Elevation of Defense Network in Chilli

Against Colletotrichum capsici by

Phyllospheric Trichoderma Strain
Amrita Saxena, Richa Raghuwanshi &
Harikesh Bahadur Singh

Journal of Plant Growth Regulation

ISSN 0721-7595
Volume 35
Number 2
J Plant Growth Regul (2016) 35:377-389
DOI 10.1007/s00344-015-9542-5

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Author's personal copy

J Plant Growth Regul (2016) 35:377389
DOI 10.1007/s00344-015-9542-5

Elevation of Defense Network in Chilli Against Colletotrichum

capsici by Phyllospheric Trichoderma Strain
Amrita Saxena1 Richa Raghuwanshi2 Harikesh Bahadur Singh3

Received: 20 August 2014 / Accepted: 13 July 2015 / Published online: 25 September 2015
Springer Science+Business Media New York 2015

Abstract Biocontrol strategies have been mainly focused

on proposing the use of biocontrol agents (BCAs) isolated
from the rhizospheric region of the plant for protection
against phytopathogens. The present study evaluates the
effectiveness of phyllospheric Trichoderma isolates in
elevating the defense responses in chilli against Colletotrichum capsici infection and comparing its efficiency
to the conventionally recommended rhizospheric Trichoderma strains. The elicitation of the defense network in the
plants was analyzed using biochemical assays for important enzymes, that is, PAL, PO, PPO, TPC, SOD along with
the total protein level in challenged plants over untreated
and unchallenged control plants. The results recorded 2.1,
5.18, 3, 0.67, and 0.5-fold increases in TPC, PAL, PO,
PPO, and total protein content in BHUF4 (phyllopsheric
Trichoderma isolate)-treated plants when compared to
control plants under C. capsici challenge. This was at par
with the increment recorded in T16A (rhizospheric Trichoderma isolate)-treated chilli plants. The increment in
growth parameters was also recorded after treatment with
the isolated Trichoderma strains. Interestingly, the

Electronic supplementary material The online version of this

article (doi:10.1007/s00344-015-9542-5) contains supplementary
material, which is available to authorized users.
& Harikesh Bahadur Singh
hbs1@rediffmail.com
1

Department of Botany, Banaras Hindu University,

Varanasi 221005, India

Department of Botany, Mahila Mahavidyalaya, Banaras

Hindu University, Varanasi 221005, India

Department of Mycology and Plant Pathology, Institute of

Agricultural Sciences, Banaras Hindu University,
Varanasi 221005, India

phyllospheric isolate (BHUF4) treatment recorded comparable growth promotion in chilli plants recording 36, 62,
and 60 % increases in one of the major parameters of plant
growth, that is, root length, no. of leaves, and dry weight,
respectively. This study proposes the use of combined
application of both rhizospheric as well as phyllospheric
Trichoderma isolates for better and all around protection of
plants against foliar as well as soil phytopathogens. This
would be a novel approach in biological control strategy
for better management of anthracnose disease of chilli.
Keywords Capsicum annuum L.
Colletotrichum
capsici
Induce defense response
Phyllosphere

Trichoderma spp.

Introduction
Plants akin to animals have been empowered with surfeit
defense responses for their protection from varied biotic
and abiotic stresses prevalent in the environment. Two
major categories of defense networking have been identified in plant defense systems against phytopathogens:
systemic acquired resistance (SAR) and induced systemic
resistance (ISR) (Contreras-Cornejo and others 2011). The
defense signaling initiated by successful colonization of
necrotizing pathogens is referred to as SAR, which requires
the intrinsic accumulation of salicylic acid (SA) for its
activation (Ryals and others 1996), whereas the defense
response induced by beneficial microbes, that is, ISR,
functions independent from SA accumulation and depends
on the plants response to phytohormones like jasmonic
acid and ethylene (van Loon and others 1998; Harman and
others 2004; Conrath 2011). In addition, the role of
antimicrobial phytoalexins such as flavonoids, terpenoids,

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indoles, phenols, and glucosinolates has also been established as vital to host defense machinery providing protection against important phytopathogens (Darvill and
Albersheim 1984; Pieterse and others 2009). The complete
protection mechanism is primed by expression of different
defense genes resulting in simultaneous synthesis of
antimicrobial compounds in response to hormonal signaling (Contreras-Cornejo and others 2011). Recent studies
have clarified the significance of defense responses induced
by beneficial microbes, that is, induced systemic resistance
(ISR) (Shoresh and others 2010; Niu and others 2011; Jain
and others 2012). Associated with the elicitation of defense
responses in spatially distant plant parts, ISR is mainly
triggered by certain elicitors that further sparks immune
responses in plants. Pathogenesis-related (PR) proteins,
phenylalanine ammonia-lyase (PAL), peroxidase (PO),
polyphenol oxidase (PPO), superoxide dismutase (SOD),
accumulation of proline and phenols have been reported to
be associated with ISR, leading to increased resistance in
plants against various phytopathogens (Jetiyanon 2007;
Magnin-Robert and others 2007; Naveen and others 2012;
Singh and others 2013).
Chilli (Capsicum annuum L.) has been an important
constituent of almost all cuisines worldwide with an
added benefit of being medicinally important. Having
high vitamin C and A content, it has been one of the
four most important agriculturally grown crops in tropical and subtropical countries. Anthracnose or fruit rot
caused by Colletotrichum capsici (Sydow) has been a
major restraint in chilli production causing noteworthy
loss in profitable cultivation and seed production among
the major chilli producing Asian countries including
India (Than and others 2008; Saxena and others 2014).
Being one of the ten most destructive pathogens of chilli
affecting the crop yield worldwide (Dean and others
2012), C. capsici has been linked with anthracnose of
chilli leading to both pre- and post-harvest losses (Bosland and Votava 2003). Characteristic disease symptoms
include formation of sunken circular or angular lesions
with the presence of dark-colored black spots in concentric rings representing the acervuli structures that
contain the conidia entrapped between different lengths
of setae (Than and others 2008). Symptoms are generally
found on leaves and stems of the plants apart from
prominently affecting the fruits both at the ripe and
unripe stages. The disease is seed borne, air borne, and
water borne affecting seed germination and plant vigor
to great extent. Also damage to foliar plant parts further
reduces the photosynthetic ability of the plant thereby
leads to reduction in fruit and seed yield.
Mainly managed through periodic use of fungicides in
the form of foliar spray and seed treatment, control of

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anthracnose disease has been difficult due to developing

resistance in the pathogen against commonly used fungicides. Moreover, the deposition of toxic residues and
breakdown of host resistance further adds to the persisting
dilemma (Beckman 1987; Thakur and others 1999).
Exploiting the eco-friendly approach of inducing a defense
response in plants is required for developing an effective
management strategy. Treatment with Psuedomonas spp.
(Anand and others 2009), Burkholderia (Madhavan and
others 2011), yeast Pichia guilliermondii (Nantawanit and
others 2010), Bacillus spp. (Intanoo and Chamswarng
2007), Trichoderma spp. (Oanh and others 2006) and
cerebroside derived from pathogenic fungus (Naveen and
others 2012) as biocontrol agents (BCAs) has been used to
induce defense responses in chilli plants against Colletotrichum spp.
Well documented for its biocontrol ability, Trichoderma
is a free living saprophytic fungus and is a potent root
colonizer (Harman and others 2004; Mukherjee and others
2013) found in most ecosystems. The colonization of roots
by Trichoderma promotes plant growth by producing
indole-3-acetic acid and auxin precursors (Contreras-Cornejo and others 2009, 2014a) and leads to a state of induced
resistance (Yedidia and others 1999). The defense
responses elicited are complex and, depending on the
amount of inoculum, may involve the canonical defense
hormones SA and JA. The defense responses also involve
biosynthesis of volatile compounds and camalexin, the
major phytoalexin in Arabidopsis (Segarra and others
2007; Contreras-Cornejo and others 2011, 2014b; Velazquez-Robledo and others 2011; Mathys and others 2012).
Trichoderma has also been reported to positively enhance
stem lignification thereby imparting strength to the plant
against pathogen attack (Saxena and others 2015). The
mechanisms by which novel strains of Trichoderma interact with roots are important in deciphering the aspects of
plant immunity protecting the plants from pathogens.
Selective isolation from the rhizospheric region of soil
has delimited the probability of obtaining proficient strains
from additional sources like the phyllospheric region of
plants. To obtain equally competent strains from the not so
explored phyllospheric region, the phyllosphere of healthy
chilli plant leaves was considered for microbial isolation.
The selectively isolated Trichoderma strains were then
compared with the conventionally recommended isolates
obtained from rhizospheric region of the soil for their
probable ability to induce defense responses in chilli plants
against C. capsici infestation. The present study shows the
probable effectiveness of Trichoderma strains isolated
from the phyllosphere region in combating the pathogen at
the foliar surface, thus providing a fortified first line of
defense against Colletotrichum spp.

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Trichoderma isolates were selectively isolated from the

phyllosphere and rhizosphere of agriculturally important
crops from the farms of BHU on PDA medium and Trichoderma Selective Medium (TSM) (Elad and others 1981),
respectively, by the serial dilution technique. One gram of
soil was used for isolation from the rhizosphere whereas 1 g
of fresh leaves was considered for isolation from the phyllosphere of plants. The culture plates at 106 dilution were
incubated at 27 C for 45 days and characteristic colonies
developing green spores were isolated on separate PDA
plates. The isolated strains were morphologically characterized as Trichoderma spp. and were stored on PDA slants at
4 C until further use. The isolates were further identified by
sequencing the universally conserved ITS region and the
sequences were submitted to NCBI Genbank.

as for their probable growth promoting ability (Saxena and

others 2015).
Pectate lyase [pectate transeliminase, E.C. 4.2.2.2]
(Hankin and others 1971), lipase (Sierra 1957), amylase
and cellulase (Hankin and Anagnostaksis 1975) enzyme
production were accessed on solid plate medium where
specific media were used for each assay.
Chitinase [E. C. 3.2.1.14] and b-1,3-glucanase [E. C.
3.2.1.39] production were estimated quantitatively using the
method described by De Marco and others (2000) and De
Marco and Felix (2007), respectively. The cultures were
grown in Trichoderma liquid medium for obtaining the
supernatant. The enzymatic activity of each isolate for both
the enzymes was expressed in enzyme units (E.U.) where
each enzyme unit corresponded to the amount of protein
required to produce 1 mg of N-acetyl glucosamine in case of
chitinase and glucose in case of b-1,3-glucanase, in 1 h.
The phosphate solubilization ability of the isolates was
accessed according to the method described by Mehta and
Nautiyal (2001) using NBRI-BPB medium. For recording
the IAA producing ability, colorimetric estimations were
made according to Gravel and others (2007) using tryptophan (200 lg ml-1) amended PDB. Siderophore producing
ability was determined qualitatively using the Chromazural
S (CAS) assay as described by Schwyn and Neilands (1987).
For all in vitro extracellular enzyme production assays,
that is, chitinase, b-1,3-glucanase, IAA and phosphate
solubilization, the supernatant of Trichoderma cultures was
considered. Spores (106 ml-1) of each isolate were inoculated in 50 ml of specific medium and was incubated for
1 week at 27 2 C at 200 rpm. One ml of supernatant
from the obtained Trichoderma cultures was considered for
the reactions. The enzymatic activity of each Trichoderma
isolate for the assays, expressed as specific units, denotes
the activity of 1 ml of the Trichoderma culture in specified
medium under definite conditions.

In Vitro Determination of Mycoparasitism

Compatibility Between Antagonists

The mycoparasitic ability of Trichoderma isolates against C.

capsici was accessed by inoculating the Trichoderma isolates and pathogen simultaneously at the opposite edges of
the Petri plate. The % inhibition of the pathogen was calculated according to the method described by Dennis and
Webster (1971) and the experiment was repeated three times.

The selected isolates of Trichoderma were checked for

their probable compatibility with each other. A mycelial
plug (5 mm diameter) from an actively growing culture of
both the isolates was placed on opposite edges of PDAcontaining plates and allowed to grow at 27 2 C for
45 days. Overgrowing Trichoderma isolates showing no
zone of inhibition were considered as compatible isolates.

Materials and Methods

Isolation and Multiplication of Pathogen
Colletotrichum capsici was isolated from infected chilli
fruits collected from the vegetable farm of Banaras Hindu
University (BHU), Varanasi, India (Latitude/Longitude
25.2644N, 82.9950E). The infected portion was cut into
small pieces of 34 mm and was surface sterilized using 1 %
NaOCl with three consequent washings in sterile distilled
water. The pieces were placed on potato dextrose agar (PDA,
MO96; Himedia, Mumbai, India) plates after tap drying on
filter paper to remove excess water. The plates were incubated at 27 2 C for 57 days in 12 h alternate dark and
light conditions. The pure culture of the pathogen obtained
was maintained on PDA slants and was stored at 4 C. The
culture was timely revived after 30 days.
Isolation and Identification of Antagonistic
Microbes

Physiological and Biochemical Characterization

of Selected Antagonists
The Trichoderma isolates obtained from the rhizosphere
and phyllosphere were biochemically and physiologically
characterized for extracellular enzyme production as well

Inoculum Preparation for Treatment of Chilli Plants

with Selected Bioagents
Two Trichoderma isolates BHUF4 (phyllospheric isolate)
and T16A (rhizospheric BCA) were selected (based on

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their biochemical and antagonistic performance). Sevenday-old cultures of the two selected Trichoderma isolates
were used for preparing the spore suspension. The plates
with fully grown cultures of the isolates were washed
with sterile distilled water and filtered through layers of
muslin cloth. A spore suspension with CFU of 2 9 107
ml-1 was maintained using a haemocytometer for both
the Trichoderma isolates used for treating chilli plants.
Twenty-one-day-old culture of C. capsici was taken
for preparing the conidial suspension of the pathogen.
Fully grown culture of C. capsici was flooded with
sterile distilled water and the mycelia were scraped using
a sterile spatula. The suspension formed was filtered
through layers of cheese cloth and the conidial count
was adjusted to 106 conidia ml-1 using a
haemocytometer.

Testing Plant Growth Promotion Activity

The plant experiments were carried out under greenhouse conditions at the Department of Mycology and
Plant Pathology, Institute of Agricultural Sciences,
BHU, Varanasi. The following treatments were studied
for their growth promotion activity: C = Control, 1:
Phyllospheric BCA (BHUF4) treated plants, 2: Rhizospheric BCA (T16A) treated plants, 3: BHUF4 ? T16A
treated plants.
Twenty-one-day-old chilli seedlings (Var: Surajmukhi)
were transplanted in pots containing a sterile soil mixture
(sandy soil: vermicompost: farmyard manure = 2:1:1).
For the 1st treatment, seedlings were sprayed with spore
suspension of the BHUF4 isolate until complete drenching of the plant. For the 2nd treatment, the spore suspension of T16A isolate was inoculated in the pots by
drenching the soil near the roots of the chilli plants. For
the 3rd treatment, a combination of both 1st and 2nd
treatments was performed. Untreated plants sprayed with
water served as controls (C). For each treatment, five pots
containing three seedlings were maintained. The whole
experiment was carried out under green house conditions
maintained at 28 2 C with 80 % RH under 12 h
alternate light and dark conditions. The experiment was
repeated twice and was designed in a completely randomized manner.
After 30 days of growth, five plants from each treatment
were uprooted and the growth parameters, that is, shoot
length, root length, no. of leaves, and no. of nodes were
recorded. The amount of chlorophyll was also determined
by homogenizing leaf tissue in 80 % acetone followed by
measuring the absorbance at 663 and 645 nm (Arnon
1949).

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Green House Experiment for Estimating the Defense

Response
For estimating the effect of Trichoderma isolates on the
elevation of defense responses in C. capsici-challenged chilli
plants, the following treatments were considered: C = Positive Control, PC = Negative Control, 1 = BHUF4-treated
plants, 2 = T16A-treated plants, 3 = BHUF4 ? T16Atreated plants (P = Pathogen Challenged, UP = Pathogen
Unchallenged plants). The whole experiment was carried
out under green house conditions maintained at 28 2 C
with 80 % RH under twelve hours alternate light and dark
conditions. Two sets of the treatments 13 were maintained
as done while estimating the growth promotion activity of
the plants. One set was kept unchallenged representing the
UP series, whereas the other set was challenged with
conidial suspension of C. capsici representing the P series.
For C. capsici inoculation, the prepared conidial suspension
was sprayed on chilli seedlings until plants were completely
drenched. The seedlings were then covered with sterile poly
bags for 48 h to maintain humidity. Untreated unchallenged
plants served as Positive Controls (C) whereas untreated but
challenged plants served as Negative Control (PC) plants.
The control plants were sprayed with sterile water and were
then covered with sterile polybags for 48 h.
Plants were harvested at 0 h after pathogen inoculation
(hapi), 24 hapi, 48 hapi, and 72 hapi. The plant samples were
thoroughly washed under running tap water and blot dried
before storing in a deep freezer (-80 C) until the completion of experiment. Older leaves from the 3rd to 5th nodes
(from bottom) were considered for biochemical estimations.

Biochemical Estimation
The phenylalanine ammonia-lyase (PAL) assay, total
phenolic content (TPC), superoxide dismutase (SOD)
assay, peroxidase (PO) assay, and polyphenol oxidase
(PPO) assays were carried out as described previously by
Jain and others (2012). Total protein content in the samples
was accessed according to Lowry and others (1951).

Disease Reduction Analysis

For estimating the effectiveness of the different treatments
with Trichoderma isolates, green house experiments with
four-week-old transplanted chilli plants were considered
(28 2 C with 80 % RH under 12 h alternate light and
dark conditions). Three replicates for each set of treatments, that is, untreated control plants, BHUF4-treated
plants, T16A-treated plants and BHUF4 ? T16A-treated

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plants were prepared according to the procedure discussed

above. Two weeks after the treatment of plants with
selected Trichoderma strains, the plants were sprayed with
a conidial suspension of Colletotrichum capsici (106 spores
ml-1) and were covered with sterile polybags for 48 h to
allow establishment of the pathogen on the leaves.
The disease incidence was recorded after 2 weeks as %
reduction in lesions on leaves in comparison to control
untreated plants for all the treatments. Ten randomly
selected plants from each treatment set were taken and the
number of lesions on leaves was recorded. The effectiveness of the treatments was decided on the basis of the
reduction in number of lesions on leaves in comparison to
control untreated plants.
Statistical Analysis
All the experiments were repeated twice unless otherwise
stated. The values from different experiments are shown in
figures as the mean of five replications standard deviation (SD). The data was subjected to analysis of variance
(ANOVA) using SPSS Ver. 16 (SPSS Inc., Chicago, IL).
The treatment mean values were compared with Duncans
multiple range tests at P B 0.05 significance level.

Results
Seventy-six Trichoderma isolates were obtained from the
rhizospheric region and 28 Trichoderma isolates were
obtained from the phyllosphere of healthy chilli plants.
Maximum pathogen inhibition against the isolated C.
capsici isolate was exhibited by Trichoderma isolate T16A
(70 2 %) obtained from the rhizosphere and BHUF4
(77.67 1.53 %) obtained from phyllopshere (Fig. 1).
Also, the most effective biochemical screening in terms of
extracellular enzyme production was recorded for the
above said two isolates as mentioned in Table 1. For better
comparison, two strains of Trichoderma (Control strain 1
from the phyllosphere region of chilli plants and Control
strain 2 isolates from the rhizospheric region of chilli
plants) having poor extracellular enzyme production ability
have been mentioned.
The selected isolates T16A and BHUF4 were identified
at the molecular level by amplifying the ITS region of the
universally conserved rDNA region of the genome. The
product obtained (500600 bp) was sequenced and the
sequences obtained were submitted to NCBI, Genbank
with accession numbers KC609758 (T16A) and KJ636986
(BHUF4). The sequencing results indicated that T16A and
BHUF4 showed the closest phylogenetic homology of
99 % with T. asperellum and T. harzianum respectively
(Fig. 2).

Fig. 1 Antagonism of Trichoderma isolates against C. capsici in dual

culture plate assay by a BHUF4 isolate and b T16A isolate

The selected isolates were further checked for their

compatibility. An overlapping mycelial growth with no
zone of inhibition demonstrated complete compatibility
among the selected Trichoderma isolates (T16A and
BHUF4) as shown in Fig. S1.
Plant growth promotion experiments exhibited interesting
results (Fig. 3). Treatment with T. harzianum (BHUF4)
recorded 36, 62, and 60 % increase in root length, number of
leaves and dry weight, respectively, which was on par with
the % increase obtained on treatment with T. asperellum
(T16A). Chilli plants treated with T16A recorded the highest
% increase in shoot length (41 %), number of nodes (32 %)
and overall increase in plant height (25 %). On combined
treatment of both the isolates, augmented plant growth was
recorded in all the parameters with the maximum increment
observed in root length (47 %).
A significant increment was recorded in total chlorophyll content along with amount of chl a and chl b in plants
treated with BHUF4 isolate. Comparable elevation in the
amount of chlorophyll was recorded in BCA-treated plants
irrelevant of their source of isolation (Fig. 3).
The maximum amount of phenol accumulation in leaves
of chilli plants was recorded at 48 h after pathogen inoculation (Fig. 4a) in the treatments. The increasing trend
continued until 48 h after pathogen inoculation after which
a decrease was recorded. When compared to positive
controls (C), a 2.1-, 4.6-, and 3.1-fold increase was recorded in plants treated with BHUF4, T16A and a combination of both BHUF4 and T16, respectively.

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Table 1 Biochemical and physiological characterization of Trichoderma isolates BHUF4 and T16A
Biochemical parameters

Control

IAA production (lg/ml)

8.27 0.29a

Phosphate solubilisation (lg/ml)

117.65 6.0
a

b-1,3 Glucanases (E.U.)

2.56 1.19

Chitinase (E.U.)

2.60 0.13a

% inhibition against C. capsici

0.0

Pectate lyase

Amylase

Lipase

Control strain 1

Control strain 2

BHUF4

20.53 1.13b

96.47 3.96e

68.84 2.24d

55.33 1.54c

301.2 0.13

3.99 1.08

988.97 3.96

24.04 0.95

28.2 1.2d
37.7 1.7

T16A

20.2 1.7

52.13 4.99

26.02 1.2c

30.19 2.78e
b

1047.04 23.26e
28.211 4.37d
23.12 1.64b
70 2d

33.33 2.22

77.67 1.53

Cellulase

Protease

Siderophore production

? denotes good activity

Fig. 2 The phylogenetic tree of

BHUF4 and T16A isolates
constructed on the basis of ITS
region of 18S rDNA sequence
(500600 bp). Bootstrap values
are based on 1000 replications.
Asterisk indicates strains whose
18S rDNA sequence was
determined in this study with
the accession numbers
KJ636986 and KC809758
respectively. Bar 0.005
substitutions per site

Comparable elevation in PAL activity in BHUF4- and

T16A-treated plants was recorded with a maximum value
at 48 hapi (Fig. 4b). When compared to positive controls
(C), a 5.18-, 6.02-, and 4.72-fold augmentation was
recorded in BHUF4, T16A and dual species consortia,
respectively. A decrease was recorded at 72 hapi, which
however was higher than the amount at 0 and 24 hapi. The
treated and unchallenged plants exhibited elevated PAL

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levels in all the treatments when compared with control

untreated and unchallenged plants.
Similar to the trend as recorded for PAL activity in the
treatments, maximum peroxidase activity was obtained at
48 hapi (Fig. 5a). The highest PO activity was supported by
the dual species consortia treatment whereas the single
species treatment showed comparable values with one
another. Application of both the antagonist in combination

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Fig. 3 Plant growth promotional activity of selected Trichoderma

isolates BHUF4 and T16A on chilli plants, either singly or in
combination. a The effect of treatment with Trichoderma isolates on

various growth parameters, b the effect of different treatments on the

growth of chilli plants and c root development of chilli plants

recorded a fourfold increment in PO activity, 24 hapi on

comparison to their untreated and unchallenged counterparts. Single treatment with BHUF4 and T16A recorded a 3
and 2.83 fold increase, respectively. The decline observed
after 24 h was gradual until 72 hapi in all the treatments.
PPO activity in all the treatments showed an increasing
trend until 48 hapi and then gradually decreased (Fig. 5b).
Maximum PPO activity was found in dual species consortia-treated plants followed by the single species treated
plants on the pathogen challenge. BHUF4- and T16Atreated plants recorded a 1.5-fold elevated level in PPO in
the challenged plants whereas BHUF4-treated plants
recorded a 0.67-fold increase and T16A-treated plants
exhibited a 1.0-fold increase when compared to untreated
and unchallenged plants. An elevated level of PPO

activity was also recorded in the treated and unchallenged

plants.
An elevated level of SOD activity (0.38-fold increase)
was recorded in all treatments when compared to untreated
and unchallenged control plants (Fig. 6a). However,
insignificant differences were recorded for the change in
the SOD activity in all the treatments, which was almost
equal for all time intervals (24, 48, and 72 hapi).
Total protein content in the leaves of the treated and
untreated plants showed an increasing trend until 72 hapi
(Fig. 6b). Maximum protein content was supported by the
dual species consortia treatment at 72 hapi (5670 lg/g
fresh weight). Plants treated with BHUF4 and T16A in
combination recorded a 0.74-fold increase whereas treating
the plants singly with BHUF4 or T16A recorded 0.5- and

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Fig. 4 Total phenolic content

(a) and phenylalanine ammonialyase activity (b) at different
time intervals in chilli plants
treated with Trichoderma
isolates either singly or in
combination and challenged
with Colletotrichum capsici.
Results are expressed as means
of five replicates, and vertical
bars indicate standard
deviations of the means.
Different letters indicate
significant differences among
treatment results taken at the
same time interval according to
Duncans multiple range test at
P B 0.05. C = normal control,
PC = pathogen control,
1 = phyllospheric BCA-treated
plants, 2 = Rhizospheric BCAtreated plants,
3 = Phyllospheric
BCA ? Rhizospheric BCAtreated plants, (P = pathogen
challenged, UC = unchallenged
plants)

0.6-fold increment when compared with untreated and

unchallenged control plants.
Control untreated plants recorded maximum lesion
development. The reduction in lesion development under
different treatments is visible in Fig. 7ad. Treatment with
phyllospheric Trichoderma isolate BHUF4 recorded a
49.6 7.74 % reduction in lesion development when
compared to control untreated plants while rhizospheric
Trichoderma isolate T16A recorded a 44.44 9.6 %
reduction in the number of lesions. The consortium of both
BHUF4- and T16A-treated plants recorded the maximum
reduction in lesion development in comparison to control
untreated plants, that is, 66.27 8.93 % (Fig. 7e).

Discussion
Plants are equipped with a plethora of defense mechanisms with various lines of defenses for protecting them
from copious phytopathogens. Phenols play an important

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role in the defense structure of the plants. They act as

antimicrobial compounds as well as precursors to structural polymers like lignin and also as signal molecules
for expression of defense related genes (Madhavan and
others 2011; Dakora 1996). Elevation in the level of
phenols in Capsicum annum against different pathogens
has been reported previously (Anand and others 2009;
Madhavan and others 2011; Naveen and others 2012) on
treatment with biocontrol agents or their byproducts.
Maximum studies have been focused on elevations on
treatment with BCAs at the rhizospheric region; only a
few have been attempted to study the direct impact of
their treatment at the phyllospheric region of the plants
(Nantawanit and others 2010; Madhavan and others
2011).
We studied the effect of using Trichoderma isolate
obtained from the phyllospheric region for their probable
role in fortifying the defense network in chilli plants
challenged with C. capsici. The proposed strategy could be
beneficial for managing foliar diseases more effectively in

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385

Fig. 5 Peroxidase activity

(a) and polyphenol oxidase
activity (b) at different time
intervals in chilli plants treated
with Trichoderma isolates either
singly or in combination and
challenged with Colletotrichum
capsici. Results are expressed as
means of five replicates, and
vertical bars indicate standard
deviations of the means.
Different letters indicate
significant differences among
treatment results taken at the
same time interval according to
Duncans multiple range test at
P B 0.05. C = normal control,
PC = pathogen control,
1 = phyllospheric BCA-treated
plants, 2 = rhizospheric BCAtreated plants,
3 = phyllospheric
BCA ? rhizospheric BCAtreated plants (P = pathogen
challenged, UC = unchallenged
plants)

chilli crops, which has been a matter of concern for chilli

growers.
Trichoderma spp.-mediated plant growth promotion has
been extensively studied in different plant pathogen systems (Harman and others 2004; Jain and others 2012;
Singh and others 2013). Few studies have been carried out
on the chilli-C. capsici system. In accordance with previous reports, enhanced growth promotion was recorded in
the chilli plants treated with Trichoderma isolates with 36,
62 and 60 % increases in root length, no. of leaves and dry
weight along with 45 and 25 % increases in shoot length
and plant height. Interestingly, plants treated with the
BHUF4 isolate had enhanced root and shoot lengths along
with numbers of leaves with a significant increase in the
dry weight of the plant. This could be attributed to the well
studied systemic effect of Trichoderma isolates capable of
augmenting plant growth through enhancing nutrient
uptake (Harman and others 2004) along with induction of
root development through production of auxins and indoles
(Contreras-Cornejo and others 2009, 2014a, b). However,

this study could support the use of foliar sprays of Trichoderma isolates from both the phyllosphere and rhizosphere for better plant growth. Moreover, a study by Bae
and others (2011) has reported interesting results regarding
the efficient colonization of Trichoderma isolates obtained
from the aerial parts in the roots of chilli plants.
Increased phenolics in plants have been directly linked
to an increased defense response against pathogenic attack
(Jain and others 2012; Singh and others 2013). Also,
infected chilli fruits have been reported with increased
phenolic content when compared to healthy fruits (Bharathi
and others 2004). In the present study, the increase in total
phenolic content in the leaves of the treated plants under
pathogen attack was comparable in both BHUF4 and T16A
treatment. Seed or seedling treatment with Trichoderma
isolates has shown promising results in increasing the
phenolic content of the plants under pathogen attack
(Harman and others 2004). There are reports that have
obtained comparable results with the use of BCAs as foliar
sprays (Madhavan and others 2011; Nantawanit and others

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J Plant Growth Regul (2016) 35:377389

Fig. 6 Superoxide dismutase

activity (a) and total protein
content (b) at different time
intervals in chilli plants treated
with Trichoderma isolates either
singly or in combination and
challenged with Colletotrichum
capsici. Results are expressed as
means of five replicates, and
vertical bars indicate standard
deviations of the means.
Different letters indicate
significant differences among
treatment results taken at the
same time interval according to
Duncans multiple range test at
P B 0.05. C = normal control,
PC = pathogen control,
1 = phyllospheric BCA-treated
plants, 2 = rhizospheric BCAtreated plants,
3 = phyllospheric
BCA ? rhizospheric BCAtreated plants (P = pathogen
challenged, UC = unchallenged
plants)

2010). However to our knowledge, this has been the first

report showing the efficiency of the phyllospheric Trichoderma isolate in chilli plants against C. capsici infestation.
For supporting the hypothesis of an elevated defense
response in chilli plants after foliar spraying with BCA, PAL,
PO, PPO, SOD enzyme assays were performed. PAL is the
first enzyme required for the phenylpropanoid biosynthesis
pathway leading to synthesis of phytoalexins or phenols
which are indispensable for the plant defense network (Nicholson and Hammerschmidt 1992) and synthesis of the
signaling compound salicylic acid (Wen and others 2005).
Increase in PAL content was on par in plants treated with
both BHUF4 and T16A Trichoderma isolates highlighting
the systemic ability of Trichoderma sp. in enhancing the
defense response of plants, independent of the mode of its
application. The decrease in the phenolic content and hence
the PAL content could be attributed to the oxidative polymerization of phenols into melanin or their subsequent
incorporation as lignin (Thompson 1964).
Like phenols, peroxidases have also been linked to
numerous physiological functions contributing to the

123

defense system of plants, for example, cross linking of

extension polymers (Everdeen and others 1988), lignifications (Walter 1992) and also for deposition of phenols in
the cell wall of plants under pathogen attack (Graham and
Graham 1991). PO and SOD work concurrently with the
enzymes of the ascorbate-gluthione cycle for scavenging
free radicals (Hernandez and others 2001). In the present
study, though the level of SOD remained indifferent to the
treatments in the plants, the level of PO was highest in the
combined treatment of BHUF4 and T16A isolates. Anand
and others (2009) have reported similar results with an
increased PO level on treatment with Trichoderma isolates
against C. capsici infection. Similarly, level of PPO was
also maximum in dual species consortia in the study. The
increased PPO level has been linked to plant pathogen
interaction for various plant systems (Bharathi and others
2004; Dutta and others 2008; Anand and others 2009;
Madhavan and others 2011). It has been reported to catalyze the oxidation of monophenolic and o-diphenolic
compounds as a terminal oxidase in infected plant tissues
(Jennings and others 1969). The increased phenolic content

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387

Fig. 7 Efficacy of different treatments in lesion development on

leaves of chilli plants after C. capsici challenge (a control; b
phyllospheric BHUF4-treated; c rhizospheric T16A-treated; d phyllospheric BHUF4 ? rhizospheric T16A-treated plants. e The %
reduction in lesions of leaves in comparison with control untreated

plants. Results are expressed as means of three replicates, and vertical

bars indicate standard deviations of the means. Different letters
indicate significant differences among treatment results taken at the
same time interval according to Duncans multiple range test at
P B 0.05

in the treated plants could be attributed to increased PO and

PPO levels as phenols may serve as substrate for these
enzymes (Lattanzio and others 2006). The overall increase
in defense related enzymes may thereby be linked with the
overall increment in the total protein content of the chilli
leaves relating the subsequent beneficial effect of treating
chilli plants with Trichoderma isolates suggesting their
possible role in modulating the overall proteome of the host
plant as well.
The effectiveness of treatment with Trichoderma isolates in reduction of disease incidence in different plantpathogen systems is well established (Harman and others
2004; Shoresh and others 2010; Saxena and others 2015).
The elevated levels of defense-related enzymes could be
linked to the reduction of the lesions of phyllosphere and
rhizospheric Trichoderma isolates. The performance of a

consortium utilizing more than one beneficial microbe was

more proficient in reducing disease incidence which has
been previously reported as well (Jain and others 2012;
Singh and others 2013) when compared to single BCA
treated plants.
The production of chilli is majorly affected by the
anthracnose disease caused by C. capsici. Both post- and
pre-harvest losses are reported from this disease leading to
significant reduction in the market value of the crop in
tropical and sub-tropical countries (Bosland and Votava
2003). Anthracnose may affect the aerial parts of the crop
which may then act as inoculums for the spread of the
disease in the field either through water splashes or through
winds. Many management strategies have been employed
for its control yet maximum reduction has been obtained
only through the use of fungicides (Than and others 2008).

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The use of BCAs for its control has not been so effective.
The prevailing concept of sustainable agriculture further
necessitates the need of developing integrated management
strategies that use BCAs (Saxena and others 2013). The
study thus proposes the use of foliar sprays of phyllospheric competent Trichoderma strains in combination
with the seed treatment of the plants for a fortified defense
network against the pathogen. This strategy could prove
useful in managing the spread of the disease as the plants
would be better reinforced with the beneficial cover of
Trichoderma spp. even at the aerial parts of the plants.
Also, better prevention could be obtained by a direct check
on the growth of the pathogen on the leaf and fruit surfaces.
Acknowledgments A.S. is grateful to Department of Science and
Technology, Govt. Of India for providing INSPIRE Fellowship under
the AORC Scheme. H.B.S. is thankful to Department of Biotechnology, New Delhi, India for providing financial assistance (BT/
PR5990/AGR/21/302/2009).
Compliance with Ethical Standards
Conflict of interest
of interest.

The authors declare that they have no conflict

References
Anand T, Ghaskaran R, Raguchander T, Samiyappan R, Prakasan V,
Gopalakrishnan C (2009) Defence responses of chilli fruits to
Colletotrichum capsici and Alternaria alternata. Biol Plant
53:553559
Arnon D (1949) Copper enzymes in isolated chloroplasts, phytophenoloxidase in Beta vulgaris. Plant Physiol 24:115
Bae H, Roberts DP, Lim HS, Strem MD, Park SC, Ryu CM, Melnick
RL, Bailey BA (2011) Endophytic Trichoderma isolates from
tropical environments delay disease onset and induce resistance
against Phytophthora capsici in hot pepper using multiple
mechanisms. Mol Plant Microbe Interact 24(3):336351
Beckman CH (1987) The nature of wilt diseases of plants. American
Phytopathological Society press, St. Paul
Bharathi R, Vivekananthan R, Harish S, Ramanathan A, Samiyappan
R (2004) Rhizobacteria-based bio-formulations for the management of fruit rot infection in chillies. Crop Prot 23:835843
Bosland PW, Votava EJ (2003) Peppers: Vegetable and Spice
Capsicums. CAB International, England, p 233
Conrath U (2011) Molecular aspects of defence priming. Trends Plant
Sci 16:524531
Contreras-Cornejo H, Macas-Rodrguez L, Cortes-Penagos C,
Lopez-Bucio J (2009) Trichoderma virens, a plant beneficial
fungus, enhances biomass production and promotes lateral root
growth through an auxin-dependent mechanism in Arabidopsis.
Plant Physiol 149:15791592
Contreras-Cornejo HA, Macas-Rodrguez L, Beltran-Pena E, Herrera-Estrella A, Lopez-Bucio J (2011) Trichoderma-induced
plant immunity likely involves both hormonal and camalexindependent mechanisms in Arabidopsis thaliana and confers
resistance against necrotrophic fungi Botrytis cinerea. Plant
Signal Behav 6:15541563

123

Contreras-Cornejo HA, Macas-Rodrguez LI, Alfaro Cuevas R,

Lopez-Bucio J (2014a) Trichoderma improves growth of Arabidopsis seedlings under salt stress through enhanced root
development, osmolite production and Na? elimination through
root exudates. Mol Plant Microbe Interact 27:503514
Contreras-Cornejo HA, Macas-Rodrguez LI, Herrera-Estrella A,
Lopez-Bucio J (2014b) The 4-phosphopantetheinyl transferase
of Trichoderma virens plays a role in plant protection against
Botrytis cinerea through volatile organic compound emission.
Plant Soil 379:261274
Dakora FD (1996) Diverse functions of isoflavonoids in legumes
transcend anti-microbial definitions of phytoalexin. Physiol Mol
Plant Pathol 49:120
Darvill AG, Albersheim P (1984) Phytoalexins and their elicitors a
defense against microbial infection in plants. Annu Rev Plant
Physiol 35:243275
De Marco JL, Felix CR (2007) Purification and characterization of a
b-glucanase produced by Trichoderma harzianum showing
biocontrol potential. Brazil Arch Biol Technol 50(1):2129
De Marco JL, Lima LH, Sousa MV, Felix CR (2000) A Trichoderma
harzianum chitinase destroys the cell wall of the phytopathogen
Crinipellis perniciosa, the causal agent of witches broom
disease of cocoa. World J Microbiol Biotechnol 16:383386
Dean R, Van Kan JAL, Pretorius ZA, Hammond-Kosack KE, Di
Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J,
Foster GD (2012) The Top 10 fungal pathogens in molecular
plant pathology. Mol Plant Pathol 13(4):414430
Dennis C, Webster J (1971) Antagonistic properties of species groups
of Trichoderma III. Hyphae interaction. Trans Br Mycol Soc
57:363369
Dutta S, Mishra AK, Kumar BSD (2008) Induction of systemic
resistance against fusarial wilt in pigeon pea through interaction
of plant growth promoting rhizobacteria and rhizobia. Soil Biol
Biochem 40:452461
Elad Y, Chet I, Henis Y (1981) A selective medium for improving
quantitative isolation of Trichoderma spp. from soil. Phytoparasitica 9:5967
Everdeen KE, Kiefer S, Willard JJ, Muldoon EP, Dey PM, Li XB,
Lamport DTA (1988) Enzymic cross-linkage of monomeric
extensin precursors in vitro. Plant Physiol 87:616621
Graham MY, Graham TL (1991) Rapid accumulation of anionic
peroxidases and phenolic polymers in soybean cotyledon tissues
following treatment with Phytophthora megasperma f. sp.
glycinea wall glucan. Plant Physiol 97:14451455
Gravel V, Antoun H, Tweddell RJ (2007) Growth stimulation and
fruit yield improvement of greenhouse tomato plants by
inoculation with Pseudomonas putida or Trichoderma atroviride: possible role of Indole Acetic Acid (IAA). Soil Biol
Biochem 39:19681977
Hankin L, Anagnostaksis L (1975) The use of solid media for
detection of enzyme production by fungi. Mycologia
47:597607
Hankin L, Zucker M, Sands DC (1971) Improved solid medium for
the detection and enumeration of pectolytic bacteria. Appl
Microbiol 22:205209
Harman GE, Howell CR, Viterbo A, Chet I, Lorito IM (2004)
Trichoderma species-Opportunistic, avirulent plant symbionts.
Nature Rev 2:4356
Hernandez JA, Ferrer MA, Jimenez A, Barcelo AR, Sevilla F (2001)
Antioxidant systems and O2/H2O2 production in the apoplast of
pea leaves: its relation with salt induced necrotic lesions in minor
veins. Plant Physiol 127:827831
Intanoo W and Chamswarng C (2007) Effect of antagonistic bacterial
formulations for control of anthracnose on chilli fruits. In:
Proceeding of the 8th national plant protection conference.
Naresuan University, Phisanulok, pp 309322

Author's personal copy

J Plant Growth Regul (2016) 35:377389
Jain A, Singh S, Sarma BK, Singh HB (2012) Microbial consortium
mediated reprogramming of defence network in pea to enhance
tolerance against Sclerotinia sclerotiorum. J Appl Microbiol
112:537550
Jennings PH, Braunaman BL, Zscheille FP (1969) Peroxidase and
polyphenol oxidase activity associated with Helminthosporium
leaf spot of maize. Phytopathology 59:963967
Jetiyanon K (2007) Defensive-related enzyme response in plants
treated with a mixture of Bacillus strains (IN937a and IN937b)
against different pathogens. Biol Control 42:178185
Lattanzio V, Lattanzio VMT, Cardinali A (2006) Role of phenolics in
the resistance mechanisms of plants against fungal pathogens
and insect. Phytochem Adv Res 2:367
Lowry OH, Nira J, Rosenburgh A, Lewis F, Rose JR (1951) Protein
measurement with the folin phenol reagent. J Biol Chem
193:265275
Madhavan S, Paranidharan V, Velazhahan R (2011) Foliar application
of Burkholderia sp. strain TNAU-1 leads to activation of defense
responses in chilli (Capsicum annuum L.). Braz J Plant Physiol
23(4):261266
Magnin-Robert M, Trotel-Aziz P, Quantinet D, Biagianti S, Aziz A
(2007) Biological control of Botrytis cinerea by selected
grapevine-associated bacteria and stimulation of chitinase and
b-1,3 glucanase activities under field conditions. Eur J Plant
Pathol 118:4357
Mathys J, De Cremer K, Timmermans P, Van Kerckhove S, Lievens
B, Vanhaecke M, Cammue BP, De Coninck B (2012) Genome
wide characterization of ISR induced in Arabidopsis thaliana by
Trichoderma hamatum T382 against Botrytis cinerea infection.
Front Plant Sci 3:108
Mehta S, Nautiyal CS (2001) An efficient method for qualitative
screening of phosphate solubilizing bacteria. Curr Microbiol
43:5156
Nantawanit N, Chanchaichaovivat A, Panijpan B, Ruenwongsa P
(2010) Induction of defense response against Colletotrichum
capsici in chilli fruit by the yeast Pichia guilliermondii strain
R13. Biol Control 52:145152
Naveen J, Hariprasad P, Nayaka SC, Niranjana SR (2012) Cerebroside mediated elicitation of defense response in chilli
(Capsicum annum L.) against Colletotrichum capsici infection.
J Plant Interact 8(1):6573
Nicholson RL, Hammerschmidt R (1992) Phenolic compounds and
their role in disease resistance. Annu Rev Phytopathol
30:369389
Niu DD, Liu HX, Jiang CH, Wang YP, Wang QY, Jin HL, Guo JH
(2011) The plant growth promoting rhizobacterium Bacillus
cereus AR156 induces systemic resistance in Arabidopsis
thaliana by simultaneously activating salicylate and jasmonate/
ethylene-dependent signaling pathways. Mol Plant Microbe
Interact 24:533542
Oanh LTK, Korpraditskul V, Rattanakreetakul C, Wasee S (2006)
Influence of biotic and chemical plant inducers on resistance of
chili to anthracnose. Kasetsart J Nat Sci 40:3948
Pieterse CM, Leon-Reyes A, Van der Ent S, Van Wees SC (2009)
Networking by small-molecule hormones in plant immunity. Nat
Chem Biol 5:308316
Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner HY,
Hunt MD (1996) Systemic acquired resistance. Plant Cell
8:18091819

389
Saxena A, Mishra S, Raghuwanshi R, Singh HB (2013) Biocontrol
agents: basics to biotechnological applications in sustainable
agriculture. In: Tiwari SP, Sharma R, Gaur R (eds) Recent
advances in microbiology, vol 2. Nova Publishers, USA,
pp 141164
Saxena A, Raghuvanshi R, Singh HB (2014) Molecular, phenotypic
and pathogenic variability in Colletotrichum isolates of subtropical region in North Eastern India, causing fruit rot of chillies.
J Appl Microbiol 117:14221434
Saxena A, Raghuwanshi R, Singh HB (2015) Trichoderma species
mediated differential tolerance against biotic stress of phytopathogens in Cicer arietinum L. J Basic Microbiol 55:195206
Schwyn B, Neilands JB (1987) Universal chemical assay for the
detection & determination of siderophores. Anal Biochem
160:4756
Segarra G, Casanova E, Bellido D, Odena MA, Oliveira E, Trillas I
(2007) Proteome, salicylic acid, and jasmonic acid changes in
cucumber plants inoculated with Trichoderma asperellum strain
T34. Proteomics 7:39433952
Shoresh M, Harman GE, Mastouri F (2010) Induced systemic
resistance and plant responses to fungal biocontrol agents. Annu
Rev Phytopathol 48:123
Sierra G (1957) A simple method for the detection of lipolytic activity
of micro-organisms and some observations on the influence of
the contact between cells and fatty substrates. A Van Leeuw J
Microb 23:1522
Singh A, Sarma BK, Upadhyay RS, Singh HB (2013) Compatible
rhizosphere microbes mediated alleviation of biotic stress in
chickpea through enhanced antioxidant and phenylpropanoid
activities. Microbiol Res 168:3340
Thakur RP, Rao VP, Sastry JG, Sivaramakrishnan S, Amruthesh KN,
Barbind LD (1999) Evidence for a new virulent pathotype of
Sclerospora graminicola on pearl millet. J Mycol Plant Pathol
29:6169
Than PP, Jeewon R, Hyde KD, Pongsupasamit S, Mongkolporn O,
Taylor PWJ (2008) Characterization and pathogenicity of
Colletotrichum species associated with anthracnose disease on
chilli (Capsicum spp.) in Thailand. Plant Pathol 57:562572
Thompson RH (1964) Structure and reactivity of phenolic compounds. In: Harborne EB (ed) Biochemistry of phenolic compounds. Academic Press, New York, pp 132
van Loon LC, Bakker PAHM, Pieterse CMJ (1998) Systemic
resistance induced by rhizosphere bacteria. Annu Rev Phytopathol 36:453483
Velazquez-Robledo R, Contreras-Cornejo HA, Macas-Rodrguez L,
Hernandez-Morales A, Aguirre J, Casas-Flores S, Lopez-Bucio
J, Herrera-Estrella A (2011) Role of the 4-phosphopantetheinyl
transferase of Trichoderma virens in secondary metabolism and
induction of plant defense responses. Mol Plant Microbe Interact
24:14591471
Walter MH (1992) Regulation of lignification in defense. In: Boller T,
Meins F (eds) Genes involved in plant defences. SpringerVerlag, New York, pp 327352
Wen PF, Chen JY, Kong WF, Pan QH, Wan SB, Huang WD (2005)
Salicylic acid induced the expression of phenylalanine ammonialyase gene in grape berry. Plant Sci 169:928934
Yedidia I, Benhamou N, Chet I (1999) Induction of defense responses
in cucumber plants (Cucumis sativus L.) by the biocontrol agent
Trichoderma harzianum. Appl Environ Microbiol 65:10611070

123