1993). EST F12 is most closely related to genes
encoding Delta3, Delta2-enoyl-CoA isomerase, an
enzyme that is essential for the beta-oxidation of
unsaturated fatty acids (Geisbrecht et al. 1998). EST
F19 encodes an alcohol oxidase (Table 3). Alcohol
oxidase is expressed by Cladosporium fulvum in to-
mato during the disease process (Segers et al. 2001).
Coleman et al. (1997) observed that many genes ex-
pressed by fungi under starvation conditions, including
the gene encoding alcohol oxidase, were also expressed
by fungi during the plant disease process.
The Trichoderma/cacao interaction involves genetic
cross talk
The Trichoderma isolates studied were able to grow on
and in all cacao seedling tissues examined. During this
colonization process the Trichoderma isolates produce
enzymes that function in the growth and development
of the fungus through interactions with the T. cacao
seedling. Some of the fungal genes being preferentially
expressed may also function in the biocontrol activity
of the Trichoderma isolates. The Trichoderma isolate/
cacao interaction was not detrimental to cacao since
colonized cacao seedlings grew normally when trans-
planted into sterile soil. The result of this interaction
on the plant side is the discoloration of roots and the
induction of genes potentially involved in resistance to
biotic and abiotic stresses. The pattern of altered gene
expression observed in cacao and Trichoderma sug-
gests a complex system of communication involving
genetic cross talk. The interaction is Trichoderma iso-
late/species specific and results in the establishment of
an endophytic association potentially beneficial to both
participants in the plant microbe interaction. This is
the first report, to our knowledge, showing complex
isolate specific genetic cross talk between endophytes
and host plants during colonization.
References
Adejumo TO (2005) Crop protection strategies for major dis-
eases of cocoa, coffee and cashew in Nigeria. Afr J Bio-
technol 4:143–150
Arnold AE, Mejia LC, Kyllo D, Rojas EI, Maynard Z, Robbins
N, Herre EA (2003) Fungal endophytes limit pathogen
damage in a tropical tree. Proc Natl Acad Sci USA
100:15649–15654
Bailey BA, Bae H, Strem MD, Antunez de Mayolo G, Guiltinan
MJ, Verica JA, Maximova SN, Bowers JH (2005a) Devel-
opmental expression of stress response genes in Theobroma
cacao leaves and their response to Nep1 treatment and a
compatible infection by Phytophthora megakarya. Plant
Physiol Biochem 43:611–622
Planta
123
Bailey BA, Strem MD, Bae H, Antunez de Mayolo G, Guiltinan
MJ (2005b) Gene expression in leaves of Theobroma cacao
in response to mechanical wounding, ethylene, and/or me-
thyl jasmonate. Plant Sci 168:1247–1258
Bastos CN (1996) Potential de Trichoderma viride no controle da
vassoura-de-bruxa (Crinipellis perniciosa) do cacaueiro.
Fitopatol Bras 21:509–512
Belsky JM, Siebert SF (2003) Cultivating cacao: implications of
sun-grown cacao on local food security and environmental
sustainability. Agric Human Values 20:277–285
Bowers JH, Bailey BA, Hebbar PK, Sanogo S, Lumsden RD
(2001) The impact of plant disease on world chocolate
production. Plant Health Prog. DOI 10.1094/PHP-2001-
0709-01-RV (online)
Capell T, Bassie L, Christou P (2004) Modulation of the poly-
amine biosynthetic pathway in transgenic rice confers tol-
erance to drought stress. Proc Natl Acad Sci USA 101:9909–
9914
Carter CJ, Thornburg RW (2004) Tabacco nectarin V is a flavin-
containing berberine bridge enzyme-like protein with glu-
cose oxidase activity. Plant Physiol 134:460–469
Chavira R, Burnett TJ, Hageman JH (1984) Assaying protein-
ases with azocoll. Anal Biochem 136:446–450
Chet I, Benhamou N, Haran S (1998) Mycoparasitism and lytic
enzymes. In: Harman GE, Kubicek CP (eds) Trichoderma
and Gliocladium, vol 2. Taylor and Francis, London, pp
153–171
Coleman M, Henricot B, Arnau J, Oliver RP (1997) Starvation-
induced genes of the tomato pathogen Cladosporium ful-
vum are also induced during growth in planta. Mol Plant
Microbe Interact 10:1106–1109
Dean JD, Goodwin PH, Hsiang T (2005) Induction of glutathi-
one S-transferase genes of Nicotiana benthamiana following
infection by Colletotrichum destructivum and C. orbiculare
and involvement of one in resistance. J Exp Bot 56:1525–
1533
Dixon DP, Davis BG, Edwards R (2002) Functional divergence
in the glutathione transferase superfamily in plants. J Biol
Chem 277:30859–30869
Dixon DP, Skipsey M, Grundy NM, Edwards R (2005) Stress-
induced protein S-glutathionylation in Arabidopsis. Plant
Physiol 138:2233–2244
Evans HC, Holmes KA, Thomas SE (2003) Endophytes and
mycoparasites associated with an indigenous forest tree,
Theobroma gileri, in Ecuador and a preliminary assessment
of their potential as biocontrol agents of cocoa diseases.
Mycol Prog 2:149–160
Geisbrecht BV, Zhu D, Schulz K, Nau K, Morrell JC, Geraghty
M, Schulz H, Erdmann R, Gould SJ (1998) Molecular
characterization of Saccharomyces cerevisiae Delta3, Del-
ta2-enoyl-CoA isomerase. J Biol Chem 273:33184–33191
Geremia RA, Goldman GH, Jacobs D, Ardiles W, Vila SB, Van
Montagu M, Herrera-Estrella A (1993) Molecular charac-
terization of the proteinase-encoding gene, prb1, related to
mycoparasitism by Trichoderma harzianum. Mol Microbiol
8:603–613
Gielkens MM, Dekkers E, Visser J, de Graaff LH (1999) Two
cellobiohydrolase-encoding genes from Aspergillus niger
require D-xylose and the xylanolytic transcriptional activa-
tor XlnR for their expression. Appl Environ Microbiol
65:4340–4345
Grishutin SG, Gusakov AV, Markov AV, Ustonov BB, Seme-
nova MV, Sinitsyn AP (2004) Specific xyloglucanases as a
new class of polysaccharide-degrading enzymes. Biochim
Biophys Acta 1674:263–281
Harman GE (2000) Myths and Dogmas of biocontrol: changes in
perception derived from research on Trichoderma harzia-
num T-22. Plant Dis 84:377–393
Harman GE, Howell CR, Viterbo A, Chet I (2004) Trichoderma
spp.—opportunistic avirulent plant symbionts. Nat Rev
2:43–56
Holmes KA, Schroers H-J, Thomas SE, Evans HC, Samuels GJ
(2004) Taxonomy and biocontrol potential of a new species
of Trichoderma from the Amazon basin in South America.
Mycol Prog 3:199–210
Hotze M, Waitz A, Schroder J (1994) cDNA for a 14-kilodalton
polypeptide from madigascar periwinkle (Catharanthus ro-
seus). Plant Physiol 104:1097–1098
Howell CR (2003) Mechanisms employed by Trichoderma spe-
cies in the biological control of plant diseases: The history
and evolution of current concepts. Plant Dis 87:4–10
Howell CR, Hanson LE, Stipanovic RD, Puckhaber LS (2000)
Induction of terpenoid synthesis in cotton roots and control
of Rhizoctonia solani by seed treatment with Trichoderma
virens. Phytopathology 90:248–253
Hu X, Bidney DL, Yalpani N, Duvick JP, Folkerts O, Lu G
(2003) Overexpression of a gene encoding hydrogen per-
oxide-generating oxalate oxidase evokes defense responses
in sunflower. Plant Physiol 133:170–181
Jang JY, Kim DG, Kim YO, Kim JS, Kang H (2004) An
expression analysis of a gene family encoding plasma
membrane aquaporins in response to abiotic stresses in
Arabidopsis thaliana. Plant Mol Biol 54:713–725
Jimenez-Zurdo JI, Frugier F, Crespi MD, Kondorosi A (2000)
Expression profiles of 22 novel molecular markers for
organogenetic pathways acting in alfalfa nodule develop-
ment. Mol Plant Microbe Interact 13:96–106
Jones RW, Hancock JG (1987) Conversion of viridian to viridiol
by viridan-producing fungi. Can J Microbiol 33:963–966
Kim SH, Hong JK, Lee SC, Sohn KH, Jung HW, Hwang BK
(2004) CAZFP1, Cys
2
/His
2
-type zinc-finger transcription
factor gene functions as a pathogen-induced early-defense
gene in Capsicum annuum. Plant Mol Biol 55:883–904
Lindstrom JT, Sun S, Belanger FC (1993) A novel fungal pro-
tease expressed in endophytic infection of Poa species. Plant
Physiol 102:645–650
McCormack E, Tsai YC, Braam J (2005) Handling calcium sig-
naling: Arabidopsis CaMs and CMLs. Trends Plant Sci
10:383–389
Nelson N (1944) A photometric adaptation of the Somogyi
method for determination of glucose. J Biol Chem 153:375–
380
Parsons BL, Mattoo AK (1991) Wound-regulated accumulation
of specific transcripts in tomato fruit: interactions with fruit
development, ethylene, and light. Plant Mol Biol 17:453–464
Perl-Treves R, Foley RC, Chen W, Singh KB (2004) Early
induction of the Arabidopsis GSTF8 promoter by specific
strains of the fungal pathogen Rhizoctonia solani. Mol Plant
Microbe Interact 17:70–80
Pfaffl MW (2001) A new mathematical model for relative
quantification in real-time RT-PCR. Nucleic Acids Res
29:2002–2007
Purdy LH, Schmidt RA (1996) Status of cacao Witches’ Broom,
biology, epidemiology, and management. Annu Rev Phy-
topathol 34:573–594
Rubini MR, Silva-Ribeiro RT, Pomella AWV, Maki CS, Araujo
WL, dos Santos DR, Azevedo JL (2005) Diversity of
endophytic fungal community of cacao (Theobroma cacao
L.) and biological control of Crinipellis perniciosa, causal
agent of Witches’ Broom Disease. Int J Biol Sci 1:24–33
Planta
123
Samuels GJ, Pardo-Schultheiss RA, Hebbar KP, Lumsden RD,
Bastos CN, Costa JC, Bezerra JL (2000) Trichoderma
stromaticum sp. nov., a parasite of the cacao witches broom
pathogen. Mycol Res 104:760–764
Schardl CL, Leuchtmann A, Spiering MJ (2004) Symbioses of
grasses with seedborne fungal endophytes. Annu Rev Plant
Biol 55:315–340
Segers G, Bradshaw N, Archer D, Blissett K, Oliver RP (2001)
Alcohol oxidase is a novel pathogenicity factor for Clado-
sporium fulvum, but aldehyde dehydrogenase is dispens-
able. Mol Plant Microbe Interact 14:367–377
Smart LB, Moskal WA, Cameron KD, Bennett AB (2001) MIP
genes are down-regulated under drought stress in Nicotiana
glauca. Plant Cell Physiol 42:686–693
Srinivasan U, Staines HJ, Bruce A (1992) Influence of media
type on an antagonistic modes of Trichoderma spp.
against wood decay basidiomycetes. Mater Organismen
27:301–321
Steyaert JM, Stewart A, Ridgway HJ (2004) Co-expression of
two genes, a chitinase (Chit42) and proteinase (prb1),
implicated in mycroparasitisim by Trichoderma hamatum.
Mycologia 96:1245–1252
Walden R, Cordeiro A, Tiburcio AF (1997) Polyamines: small
molecules triggering pathways in plant growth and devel-
opment. Plant Physiol 113:1009–1013
Walters DR (2000) Polyamines in plant-microbe interactions.
Physiol Mol Plant Pathol 57:137–146
Wilson M (1997) Biocontrol of aerial plant diseases in agricul-
ture and horticulture: current approaches and future pros-
pects. J Ind Microbiol Biotechnol 19:188–191
Wood GAR, Lass RA (2001) Cacao, 4th edn. Blackwell, Oxford,
620 pp
Yoo TH, Park CJ, Ham BK, Kim KJ, Paek KH (2004) Ornithine
decarboxylase gene (CaODC1) is specifically induced dur-
ing TMV-mediated but salicylate-independent resistant re-
sponse in hot pepper. Plant Cell Physiol 45:1537–1542
Planta
123
