Physiological and Molecular Plant Pathology 74 (2009) 111–120

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Physiological and Molecular Plant Pathology

journal homepage: www.elsevier.com/locate/pmpp

Biological and molecular characterization of the response of tomato

plants treated with Trichoderma koningiopsis
C.A. Moreno a, F. Castillo b, A. González b, D. Bernal c, Y. Jaimes a, M. Chaparro a, C. González a,
F. Rodriguez a, S. Restrepo b, A.M. Cotes a, *
a
Centro de Biotecnologı́a y Bioindustria – Corpoica, Km 14 vı́a Occidente Bogotá-Mosquera, Colombia
b
Laboratorio de Micologı́a y Fitopatologı́a Uniandes – LAMFU, Universidad de los Andes, Carrera 1 No. 18a-10, Bogotá, Colombia
c
Centro Internacional de Agricultura Tropical, Unidad de Biotecnologı́a, Recta Cali-Palmira Km 17, Cali, Colombia

a r t i c l e i n f o a b s t r a c t

Article history: Tomato-Fusarium oxysporum f.sp. radicis-lycopersici pathosystem was used to study induced systemic
Accepted 2 October 2009 resistance elicited by Trichoderma koningiopsis (Th003) using the split root model. The ability of the
antagonist to promote plant growth was also established. Stem colonization by the pathogen was
Keywords: significantly reduced in treated plants. The induction of resistance was enhanced 6 days after elicitation
Solanum lycopersicum and when the antagonist was used in a concentration of 105 conidia per ml. Th003 application in seed
Gene expression
priming and nursery significantly stimulated plant growth. Gene expression induced by Th003 was
Induced systemic resistance
evaluated using the tomato TOM1 microarray. Plant treatment with T. koningiopsis affected mRNA levels
Trichoderma koningiopsis
Fusarium oxysporum f.sp. radicis-lycopersici of 45 genes: 41 in roots and 4 in leaves. Of particular interest was the induction of genes involved in the
jasmonic and ethylene transduction pathways found in the microarray analysis and qRT-PCR, which
suggest a temporary increment of defense related gene expression response to T. koningiopsis Th003.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction plant growth promotion [4,5]. An important mode of action for

The Solanaceae botanical family comprises more than 3000
species, including potatoes, peppers, eggplants and tomatoes
among others. Nearly 18% of the members of this family are used by
humans as vegetable crops providing a significant dietary resource
of vitamins and antioxidants [1]. Within this family, tomato
(Solanum lycopersicum L.) represents 14% of the vegetable world
production, with 120,663,000 tons produced per year. In Colombia,
this crop represents 23% of the total vegetable production;
however, production is constantly threatened by biotic constraints,
mainly fungal diseases produced by eighty-two different species of
fungi and oomycetes [2] being Fusarium oxysporum f.sp. radicis-
lycopersici one of the most limiting. Although chemical fungicides
are still considered by farmers as the major control method of these
diseases, some of them already use preparations based on biolog-
ical control agents (BCA) which is of great interest due to the
undesirable side effects of pesticides.
The use of Trichoderma spp. is considered nowadays as one of
the most promising alternatives for plant disease control [3] and
* Corresponding author. Tel.: þ57 1 4227373; fax: þ57 1 4227328.
E-mail addresses: d.bernal@cgiar.org (D. Bernal), srestrep@uniandes.edu.co
(S. Restrepo), amcotes@corpoica.org.co (A.M. Cotes).
Trichoderma spp. occurs when the biocontrol agent affects the plant
rather than the pathogen, inducing systemic resistance [6–8].
Previous studies developed with the native isolate Th003 of
Trichoderma koningii (recently re-identified as Trichoderma konin-
giopsis) have shown its ability to control different pathogens
including the system tomato-F. oxysporum and to stimulate growth
in a great variety of crops [9–12]. Additionally T. koningiopsis Th003
has the ability to induce the activity of proteins in plants, as
b-1,3-glucanases and endochitinases [13], suggesting an inducer
effect of defense responses. The use of relatively novel tools to
investigate these complex processes such as proteomic analysis,
gene expression reporter systems, and high throughput methods to
study gene function, has demonstrated that a molecular cross-talk
is established between Trichoderma spp., the plant, and the path-
ogen, and has enabled the identification of a variety of signaling
molecules that may strongly influence the life and physiology of
many crops [3].
Recently, by using an array of 15,925 genes, it was shown that
Trichoderma hamatum 382 induced foliar systemic changes in plant
physiology and disease resistance, which determined the protec-
tion phenotype of tomato plants against Xanthomonas euvesicatoria
110c [14]. However, to the best of our knowledge gene induction in
the root system has never been studied in T. koningiopsis treated
tomato plants.

0885-5765/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pmpp.2009.10.001
112 C.A. Moreno et al. / Physiological and Molecular Plant Pathology 74 (2009) 111–120
The goal of this study was to confirm that T. koningiopsis Th003
induces systemic resistance in tomato by using a split root model,
and that it enhances plant growth. Additionally, we wanted to gain
a better understanding of how T. koningiopsis enhances plant
growth and defenses using global gene expression analyses with
the EST microarray TOM1.
2. Materials and methods
2.1. Fungal isolates
T. koningiopsis Th003 and F. oxysporum f.sp. radicis-lycopersici
Fu040 were supplied by the Microorganisms Collection of the
Biological Control Laboratory (Biotechnology and Bioindustry
Center, Corpoica). The biocontrol agent was cultured 7 days at 22  C
on potato dextrose agar (PDA) medium, conidia were gently scra-
ped from PDA with Tween80 sterile solution (0.01% v/v) and filtered
through nylon mesh (38 mm). The conidial suspension was counted
by hemacytometer and adjusted to 105, 106 and 107 conidia per ml.
F. oxysporum Fu040 was subcultured on PDA and incubated at 22  C.
After fifteen days of growth the pathogen inoculum was prepared
from F. oxysporum colony by transferring 5 discs (0.5 cm diameter)
to 100 g of wheat bran supplemented with 100 ml of hydrolyzed
casein medium (glucose, 15 g; yeast extract, 1 g; hydrolyzed casein,
1 g; KH2PO4, 1.5 g; MgSO4.7H2O, 1 g; FeSO4.7H2O, 0.01 g;
MnCl2.4H2O, 0.01 g; ZnSO4.7H2O, 0.01 g; distilled water, 1000 ml)
[15]. The inoculated substrate was incubated at 22  C during 15
days with constant light.
2.2. Plant material
Tomato seeds (S. lycopersicum L. cv. Rocio ‘Syngenta Seeds INC.’)
were used for all experiments. Seeds were sown in seedling-type
preformed trays containing sterile peat and placed under green-
house conditions.
2.3. Induced resistance bioassays
Two experiments were carried out to evaluate the induced
systemic response by T. koningiopsis Th003 by using 6-week-old
tomato seedlings transplanted under the split root system
described by Fuchs et al. [16] and Duijff et al. [17] in two 432-ml
pots filled with sterile vermiculite (pH 8.4). This system was
modified since the vertical cutting in crown was not included
(Fig. 1). In the first experiment, the plants were inoculated on the
substrate around one root portion of each split plant by spreading
10 ml of a T. koningiopsis Th003 conidial suspension (105, 106 and
Fig. 1. Description of the tomato split root system method used to demonstrate the induction of resistance to Fusarium oxysporum f. sp. radicis-lycopersici Fu040 by Trichoderma
koningiopsis Th003. Details described in text. Th003 was only applied in one (cross patterns) and the pathogen was then inoculated in the other pot. (circle patterns).
107 conidia per ml) 4 days before pathogen inoculation. The other
root portion was inoculated with Fu040, for which the pathogen’s
inoculum was mixed at a proportion of 5% w/w (103 CFU/g dried
substrate) with the sterile vermiculite previously moistened and
then the initial substrate was replaced by this mixture.
In the second experiment, the effect of the inoculation time of
the biocontrol agent was evaluated. T. koningiopsis was inoculated
2, 4 and 6 days prior to F. oxysporum application. For these tests
a concentration of 106 conidia per ml of T. koningiopsis was used and
pathogen inoculation was performed as described above (Fig. 1). In
both experiments three replicates per treatment were considered,
with 12 plants per replicate, arranged in a completely randomized
design; the control plants were not inoculated with T. koningiopsis
Th003. The sample consisted in one and three plants per replicate
for the first and second experiment respectively. Plants were irri-
gated daily and fertilized with a standard nutritive solution every 3
days.
Progress of F. oxysporum f. sp. radicis-lycoperisci in stem was
recorded to determine the induced systemic resistance ISR effect by
T. koningiopsis Th003. Samples were taken along the stem from 1 to
6 cm above ground, seven, fourteen, twenty-one and twenty-eight
days after pathogen inoculation. The entire stem segment was
harvested and washed with sterile water, then it was surface-
sterilized with ethanol (75%) for 30 s and then with NaOCl (2.5%) for
2 min and rinsed twice in sterile water for 2 min, and finally cut into
1 cm pieces. Each piece was longitudinally cut into two portions
and placed on Petri dishes containing rose Bengal medium [15].
Plates were incubated at 23  C for 7 days and examined for the
presence of Fusarium colonies. In both experiments, the proportion
of the stem segments colonized by Fusarium was measured from
the total number of plant segments assessed.
2.3.1. Induced resistance bioassays data analysis
In the first induced systemic resistance experiment, the effect of
Th003 conidial concentration on the proportion of tissue colonized
by F. oxysporum f. sp. lycopersici was determined by using an
analysis of variance. In the second experiment, the colonization
frequency by the pathogen in the stem segments was calculated
and analysis of variance was used to determine the effect of the
biocontrol agent and time of application on disease rating. In both
cases, the analysis was performed using the SAS software package
(version 8; SAS Institute, Cary, NC).
2.4. Plant growth promotion bioassay
The effect of T. koningiopsis Th003 on plant growth was evalu-
ated in a nursery using the biopriming seeds technique [9]. Tomato
 C.A. Moreno et al. / Physiological and Molecular Plant Pathology 74 (2009) 111–120
seeds were immersed for 10 min in 107 T. koningiopsis conidia per
ml suspension, and then seeds were primed in a solid matrix
consisting of wheat husks moistened to 80% under a layer of nylon
mesh for 48 h at 20  C. After this time, seeds were sown in seedling-
type preformed trays (72 conical compartments each measuring
2.5  2.5  5 cm) filled with sterile peat. Plants were again inoc-
ulated seven and forty-eight days after sown by spreading 5 ml of
a 106 conidia per ml suspension around the plants (TBI). In other
three treatments, seeds were primed without Th003 but they were
inoculated after sown (BI), seeds were not primed but inoculated
with Th003 after sown (I); seeds were primed without Th003 and
were not inoculated after sown (B). A control treatment consisting
in unprimed seeds and non-inoculated with Th003 was included
(C). Stands were counted daily and counts were expressed as
percentage of emergence, the plant height was measured from the
two true leaf stages. Dry weight of root and foliar tissues was
determined by drying them in the oven during 72 h, and then
weighting them on analytical balance (Kern & Sohn, Balingen,
Germany).
In another experiment two treatments were evaluated on plant
growth after transplant. In the first treatment, tomato seeds were
primed in the absence of Th003 in a solid matrix as described
above, and in the second treatment seeds were immersed in Th003
conidial suspension before introduction in the priming matrix.
Primed seeds in the absence of T. koningiopsis were considered as
control. Seeds were sown in seedling-type preformed trays filled
with sterile peat. Trichoderma-treated plants were again inoculated
by spreading 5 ml of a 106 conidia per ml suspension in the
substrate around the plants at seven and forty-two days after sown.
The forty-nine day-old tomato seedlings were transplanted in
2-liter pots filled with a mixture of soil (15.3% organic matter; pH
5.2) and rice husks (2:1 v/v). The stem plant diameter of the first
and second internodes, leaf area, the root and foliar dry weight
were measured at seven, fifteen and thirty-three days after trans-
plant. Element concentration in foliar tissue was evaluated at seven
(vegetative stage) and thirty-three (flowering stage) days after
transplant.
In the first experiment, 20 seeds were used for each of three
replicates; in the second one the experimental unit consisted of 12
plants per each of three replicates per treatment. Both experiments
were arranged in a completely randomized blocks design. The
sample consisted in three plants per replicate.
2.4.1. Plant growth bioassays data analysis
The effect of T. koningiopsis Th003 on seedlings emergence,
plant height, stem diameter, leaf area, plant dry weight and foliar
nutrient concentration was determined by using analysis of vari-
ance and Tukey’s least significant test (a ¼ 0.05) was applied for
mean comparisons using the SAS software package (version 8).
2.5. Gene expression bioassay
In order to evaluate the gene expression, fifteen tomato seeds
were primed in the presence of Th003 as described previously and
another group of fifteen seeds were primed without Trichoderma as
control. Forty-eight hours after priming, the seeds were sown in
seedling-type preformed trays consisting in cubical compartments
each measuring 6  6  12 cm filled with sterile peat (one seed per
compartment) and maintained for 6 weeks, and then transplanted
in 3.4-liter pots (30 cm high) filled with a soil-rice husks mixture
(2:1 v/v) for two weeks. Th003 treated plants were inoculated
twice by spreading 10 ml of T. koningiopsis Th003 suspension (106
conidia per ml) around the plants the first and second week after
transplant. Twenty-four and forty-eight hours after the last inoc-
ulation, tissue was collected from roots and leaves respectively, and
113
immediately frozen in liquid nitrogen and stored at 80  C until
RNA extraction. Samples were collected at 24 and 48 h based on
Shoresh et al. procedures [18]. The whole experiment was repeated
twice (three biological replicates) and two replicates were selected
for the microarray experiments based on the effect on seed
germination percentage (data not shown).
2.5.1. RNA extraction and cDNA labeling
Total RNA extraction was performed using the SV total RNA
isolation kit (Promega) following manufacturer instructions. One-
hundred and 20 mg of sample tissue were used as extraction
starting material for each of the microarray hybridizations. RNA
amplification and labeling were done following the protocols
form the Biotechnology lab at CIAT (http://www.ciat.cgiar.org/
biotechnology/genomics.htm). Briefly, the RNA was amplified in
the sense orientation as previously described [19]. 5-(3-amino-
allyl)-dUTP (Ambion) was incorporated during cDNA synthesis by
reverse transcription of 2 mg of amplified sense RNA, and coupled to
Cy3 or Cy5 NHS ester (Amersham) respectively. Finally, the labeled
cDNA was cleaned with WizardÒ SV Gel and PCR Clean-Up system
(Promega).
2.5.2. Microarray hybridizations and scanning
Tom1 array provided by the Center of Gene Expression Profiling
(CEGP, Boyce Thompson Institute, Ithaca, NY, USA) was used in this
study; detailed information of the construction of the array,
annotation and sequence information is available online at http://
bti.cornell.edu/CGEP/CGEP.html. Ten hybridizations were per-
formed, four for each of the biological replicates: roots and leaves
with a dye – swap design and two self–self hybridizations.
Arrays were processed following the protocols from the
Biotechnology laboratory at CIAT http://www.ciat.cgiar.org/
biotechnology/gene_expression.htm. Briefly, each slide was pre-
hybridized in order to block nonspecific background during
hybridization. Then, slides were washed in sterile distilled water.
Meanwhile, Cy-3 and Cy-5 probes were combined together with
10 mg of Poly A and 20 mg of Salmon Sperm DNA. The slide was
covered with a lifter slip and the ready to hibridize probe was
located by capillarity under the lifter slip. Arrays were placed into
hybridization chambers (Corning Inc., Corning, NY, U.S.A.) and
hybridized overnight at 42  C in a water bath. The hybridization
solution consisted of 50% formamide, 10 SSC and 0.2% SDS. The
slides were removed from the chambers and washed first in
2  SSC, 1% SDS at 42  C for 5 min, then in 1 SSC at room
temperature for 5 min and finally twice in 0.1 SCC at room
temperature for 5 min, and then dried. Slides were scanned using
a VersArray ChipReader (BioRad, U.S.A.). The photomultiplier
settings were adjusted in each channel to result in nonsaturation of
the most highly expressed genes.
2.5.3. Array data analysis
VersArray Analyzer software was used to isolate spots on the
array. The median pixel intensity within a given spot was used as
the signal intensity. For the Cy-3 dye, the background signal
intensity was occasionally higher than that of genes that were not
highly expressed but had previously shown to be differentially
expressed after the Trichoderma treatment [14]. Therefore, to avoid
negative values, we used the strategy proposed by Frick and Shaller
[20] and did not subtract the background signal. Data was
normalized using the local regression technique, LOWESS (locally
weighted scatterplot smoothing), and scale normalization within
print-tip groups using the MIDAS software (TIGR). Genes with
a log2 of red/green normalized ratio greater than 1.5 standard
deviations away from the mean in all the replicates were consid-
ered unreliable and were discarded, as previously described [21].
114 C.A. Moreno et al. / Physiological and Molecular Plant Pathology 74 (2009) 111–120
Differentially expressed genes were identified using two criteria, an
intensity-dependent Z-score as described previously [21] and
a threshold defined by the self–self hybridizations. Genes showing
data greater than 2SD from the local mean (Z > 1,96) and exhibiting
a greater than 1,5 fold change in the Cy5/Cy3 ratio were considered
as being significantly expressed.
2.5.4. Database submission of microarray data
The microarray data were prepared according to MIAME
(minimum information about a microarray experiment) recom-
mendations [22] and were submitted to the tomato expression
Database (TED), with all files and images for each of the 10 arrays
available online.
2.6. Real-time qRT-PCR
Genes showing differential expression were selected according
to sequence homologies and tested by QRT-PCR. Two independent
sets of experiments were runned and used for data analysis.
Seventy two hours after treatment with T. koningiopsis Th003, total
RNA was extracted from tomato plants (leaves and roots) by using
the SV total RNA isolation kit (Promega), following manufacturer’s
recommendations. RNA quantification was performed by using the
Nanodrop ND1000 Spectrophotometer v 3.3.0 (Thermo Scientific,
USA). cDNA was synthesized using the iScript cDNA Synthesis Kit
(BioRad, CA) according to manufacturer’s instructions. All instruc-
tions outlined in the iCycler IQ multicolour real-time detection
system (Biorad, CA) were followed for setting up reactions in 96-
well plates. In short, 1 of 2 PlatinumÒ SYBRÒ Green qPCR
SuperMix-UDG (including PlatinumÒ Taq DNA polymerase, MgCL2
at a final concentration of 3 mM, uracil-DNA glycosylase (UDG),
dNTPs with dUTP instead of dTTP) (Invitrogen, CA) was incubated
with 1 of ROX, 300 nM forward and reverse primers, 250 ng of
cDNA template and water for a final volumen of 25 ml. Each reaction
in triplicate was transferred to a ninety-six well plate and sealed
with optical tape. Sequences of primers forward and reverse for the
Extensin gene were previously reported by Alfano et al. [14]
(Table 3). To determine Actin and 1-amynocyclopropane-1-
carboxylate oxidase homolog PE8 expression, the reactions in the
real-time thermal cycler were programmed as follow: an initial
denaturation step at 95  C for 2 min, followed by 60 cycles of 95  C
for 15 s, 55  C for 30 s, and a final extension of 72  C for 10 min. The
annealing temperature for Actin gene required an additional time
of 15 s. To determine the expression of Myb TF, Extensin and TR8
genes, the conditions of real time amplification were similar, except
for annealing temperature that was 57  C for Myb TF and Extensin
gene, and 61  C for TR8 gene. A dissociation analysis to ascertain if
a single amplicon was generated for each primer used, was run by
an initial denaturation step at 95  C for 2 min, 60  C for 15 s, and
95  C for 15 s. Dilutions of purified genomic DNA from tomato
leaves were used to construct calibrations curves.
2.6.1. QRT-PCR analysis
In order to set the base line, one to two baseline cycles were set
before the reporter dye signal began to increase. The threshold was
set as a halfway point in the log part of the amplification. The
amplification efficiency was determined analyzing the standard
curve, by plotting the log 10 (cDNA dilution or template dilution) vs.
mean Ct of replicate reactions. The equation E ¼ 10[–1/slope] was
used to calculate the PCR efficiency, where a value approaching
2 ¼ 100% efficiency for amplification [23,24]. Threshold (Ct) values
were also used to determine the gene expression profiles from
different cDNA samples. The expression of studied genes was
normalized by expression of a reference control (Actin gene). Ct
values were used to calculate the fold changes (FC) in each sample
with respect to the expression level found in non-treated plants
with the following formula [23]:
FC ¼ 2DDCt, where DDCt ¼ (Ct target  Ct Actin) sample  (Ct
target  Ct Actin) control.
The data are expressed as log2 of the average FC of two inde-
pendent experiments. The standard deviation of the mean (SDM)
was computed for each analysis.
3. Results
3.1. Effect of T. koningiopsis inoculation on stem colonization
by F. oxysporum f. sp. lycopersici
The ANOVA indicated significant (P < 0.05) effects on Fu040
stem colonization in both experiments, when T. koningiopsis Th003
was evaluated at different concentrations and at different inocu-
lation times before the pathogen. In both experiments, the stem
colonization by pathogen in plants inoculated with Th003 was
significantly (P < 0.05) lower as compared with Th003 untreated
plants.
In the first experiment, in the control treatment, F. oxysporum
f. sp. lycopersici colonized the entire stem segment at 14 day after
inoculation while when one root portion of plants were inoculated
with T. koningiopsis Th003 and the other portion was inoculated
with the pathogen the colonization of the stem was significantly
(P < 0.05) lower (Figs. 2 and 3). The concentration of 105 conidia per
ml of Th003 had the most pronounced effect (reduction in 89% of
the proportion of the stem segment colonized by the pathogen) as
compared with the concentrations of 106 and 107 conidia per ml
(56 and 67% respectively).
When different application times of Th003 before Fu040 inoc-
ulation were evaluated, a significant delay in the stem colonization
by F. oxysporum was also observed in plants treated with T. konin-
giopsis Th003, being more evident at 14 and 21 day after pathogen
inoculation than 7 days after (Fig. 4). In general, it was observed
that the time of Th003 inoculation before inoculation with Fu40
presented a significant (P < 0.05) effect on the proportion of stem
segments colonized by the pathogen (Fig. 4). A lower colonization
effect by the pathogen was observed when T. koningiopsis Th003
was inoculated in a portion of the root system 4 and 6 days before
the inoculation with Fu040 in the other portion of the root
(reduction of 74 and 94% respectively taking into account the mean
percentage of plant segments colonized by the pathogen at the end
of the experiment) as compared when Th003 was inoculated 2 days
before (reduction of 51%) and with the Th003 non-inoculated
Fig. 2. Effect of Trichoderma koningiopsis Th003 conidial concentration on colonization
rate in tomato plants by Fusarium oxysporum f. sp. radicis-lycopersici Fu040 at 14 days
after pathogen inoculation. Colonization of the pathogen was assessed along the stem,
1–3 cm above the substrate surface. Colonization rate is expressed as the proportion of
the stem segment colonized by the pathogen. Tomato plants were inoculated with 105,
106, 107 conidia per ml of Th003. P represents the Th003 untreated plants. Bars topped
by the same letter are not significantly different (P < 0.05) according to Tukey’s test.
 C.A. Moreno et al. / Physiological and Molecular Plant Pathology 74 (2009) 111–120
Fig. 3. Growth of F. oxysporum f. sp. radicis-lycopersici Fu040 from tomato stem segments (1–3 cm above the substrate surface) of plants untreated (A) and treated with
T. koningiopsis Th003 4 days before application of the pathogen in a portion of root system (B).
Fig. 4. Effect of Trichoderma koningiopsis Th003 application time (2, 4 and 6 days
before pathogen) on colonization rate in tomato plants by Fusarium oxysporum f. sp.
radicis-lycopersici Fu040 at 7 days (A); 14 days (B) and 21 days (C) after pathogen
inoculation. Colonization of the pathogen was assessed along the stem, 1–6 cm above
the substrate surface. Colonization rate is expressed as the percentage of plant
segments colonized by the pathogen. P represents control plants. Vertical bars
represent the standard deviation.
115
control, which presented 78% of plant segments colonization by
Fu040 21 days after pathogen inoculation.
3.2. Effect of T. koningiopsis application on plant growth promotion
In nursery, it was observed that the seeds primed with Th003
and inoculated further with this antagonist (TBI) presented the
greatest values for the evaluated variables. The ANOVA indicated
significant differences (P < 0.05) among the treatments for seed-
lings emergence at seven, eight and 9 days after sown, then the
seedlings emergence was similar for all treatments. For seeds bio-
primed in presence of Th003 and subsequent inoculations (TBI) the
emergence was 36.7% at 7 days after sown, while it was 3.3% for
seeds primed without Th003 and subsequent inoculations of the
antagonist (BI), and 1.7% for seeds primed without Th003 and not
inoculated later (B) (Fig. 5). The rest of treatments presented
a percentage of emergence lower than TBI, BI and B treatments. At
nine days after sown the BTI treatment showed an emergenece of
80%, while the rest of treatments presented an emergence inferior
to 60%. These results show that the use of seed primed in the
presence of Th003 and subsequent inoculations of this antagonist
in nursery stimulates the germination of tomato seeds.
The plant height was not significantly different (P > 0.05) among
the evaluated treatments. However, it was observed that seeds bio-
primed and inoculated subsequently with Th003 (TBI) were higher
(12.55 cm) than other treatments (11.46 cm in control treatment)
Fig. 5. Emergence of tomato seedlings as result of priming and Trichoderma konin-
giopsis Th003 application in seeds. TBI: seeds primed with Th003 and two subsequent
applications during nursery; BI: seeds primed without Th003 and two subsequent
applications during nursery; I: seeds not primed and inoculated with Th003 subse-
quently; B: seeds primed only; C: seeds not primed and not inoculated with Th003.
The asterisks represent significant differences in accordance with Tukey test at
P < 0.05.
116 C.A. Moreno et al. / Physiological and Molecular Plant Pathology 74 (2009) 111–120

at 49 days after sown (end of the experiment) (data not shown). Dry
weight of root and foliar parts were significantly different (P < 0.05) at
21 days after sown only compared with control treatment, the
following days the values of this variable were similar among treat-
ments. The greatest dry weight was obtained with the TBI treatment
(0.1 g) when compared with control treatment (0.06 g) (data no
shown). This indicates that Th003 application in seed priming and
during nursery stimulates plant growth as well.
Seven days after transplant, in the second growth promotion
experiment, significant differences (P < 0.05) were determined for
the diameter of the first stem internode when plants were primed
in the presence of Th003 and inoculated after sown, with
4.16  0.14 mm, while in the untreated control it presented
3.42  0.32 mm (Table 1). Fifteen days after transplant, significant
differences were also observed in plants treated with Th003 which
had increased diameter of the second stem internode and higher
foliar and root dry weight being 4.61  0.14 mm, 5.42  0.83 g and
1.09  0.17 g respectively, as compared with the untreated control
with values of 4.16  0.17 mm, 2.86  1.21 g and 0.57  0.20 g
respectively (Table 1). When foliar area was determined, no
significant differences were observed among treatments, while
when foliar nutrient concentration was analyzed, the only signifi-
cant difference (P < 0.05) among the treatments was observed 15
days after transplant for the calcium concentration (Caþþ) which
was approximately 20% higher in plants treated with Th003 than in
untreated control plants (data not shown).
3.3. Microarray expression analysis
In order to determine microarray technical reproducibility two
self–self hybridizations were performed. In self–self hybridizations,
the ideal result would be a Cy5/Cy3 signal ratio of 1.0 for each spot
on the array following data normalization. A total of 99.03% of the
genes were within 1.5 fold. Based on these results, only genes
exhibiting a change greater than 1.5-fold in the Cy5/Cy3 ratio were
considered biologically significant in this study. Additionally, an
intensity-dependent Z-score was used as a second criterium for
selecting differentially expressed genes.
Treatment of tomato plants with T. koningiopsis altered the
mRNA levels of several genes. To increase the confidence with
which the results could be interpreted, two biological replicates of
the entire experiment were performed, with a technical replicate
(two arrays in a dye-swap design) for each hybridization. Thus,
results from four arrays were analyzed for each tissue. A significant
change in gene expression was reported only if the gene showed
the same result (induction or repression) from both biological
experiments. Analysis of the T. koningiopsis treated plants
compared with water inoculated tomato revealed significant
differential expression for 45 genes, 41 in roots and 4 in leaves. In
roots, 17 genes were repressed and 24 induced, 24 h after the last
soil inoculation with conidia. In leaves, one gene was significantly
repressed and 3 induced, 48 h after the last treatment (Table 2).
We obtained a partial annotation of all differentially expressed
genes (Table 2). All genes belonged to a few number of functional
categories that included transport, signal transduction and
signaling, cell wall degradation and hormone responses (to auxin
and ethylene). Expression of genes previously reported to be
altered during the treatment with Trichoderma spp. were also
identified in this study [14], in particular MYB transcription factor
genes involved in the jasmonic defense pathway and a sequence
putatively similar to an extensin. The entire data set (all data and
images for each of the 10 arrays) is available at The Tomato
Expression Database (TED) website.
3.4. qRT-PCR
Four genes found to be differentially expressed during the
exposure of tomato plants to T. koningiopsis Th003 were selected
and analyzed by qRT-PCR to confirm the reliability of the arrays
results obtained. The primers and their corresponding targeted
genes used in the qRT-PCR reactions are shown in Table 3. These
genes were selected based on their homology and functional
category (hormone response, protein metabolism, signaling and
plant defense) (Table 4). To normalize the qRT-PCR data, each gene
was compared with the Actin transcript, in treated and untreated
plants. The PCR efficiency was calculated for each gene and the
obtained E value was approximately 2.0. The expression of the gene
TR8 (leaves- 48 h), PE8 and myb TF (roots – 24 h) showed to be up-
regulated after exposure to T. koningiopsis Th003 in the array
(Table 2). qRT-PCR analysis revealed that PE8 (72 h) was signifi-
cantly (P < 0.05) induced in leaves but not in roots. qRT-PCR
analysis revealed that gene TR8 was expressed in roots (72 h). In
leaves, however, this expression was not confirmed. Plants treated
with T. koningiopsis Th003 expressed elevated levels of the extensin
gene in leaves and roots. Similar results were reported by Alfano
et al. [14] in tomato leaves (Table 4).
In the array analysis performed in this study, the myb TF gene
was over expressed in roots. Alfano et al. [14] also revealed the up-
regulation of this gene when tomato plants were treated with
T. hamatum 382. According to the qRT-PCR data analysis (Table 4),
the up-regulation of myb TF gene at 72 h was not confirmed in
leaves and roots.

Table 1
Effect of T. koningiopsis Th003 on tomato growth (Solanum lycoperscicum L. cv. Rocio).

Treatment Days after transplant Dry weightc (g) Internode diameter (mm) Foliar aread (mm2)

Foliar Root First Second

Th003a 7 4.14 (0.98) 0.84 (0.81) 4.16 (0.14)* 3.97 (0.31) 2897.9(86.92)
15 5.42 (0.83)* 1.09 (0.17)* 4.59 (0.13) 4.61 (0.14)* 6406.1(10.20)
33 28.8 (11.94) 4.25 (1.46) 5.45 (0.44) 5.22 (0.21) 14360.2(170.78)

Controlb 7 3.25 (0.45) 0.40 (0.10) 3.42 (0.32) 3.35 (0.23) 2125.0(52.48)
15 2.86 (1.21) 0.57 (0.20) 3.97 (0.38) 4.16 (0.17) 4582.6(335.01)
33 20.98 (5.60) 3.05 (1.36) 4.99 (0.21) 5.2 (0.16) 12452.4(219.68)

Numbers in parenthesis represent the standard deviation of mean (n ¼ 3) and the asterisk indicate significantly differences for each variable at the same time of measure
according to Tukey’s test at P < 0.05.
a
Seeds were immersed in a suspension of Th003 containing 1  107 conidia per ml during 10 min and then were primed in a solid matrix for 48 h before sown. Later, at 7
and 42 days after, plants were again inoculated with Th003 by spreading 5 ml of a 106 conidia per ml suspension. Then 49 days-old tomato seedlings were transplanted in
2-liter pots.
b
Seeds were primed in a solid matrix for 48 h before sown. 49 days later the seedlings were transplanted.
c
Dry weight of root and foliar parts were determined after drying during 72 h.
d
The entire leaves of the plants were harvested and the leaf area was measured by a leaf-area meter (Li-Cor LI3000).
 C.A. Moreno et al. / Physiological and Molecular Plant Pathology 74 (2009) 111–120 117

Table 2
List of genes that are differentially expressed in Trichoderma – treated tomato plants versus non-treated plants and their expression ratios. SGN unigene best hit and functional
category (GO annotation) were manually re-checked at the Sol Genomics network (http://www.sgn.cornell.edu/).

Tissue SGN unigene ID SGN unigene nr best hit Functional category Expa1 Exp2
Leaves SGN-U143905 Proteinase inhibitor type II (TR8) Protein metabolism 1.7 2.8
SGN-U153109 ribosomal protein S3 Protein metabolism 1.58 1.71
SGN-U144781 Ethylene-inducible protein HEVER Hormone responses 1.5 1.99
SGN-U159125 hypothetical protein Unknown 0.64 0.66

Roots SGN-U147015 Unknown Unknown 0.51 0.56

SGN-U143394 ADP/ATP translocator Transport 0.57 0.59
SGN-U143967 aquaporin 2 Transport 0.51 0.62
SGN-U144670 MADS-box protein 15 Signaling 0.52 0.56
SGN-U152994 cryptochrome 1b Light responses 0.55 0.48
SGN-U148475 peroxisomal membrane protein family Unknown 0.56 0.61
SGN-U154494 growth regulator protein Hormone responses 0.57 0.59
SGN-U143929 putative ABC transporter Transport 0.6 0.64
SGN-U143318 Histidine decarboxylase Amino acid metabolism 0.61 0.62
SGN-U149209 Expressed protein Unknown 0.64 0.59
SGN-U153138 Expressed protein Unknown 0.65 0.66
SGN-U144121 Fructose-1,6-bisphosphatase, cytosolic Photosynthesis 0.64 0.66
SGN-U143233 60 S Ribosomal protein L2 Protein biosynthesis 0.64 0.6
SGN-U145779 expressed protein Unknown 0.65 0.62
SGN-U147969 OSJNBb0017I01.6 Transport 0.65 0.61
SGN-U156651 hypothetical protein Unknown 0.66 0.61
SGN-U149175 CDK-activating kinase Signal transduction 0.66 0.59
SGN-U143274 1-aminocyclopropane-1-carboxylate oxidase 1 Hormone responses 1.6 1.5
(ACC oxidase 1) (Ethylene-forming enzyme)
SGN-U146663 Expressed protein Unknown 1.69 1.51
SGN-U151527 Expressed protein Unknown 1.56 1.52
SGN-U148558 Expressed protein Transport 1.62 1.54
SGN-U146138 MYB family transcription factor (myb TF) Signaling 1.9 1.57
SGN-U144902 Putative polyprotein (putative homology: elastin) Cell structure 1.62 1.57
SGN-U144031 Calmodulin 2 Signal transduction 1.68 1.6
SGN-U155078 Unknown Unknown 1.54 1.6
SGN-U143387 1-aminocyclopropane-1-carboxylate oxidase homolog Hormone responses 1.7 1.6
(PE8)
SGN-U144612 Expressed protein Unknown 1.53 1.61
SGN-U150684 Aldehyde oxidase Oxygen responses 1.56 1.63
SGN-U143525 Acid beta- fructofuranosidase precusor Carbohydrate metabolism 1.68 1.65
(acid sucrose-6-phosphate hydrolase) (Acid invertase)
SGN-U146418 Putative AP2 domain transcription factor Unknown 1.66 1.69
SGN-U147085 Unknown Unknown 1.6 1.69
SGN-U144103 probable calcium-binding protein Unknown 1.53 1.7
SGN-U143408 similar to ci21A gene product encoded by the sequence Unknown 1.65 1.56
(GenBank Accession Number U76610)
SGN-U148787 protein kinase-like protein Signal transduction 1.57 1.72
SGN-U144741 ATPase – related Transport 1.57 1.73
SGN-U146990 Unknown Unknown 1.81 1.76
SGN-U146968 glycosyl hydrolase family 5/cellulase Cell wall degradation 1.96 1.77
((1–4)-beta-mannan endohydrolase)
SGN-U148554 trigger factor-related protein Unknown 1.56 1.77
SGN-U149297 VPS13 – like protein Transport 1.63 1.77
SGN-U143471 systemic acquired resistance-related protein SRE1b Unknown 1.86 1.86
SGN-U160678 expressed protein Unknown 1.6 1.55
a
Exp1 and EXp2 correspond to the two biological replicates of the expression experiment.

4. Discussion interact with the plant to induce resistance to foliar pathogens.

Induced systemic resistance in plants has been largely demon-
strated for air-borne phytopathogenic bacteria [14,25] and fungi
[26,27] but in few cases for soil-borne phytopathogenic fungi
[28–30]. Trichoderma spp. are well known antagonists against
several soil-borne plant pathogenic fungi, such as was reviewed by
Papavizas [31] and more recently by Verma et al. [32] and Vinale
et al. [33] through several mechanisms of biocontrol [34,35]. Even
tough Trichoderma spp. is a common soil-borne microorganism [3]
it has also been applied to foliar surfaces for biocontrol of foliar
plant pathogens caused by fungi [36–38]. Moreover, to control
plant pathogens by mycoparasistism, antibiosis, and competition, it
has been demonstrated that some Trichoderma species as
Trichoderma harzianum T22 [5], T. asperellum T203 [18], T. harzia-
num T39 [26] and Trichoderma hamatum 382 [27] are also able to
However, in the present work the split root system allowed to
separate spatially T. koningiopsis Th003 and F. oxysporum f. sp.
lycopersici Fu040 in tomato roots, to demonstrate a systemic
protection effect induced by Th003.
In addition to control plant pathogens several Trichoderma
strains are also able to promote plant growth, a phenomenon that
has been largely documented [4,39,40]. In the present work, the
seedling emergence results showed that the use of T. koningiopsis
Th003 in seeds priming and during early stages of growth
enhanced the germination rate and the development during the
nursery period. These effects on seedlings have important
economic implications in terms of shortening the nursery time, as
mentioned previously by Inbar et al. [39]. The data suggested that
Th003 was mainly responsible of the increased growth response
observed, since the addition of T. koningiopsis in seed priming and
118 C.A. Moreno et al. / Physiological and Molecular Plant Pathology 74 (2009) 111–120

Table 3
Primers used to measure expression of different genes using qRT-PCR.

Gen SGN unigene ID SGN unigene nr best hit Functional category Forward primer Reverse primer Tm ( C)
TR8 SGN-U143905 Proteinase inhibitor type II Protein metabolism GAATGGGTAAG TGAGGGAGAAA TTCTTAGAACAAA CTAGTGTTCCATTT 61
myb TF SGN-U146138 MYB family transcription factor Signaling TCCATGGACTG TTGAAGAAGA CAGTTCTCCCTGGC AAATGT 57
PE8 SGN-U 1433 87 1-aminocyclopropane-1-carboxylate Hormone responses AAGGCCGGTGT TAAAGGACT TGCAACGTTCTGTC CAAGAC 55
oxidase homolog
Extensin TC124404 Extensin Plant defense CACTATGTTTAC TCCTCTCCC CATATGGGAGTAG TAATAAC 57
Actin Actin Reference control gene CCAGTGGCCGT ACAACAGGT TCGAAGAATTGCG TGAGGAAG 55

twice inoculated with Th003 after sown (TBI treatment) had
greater effect than seeds primed without Th003 and twice inocu-
lated with Th003 after sown (BI treatment) (Fig. 5). Although the
other parameters (plant height and dry weight) used for measuring
growth response showed no statistical differences among treat-
ments during nursery period, Trichoderma-treated plants showed
best appearance in development and vigor than non-treated plants.
The stem diameter, root and foliar dry weight parameters sug-
gested a positive effect of T. koningiopsis Th003 on tomato growth
response after transplant as well. These results are in agreement
with an early report by Yedidia et al. [4] in cucumber plants inoc-
ulated with T. harzianum T-203.
Similar to our experiments, earlier Inbar et al. [39] and more
recently Ozbay and Newman [40] observed no significant differ-
ences among Trichoderma-treated plants and non-treated plants for
foliar concentrations of essential nutrients. However, in this study
the greatest Caþþ content in leaves of treated plants vs. non-treated
plants, could be related with the induced systemic resistance effect
stimulated by T. koningiopsis Th003 observed in this work, since it
has been demonstrated that calcium is essential in activation of
phytoalexins synthesis in the plant [41,42].
The results found in this study show that genes that encode
proteins that are involved in the JA/ethylene signal transduction
pathways were up-regulated following application of T. koningiopsis
Th003. This result is in agreement with previous studies using other
species of Trichoderma [14,18]. The plant hormone ethylene regu-
lates many processes during plant growth and development and is
also an important mediator of plant responses to biotic and abiotic
stresses [43,44]. In ethylene biosynthesis, the conversion of S-ade-
nosyl Met (Ado-Met) to 1-aminocyclopropane-1-carboxylic acid
(ACC) is catalyzed by the enzyme ACC synthase (ACS), and ACC
oxidase (ACO), which oxidizes ACC to ethylene. In tomato (S. lyco-
persicum), ACS and ACO are encoded by gene families consisting of at
least eight [45,46] and four members [47,48], respectively. These
genes show differential expression during plant growth and
development, and respond differentially to various external stimuli
[45,47–53]. Of notable interest in this study is the consistent
up-regulation of the ACC oxidase gene (PE8) after the last applica-
tion of T. koningiopsis Th003. Previous studies by Moeder et al. [54]
reported that the induction of the ACC oxidase gene, required for
hydrogen peroxide accumulation and cell death, occurred via
ethylene in ozone-exposed tomato plants, suggesting that Th003
effect may activate this gene by the same signal transduction
pathway.
Proteinase Inhibitor II (PI-II) family members have been iden-
tified in tomato (S. lycopersicum) [55,56]. TR8-like proteinase
inhibitors appeared to be essential part of the plant’s natural
defense system against herbivores, and induced by molecules like
the 18 amino acid peptide hormone systemin, the abscisic acid, the
jasmonic acid, bacterial infections and fungal elicitors [57]. Fungal
elicitors not only induced an accumulation of proteinase inhibitors
but also a stimulation of ethylene synthesis in tomato. The
messenger RNA of these PI-II class protein is inducible in roots and
hypocotyls of tomato by fungal elicitors [57]. Shoresh et al. [18]
found that T. asperellum (T203) modulated the expression of genes
involved in the jasmonate/ethylene signaling pathways of ISR in
cucumber. In this study, the expression of the gene TR8 showed to
be elicited 48 h after exposure to T. koningiopsis Th003 in the array.
In Nicotiana tabacum, for example less than 30 mg per milliliter of an
elicitor obtained from Phytophthora parasitica var. nicotianae were
necessary for maximal induction of proteinase inhibitor accumu-
lation and detection (12 h) [58].
In this study, the microarray analysis and qRT-PCR results
showed that the main signal transduction pathway through which
ISR mediated by Trichoderma was activated by using JA and
ethylene as signal molecules. However, our results also suggest
a temporary increment of defense related gene expression
response to T. koningiopsis Th003, as was demonstrated by Shoresh
et al. [18] after treatment of cucumber with T. asperellum T203. This
fact is one of the possible explanations that the extensin gene
expression, detected by microarray analysis at 24 h after treatment
of tomato with Th003, was not confirmed by qRT-PCR at 72 h after
treatment. A temporary activation of PE8 gene in Trichoderma
inoculated plants was also obtained, showing an activation of this
gene in roots at 24 h but not at 72 h. By contrast, PE8 gene was
expressed in leaves at 72 h, suggesting that activation of this gene in
roots preceded its temporary activation in leaves. Relate this when
takes about PE8 on P22-23. Otherwise, seems unrelated.
Additionally, we could confirm the effect of the fungus on altering
the general metabolism of the roots since genes acting on very
different biological processes were induced or repressed. In this way,
we could molecularly confirm the effect of T. koningiopsis on plant

Table 4
Real-time PCR profiles for the expression of genes related with tomato and T. koningiopsis Th003 interaction.

Target gene Ct

Treated Untreated FC

Leaves Roots Leaves Roots Leaves Roots

PE8 28.3 33.5 30.6 31.8 2.5* 1.7*
TR8 30.4 27.6 22.5 28 7.7* 0.4
myb TF 43.3 29.6 29 28.8 14.1* 0.8
Extensin 37.7 40.5 45.1 52.9 7.6** 1.2*
Actin 32.7 32.6 32.5 32.6 – –

*,**The target gene treatment is significantly different (P < 0.05) or highly significantly different (P < 0.01) respectively from the target gene control. The threshold (Ct) value
was set as a halfway point in the log part of the amplification. It was used to determine the gene expression profile from different cDNA samples. Ct values were used to
calculate the fold changes (FC) in each sample with respect to the expression level found in non-treated plants.
 C.A. Moreno et al. / Physiological and Molecular Plant Pathology 74 (2009) 111–120
growth as our study detected the up-regulation of a gene involved in
cell wall degradation (glycosyl hydrolase family 5/cellulase (1–4)-
beta-mannan endohydrolase) and we could confirm the differential
expression of the extensin gene [14]. The next step will be to use the
genes characterized here as molecular markers to dissect and docu-
ment the effect of the biocontrol agent under field condition to aid in
the proposal of the best strategy to use it as alternative of control.
Acknowledgements
We thank Joseph Tohme for his critical suggestions, reading and
comments. This project was supported by COLCIENCIAS competi-
tive grants program for funding.
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