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Received: 20 August 2014 / Accepted: 13 July 2015 / Published online: 25 September 2015
Springer Science+Business Media New York 2015
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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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.
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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).
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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).
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Table 1 Biochemical and physiological characterization of Trichoderma isolates BHUF4 and T16A
Biochemical parameters
Control
8.27 0.29a
117.65 6.0
a
2.56 1.19
Chitinase (E.U.)
2.60 0.13a
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
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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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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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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.
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