Rhizosphere 16 (2020) 100247

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Rhizosphere
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Impact of the alkaloid colletotrichumine A on the pathogenicity of

Colletotrichum capsici in Capsicum annum L
Manoj K. Chitara a, Chetan Keswani b, *, Kyriakos G. Varnava c, Hareram Birla b,
Hagera Dilnashin b, Surya P. Singh b, Viji Sarojini c, Jonathan Sperry c, Harikesh B. Singh a
a
Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, 221005, India
b
Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi, 221005, India
c
School of Chemical Sciences, University of Auckland, Auckland, New Zealand

A R T I C L E I N F O A B S T R A C T

Keywords: Herein we report a study examining the effects of colletotrichumine A, an indole-pyrazine alkaloid isolated from
Colletotrichum capsici, anthracnose the anthracnose pathogen Colletotrichum capsici, on chilli plants. A colletotrichumine A-pathogen combination
Pathogenicity factor was more toxic to the host than individual inoculations of colletotrichumine A or the pathogen. The colleto­
Colletotrichumine A
trichumine A-pathogen combination led to an increased activity of defense related enzymes viz. PPO and SOD,
while levels of the lignification enzymes PAL and PO decreased. Higher levels of phenols including catechin and
ferulic acid were also observed with the colletotrichumine A-pathogen combination compared to individual
treatment. The likely role of colletotrichumine A during chilli anthracnose infection is supported by histo­
chemical analysis of infected plants that showed increased cell death after infection.

1. Introduction to approximately 40 fungal phytopathogens, including Colletotrichum

Chilli (Capsicum annuum), is a prime member of the family Sol­
anaceae, due to its importance as a food as well as its medicinal prop­
erties. The domesticated chilli has been part of the human diet for
hundreds of years (Andrews, 1995). Principally regarded as a spice crop,
the plant traces its origins back to the American tropics, from where it
has spread globally (Pickersgill, 1997). Amongst the different species,
C. annuum is the most widely cultivated, followed by C. frutescens
(Bosland et al., 2012; Tong and Bosland, 1999). C. annum is mostly used
as a spice for flavoring foods, particularly in South-East Asian and Latin
American countries, but the species also has widely revered medicinal
properties due to a high vitamin and antioxidant content (Kaefer and
Milner, 2008). For example, the pungent capsaicinoids has been found
to be effective in relieving diabetic and common neuropathic pains,
preventing adipocyte differentiation, improving glucose tolerance and
glucose-stimulated insulin response and reducing diabetic hyperglyce­
mia (Head, 2006; Rashid et al., 2003; Hwang et al., 2005; Babu and
Srinivasan, 1999; Tolan et al., 2004). Chilli powder contains phenolic
diterpenes that have anticancer properties (Kaefer and Milner, 2008).
Chilli production is severely influenced by several types of fungal,
bacterial and viral diseases. Rangaswami (1979) reported chilli as a host
capsici (Rangaswami, 1979) also termed as Colletotrichum complex. In
this complex several Colletotrichum sp. were reported by researchers
including C. siamense, C. simmondsii, C. queenslandicum, C. truncatum and
a C. cairnsense sp. nov. in Australia (De Silva et al., 2017), C. fioriniae,
C. fructicola, C. gloeosporioides, C. scovillei, and C. truncatum, C. conoides,
C. grossum, and C. liaoningense, C. aenigma, C. cliviae, C. endophytica,
C. hymenocallidis, C. incanum, C. karstii, and C. viniferum in China (Diao
et al., 2017) and C. truncatum (syn. C. capsici), C. coccodes, C. karstii,
C. kahawae, C. nymphaeae, C. fructicola and C. gloeosporioides in India
(Katoch et al., 2017). This pathogen, rated among the top ten most
notorious pathogens in the world, causeing the economically devas­
tating disease anthracnose (die back or fruit rot, leaf spot, wilt, damping
off) in a wide range of hosts, including cereals, legumes, vegetables and
tree fruits (Bailey, 1992; Dean et al., 2012). Anthracnose causes an
estimated loss of about 29.5% in crop yield amounting to huge economic
losses (Garg et al., 2014; Poulos). C. capsici thrives at temperatures
around 27 ◦ C in a high humidity range of 75–80%. Generally, warm and
wet climatic conditions favor its growth. Two mechanisms have been
linked to the pathogen entry in the field, either from infected seeds or
through diseased fruits acting as inocula (O’Connell et al., 2000).
In recent years, C. capsici has proven to be a rich source of natural

* Corresponding author.
E-mail addresses: chetan.keswani4@bhu.ac.in (C. Keswani), v.sarojini@auckland.ac.nz (V. Sarojini), j.sperry@auckland.ac.nz (J. Sperry).

https://doi.org/10.1016/j.rhisph.2020.100247
Received 3 July 2020; Received in revised form 18 August 2020; Accepted 1 September 2020
Available online 6 September 2020
2452-2198/© 2020 Elsevier B.V. All rights reserved.
M.K. Chitara et al.
products, which attracted interest as targets for developing new me­
dicinal agents (Kharwar et al., 2011). C. capsici might be an excellent
alternate source to develop new anticancer drugs (Kumaran et al.,
2011). The phytotoxin acetylcolletotrichin has been isolated from the
culture filtrates of C. capsici and is frequently associated with anthrac­
nose (Kumaran et al., 2011; Hu et al., 2014; García-Pajón and Collado,
2003).
The indole-pyrazine alkaloid colletotrichumine A was isolated from
C. capsici in 2014 (Hu et al., 2014). We were keen to examine if this
natural product plays a role in chilli anthracnose; indeed, glume blotch
in wheat (inflicted by Septoria nodorum) can (at least in part) be attrib­
uted to the pyrazine alkaloid septorine causing a decoupling action on
wheat mitochondria, likely contributing to the pathogenicity of the
fungus (Devys et al., 1982).
2. Materials and methods
2.1. Pathogen’s collection and maintenance
The pure cultures of the pathogen C. capsici (strain code C56) were
procured from the Laboratory of Prof. H. B. Singh, Department of
Mycology and Plant Pathology, Institute of Agricultural Sciences,
Banaras Hindu University, Varanasi, India. Details regarding molecular
identification of the C. capsici (strain code C56) were reported previ­
ously by Saxena et al. (2014) (Saxena et al., 2014; Amrita, 2015). The
pathogen was maintained in potato dextrose agar (PDA) slants by
sub-culturing periodically on fresh media.
2.2. Pathogenicity test
2.2.1. In vitro pathogenicity test
The pathogen was tested for pathogenicity by Koch’s postulates. The
spore suspension was inoculated (10 μL) on the surface of the sterilized
chilli leaf and incubated at 27 ± 1 ◦ C for 4–5 days. The lesion produced
was aseptically cut into small pieces of 1–2 mm and placed on PDA
plates, which were incubated at 27 ± 1 ◦ C for 4–5 days. The mycelia
obtained were sub-cultured to obtain a pure colony of the pathogen on
PDA plates. Subsequently, the isolated strain of the pathogen was
compared with the mother culture for morphological confirmation.
2.2.2. In vivo pathogenicity test
The in vitro experiment was repeated in glasshouse conditions to test
the Koch’s postulate of pathogenicity on a locally available susceptible
‘G4’ variety of chilli. The spore suspension was inoculated on the
healthy plant grown in pots and the plants were observed daily for
symptom development. After lesions were observed, the pathogen was
isolated aseptically under laboratory conditions. The infected fruits
were cut into small pieces of 1–2 mm length and placed on PDA plates,
which were incubated at 27 ± 1 ◦ C for 4–5 days. The mycelia obtained
were sub-cultured to obtain a pure colony of the pathogen on PDA
plates. Subsequently, the isolated strain of the pathogen was compared
with mother culture for morphological confirmation.
2.3. Colletotrichumine A
Pure colletotrichumine A was synthesized as described previously by
Varnava and Sperry (2016) (Varnava and Sperry, 2015) and tested on
healthy plants for its role in pathogenicity.
2.4. Experimental design
The pot experiments were carried out in the greenhouse at the
Department of Mycology and Plant Pathology, Institute of Agricultural
Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India. The
following treatments were studied: A = control, B = pathogen, C =
DMSO, D = pathogen and colletotrichumine A @ 40 μg/mL, E =
2
Rhizosphere 16 (2020) 100247
pathogen with colletotrichumine A @ 50 μg/mL, F= Pathogen with
colletotrichumine A @ 60 μg/mL, G = colletotrichumine A @ 40 μg/mL,
H colletotrichumine A @ 50 μg/mL, I = colletotrichumine A @ 60 μg/
mL. For each treatment, three pots containing three seedlings were
maintained. The experiment was designed in a completely randomized
manner.
2.5. Inoculum preparation for treatment of chilli plants with
colletotrichumine A and C. capsici
The three concentrations of colleotrichumine A, i.e. 40 μg/mL, 50
μg/mL and 60 μg/mL in DMSO were prepared and tested both individ­
ually and in combination with the pathogen on chilli plants. A 7 day old
culture of C. capsici was used for preparing the conidial suspension of the
pathogen. Petri-dishes containing the 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
autoclaved filter papers and conidial count was adjusted to 106 conidia/
mL using a haemocytometer.
2.6. Treatment of chilli plants with colletotrichumine A and spore
suspension of C. capsici
21 day old chilli seedlings (Var. G4) were transplanted in pots con­
taining a sterile soil mixture. The soil mixture containing sandy soil,
vermicompost and farmyard manure (2:1:1) was sterilized in an auto­
clave at 15 psi for 20 min on three consecutive days and the mixture was
placed in each pot. Untreated plants (A) served as control. The treatment
(B) was inoculated with conidial suspension of the pathogen after one
week of transplanting by spraying (Saxena et al., 2019), treatment (C)
was inoculated with sterile DMSO, treatments (D), (E) and (F) were
inoculated with the pathogen-colletotrichumine A combination @ 40,
50, 60 μg/mL, respectively. Treatments (G), (H) and (I) were inoculated
with only colletotrichumine A @ 40, 50, 60 μg/mL. All the treated and
untreated plants A-I were kept covered with sterile polybags for 24 h to
maintain humidity up to 75–80%.
2.7. Sample collection
From each treatment, five plants were randomly uprooted and
collected in sterile polybags after 24, 48, 72 and 96 h. Nodal leaves (3rd
to 5th nodes) from the bottom were collected as samples for biochemical
analysis and other leaves for histochemical analysis. Collected leaves
were washed in running tap water, dried with blotting paper and stored
in a deep freezer (− 80 ◦ C) until required for biochemical and HPLC
analysis. For histochemical analysis, the sample was harvested in icebox
and used immediately.
2.8. Biochemical analysis
2.8.1. Phenylalanine ammonia-lyase (PAL) assay
From each treatment, 0.1 g leaf sample was homogenized in 2 mL of
0.1 M sodium borate buffer (pH 7.0; 4 ◦ C) containing 1.4 M β-mercap­
toethanol. The homogenate was centrifuged at 16,000 g for 15 min at
4 ◦ C. The supernatant was collected and used as the enzyme source. A
reaction mixture was prepared containing 0.2 mL of enzyme extract, 0.5
mL of 0.2 M borate buffer (pH 8.7) and 1.3 mL of sterile distilled water.
Addition of 1 mL of 0.1 M L-phenyalalanine (pH 8.7) to the reaction
mixture initiated the reaction. After an incubation of 30 min, the reac­
tion was stopped by addition of 0.5 mL of 1 M trichloroacetic acid (TCA).
As described by Brueske (1980) (Brueske, 1980), the PAL activity was
measured at 290 nm, after the formation of transcinamic acid and
expressed as μmol L− 1 TCA g− 1 fresh weight (FW).
2.8.2. Total free phenolic content (TPC)
The TPC was determined following the method of Zheng and Shetty
M.K. Chitara et al.
(2000). Leaf tissue (0.1 g) was placed in 5 mL of 95% ethanol and kept at
0 ◦ C for 48 h. The samples were homogenized individually and centri­
fuged at 13000 g for 10 min. To 1 mL of the supernatant, 1 mL of 95%
ethanol and 5 mL of sterile distilled water and 0.5 mL of 50% Folin-
Ciocalteau reagent was added, and the content was vortexed
throughly (Velikova et al., 2000). After 5 min, 1 mL of 5% sodium
carbonate was added, the reaction mixture was allowed to stand for 1 h
and the absorbance of the color was recorded at 725 nm. Standard
curves were prepared for each assay using various concentrations of
gallic acid (GA; Sigma-Aldrich-27645) in 95% ethanol. Absorbance
values were converted to mg GA equivalents (GAE) g− 1 FW.
2.8.3. Superoxide dismutase (SOD) assay
SOD activity was assayed following the method of Fridovich (1986)
(Fridovich, 1986) by measuring the ability of enzyme extract from
samples to inhibit the photochemical reduction of nitroblue tetrazolium
(NBT) chloride. Fresh leaves (0.1 g) from each of the treatments were
homogenized in 2.0 mL of extraction buffer (0.1 mol L− 1 phosphate
buffer containing 0.5 mmol L− 1 EDTA at pH 7.5) in a prechilled mortar
and pestle. The homogenate was centrifuged at 15,000 g for 20 min at
4 ◦ C. The reaction mixture contained 200 mmol L− 1 methionine, 2.25
mmol L− 1 NBT, 3 mmol L− 1 EDTA, 100 mmol L− 1 phosphate buffer (pH
7.8), 1.5 mmol L− 1 sodium carbonate and the aforementioned enzyme
extract. The final volume was made up to 3 mL with sterile distilled
water. The reaction was initiated by adding 2 μmol L− 1 riboflavin (0.4
mL), and the tubes were illuminated with two 15-W fluorescent lamps
for 15 min. The mixture in the absence of theenzyme served as the
control. The reaction was terminated by turning the light off and
keeping the tubes in dark until the absorbance was recorded at 560 nm.
One unit of the SOD activity was defined as the amount of enzyme
reducing the absorbance to 50% in comparison to the control lacking the
enzyme.
2.8.4. Peroxidase (PO) assay
PO activity was assayed using a slightly modification to the method
described by Hammerschmidt et al. (1982) (Hammerschmidt et al.,
1982). Leaf samples (0.1 g) were homogenized separately in 2 mL of 0.1
mol L− 1 phosphate buffer (pH 7.0) at 4 ◦ C then centrifuged at 16,000 g at
4 ◦ C for 15 min. The resulting supernatant was used as the enzyme
source. The reaction mixture consisted of 1.5 mL pyrogallol (0.05 mol
L− 1), 0.05 mL enzyme extract and 0.5 mL H2O2 (1% v/v). A reaction
mixture without the enzyme served as the control. The changes in the
absorbance at 420 nm were recorded after 30 s intervals for 3 min. The
enzyme activity was expressed as change in the unit min− 1 g− 1 FW.
2.8.5. Polyphenol oxidase (PPO) assay
Leaf samples (0.1 g) were homogenized in 2 mL of ice cold phosphate
buffer (0.1 mol L− 1, pH 6–5). The homogenate was centrifuged at 16000
g for 30 min at 4 ◦ C and the resulting supernatant was used as the
enzyme source. The reaction mixture contained 0.4 mL catechol (1
mmol L− 1) in 3 mL of 0.05 mol L− 1 sodium phosphate buffer (pH 6.5)
and 0.4 mL enzyme extract. A reaction mixture without the enzyme
served as the control. Catechol was used as substrate for PPO and the
increase in absorbance was recorded at 405 nm (Gauillard et al., 1993).
The linear portion of the activity curve was used to express PPO enzyme
activity as change in OD min− 1g− 1 FW.
2.8.6. Hydrogen peroxide (H2O2) assay
Leaf sample (0.1 g) was homogenized in 2.0 mL of 0.1% (w/v) of
trichloroacetic acid (TCA). The crushed material was centrifuged at
12000g for 10 min and 0.5 mL of the resulting supernatant was used
directly in the assay. 10 mM potassium phosphate buffer (pH 7.0) and 1
mL of 1 M potassium iodide solution was added to the supernatant and
the resulting mixture incubated at room temperature for 5 min. The
oxidation product formed was measured by UV spectrophotometer at
390 nm (Velikova et al., 2000). The amount of H2O2 formed was
3
Rhizosphere 16 (2020) 100247
determined by correlating with the standard curve made with known
concentrations of H2O2 and expressed as mmol g− 1 fresh weight (FW).
2.9. Histochemical analysis
2.9.1. In situ H2O2 localization
Histochemical localization of H2O2 production was performed by
incubating the leaves of each treatment in 3,3′ -diaminobenzidine (DAB)
solution for 4–5 h in dark followed by boiling the leaves in destaining
solution containing ethanol-acetic acid (3:1). Formation of brown spots
on the leaves indicated production of H2O2 (Jain et al., 2015a; Singh
et al., 2013).
2.9.2. In situ detection of lipid peroxidation
Aldehydes produced by peroxidation of membrane lipids were
assessed using the protocol of Airaki et al.; (2012). The leaves of the
different treatments (including control plants) were stained with Schiff’s
reagent (Sigma Aldrich) for 1 h and boiled in a destaining solution of
ethanol-acetic acid (3:1) for 30 min. Formation of a pink spot on the leaf
tissues indicated lipid peroxidation.
2.9.3. In situ detection of cell death
Cell death was determined by immersing the leaves of the different
treatments in 0.1% Evan’s blue solution for 15 min followed by depig­
mentation of the leaf tissues in 95% boiling ethanol for 30 min. Necrotic
lesions were observed as indigo blue spots on the leaves (Iannone et al.,
2015).
2.9.4. In situ superoxide localization
Superoxide (O•–2 ) production was localized following the method
described by Tewari et al. (2015) (Tewari et al., 2015). Leaves of the
treated plants were directly placed in 25 mM 4-(2-hydrox­
yethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.6) sup­
plemented with 0.1 mg mL-1 nitroblue tetrazolium chloride (NBT) and
incubated at 25 ◦ C in the dark for 2 h. Following incubation, the leaf
samples were rinsed in 80% (v/v) ethanol for 10 min at 70 ◦ C and
mounted in a lactic acid-phenol-water solution (1:1:1). Formation of
bluish spots, a characteristic of formazan deposits on the leaf surface,
indicated the production of O•– 2 .
2.9.5. In situ determination of lignin deposition
Lignin deposition around the vascular bundles of the collar region
was measured by cutting thin transverse sections of the plants, partic­
ularly in the collar region. The sections were mounted on 1%
phloroglucinol-methanol solution in 20% hydrochloric acid and
observed under a light microscope (Nikon DS-fi1, Japan) at 10× and
40× magnification. The areas of lignin deposition were indicated by a
red-violet color (Mishra et al., 2014).
2.10. High performance liquid chromatography (HPLC) analyses of
phenylpropanoid derivatives in treated chilli plant leaves
For the analysis of phenylpropanoid derivatives, 0.5 g of fresh tissue
was harvested 24 h after inoculation and subsequently extracted with
50% methanol (10 mL). The solvent was evaporated under reduced
pressure on rotary evaporator (Eyela N-Nseries, Tokyo, Japan). The
resulting residue was dissolved in HPLC grade methanol and subjected
to HPLC for the qualitative and quantitative analysis of specific pheno­
lics. The HPLC system (Shimadzu LC-10A, Japan) was equipped with
dual pump LC- 10A binary system, UV detector SPD-10 A, Phenomenex
(Torrance, USA) C18 column (RP-Hydro, 4×, 250 × 4.6 mm). The data
was integrated by Shimadzu Class VP series software. Separation of the
compounds was achived with acetonitrile/water (1:1 v/v) containing
1% acetic acid in a linear gradient program, starting with 18% aceto­
nitrile, changing to 32% at 15 min and finally to 50% at40 min (Singh
et al., 2009). The solvent flow was 1.0 mL min− 1. Results (μg g− 1 FW)
M.K. Chitara et al. Rhizosphere 16 (2020) 100247

Fig. 1. Disease symptoms on leaves (A) control, (B) and (C) anthracnose infected leaves.

Fig. 2. Characteristic symptoms of anthrocnose on Control (A) leaves (B), stem (C) and fruit of chilli plant (D and E).

Fig. 3. The morphological appearance of the Colletotrichum isolates on PDA growth medium (A) and the microscopic structures i.e. conidia (B and C), setae (D) and
acervuli (E and F).

4
M.K. Chitara et al.
were obtained by comparing the peak areas (max 254 nm) of the sam­
ples with those of standards (Class VP series software, Shimadzu, Japan).
2.11. Statistical analysis
All the experiments were repeated thrice unless otherwise stated
using a completely randomized design. The data were expressed as
means of three replications standard deviations. LSD test (p ≤ 0.05) was
used for comparing means using SPSS Version 20 software. Probability
level of 0.05 was set for analyzing the critical difference among the
isolates in each treatments and values ≤ 0.05 were considered signifi­
cantly different.
3. Results
3.1. Symptomology
Characteristic disease symptoms were observed on chilli leaves as
5
Rhizosphere 16 (2020) 100247
Fig. 4. 4.1: Characteristic symptoms of colleto­
trichumine A after inoculated both individually
and in combination with pathogen at different
concentration on healthy plants. (A) control, (B)
pathogen, (C) DMSO, (D) pathogen-
colletotrichumine A @ 40 μg/mL, (E) pathogen-
colletotrichumine A @ 50 μg/mL, (F) pathogen-
colletotrichumine A @ 60 μg/mL(G) colleto­
trichumine A @ 40 μg/mL, (H) colletotrichumine
A @ 50 μg/mL, (I) colletotrichumine A @ 60 μg/
mL, 4.2: Characteristic aerial symptoms of col­
letotrichumine A after inoculation both individ­
ually and in combination with the pathogen at
different concentration on healthy plants (A)
control, (B) pathogen treated, (C) DMSO, (D)
pathogen-colletotrichumine A @ 40 μg/mL, (E)
pathogen-colletotrichumine A @ 50 μg/mL, (F)
pathogen-colletotrichumine A @ 60 μg/mL, (G)
colletotrichumine A @ 40 μg/mL, (H) colleto­
trichumine A @ 50 μg/mL, (I) colletotrichumine
A @ 60 μg/mL.
sunken circular lesions on the leaves and fruits (Fig. 1). Generally, the
lesions were characterized with the presence of black spots in concentric
rings at maturity. The dark spots observed under a microscope were
identified as the acervuli structures containing setae hairs entrapping
the conidia of the pathogen. Symptoms were found on all parts of the
plant including leaves, fruits and stem (Fig. 2). Lesions on the stems and
leaves appeared as small sunken grayish brown spots with dark margins,
where development of acervuli in concentric rings could be easily
observed.
3.2. Pathogenicity test
The Koch’s postulates were successfully proven for the host under in
vitro conditions. The pathogen was able to produce characteristic
symptoms on the leaves under in vitro conditions as well as under field
conditions. The pathogen was re-isolated from the infected fruits and
was found to be identical to the mother culture (Fig. 3).
M.K. Chitara et al.
3.3. Colletotrichumine A
After healthy plants were inoculated with colletotrichumine A both
individually and in combination with pathogen, disease symptoms were
observed. These results infer that colletotrichumine A was able to pro­
duce disease symptoms in vivo (Figs. 4.1 and 4.2).
3.4. Disease severity
Figs. 4.1 and 4.2 show the disease severity after infection in chilli
plants at 24, 48, 72 and 96 h, as assessed by measuring the region of
necrosis around the point of infection. The combination of pathogen-
colletotrichumine A @ 40, 50, 60 μg/mL showed the highest percent
mortality (80, 85 and 90%, respectively). Individual inoculation with
colletotrichumine A @ 40, 50, 60 μg/mL led to significant levels of
mortality (60, 65 and 70%, respectively). Inoculation with the pathogen
alone led to 78% mortality, while DMSO treated plants showed only 5%
mortality in comparison to the untreated control.
3.5. Biotic stress leads to superoxide production within host leaf tissues
Production of superoxide (O•–
2 ) ions within the leaf was indicated by
6
Rhizosphere 16 (2020) 100247
Fig. 5. 5.1: Production of superoxide ions in leaves of
chilli plant. A represents production of superoxide ions in
untreated control after 24, 48, 72 and 96 h, respectively.
B, C, D, E, F and G, H, I represent superoxide ion pro­
duction in the plants treated with pathogen, DMSO,
pathogen-colletotrichumine A @ 40, 50, 60 μg/mL and
colletotrichumine A @ 40, 50, 60 μg/mL 5.2: Comparison
of amount of superoxide production in uninfected control
(A), pathogen infected (B), DMSO (C), pathogen-
colletotrichumine A @ 40 μg/mL (D), pathogen-
colletotrichumine A @ 50 μg/mL (E), pathogen-
colletotrichumine A @ 60 μg/mL (F), colletotrichumine
A @ 40 μg/mL (G), colletotrichumine A @ 50 μg/mL (H),
colletotrichumine A @ 60 μg/mL (I). 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 Duncan’s multiple
range test at P ≤ 0.05.
the formation of blue formazan deposits as a result of reaction of NBT
with O•–2 ions. The untreated control and DMSO treated plants expressed
minimum blue formazan deposition, indicating the least accumulation
2 ions. Colletotrichumine A, both individually and in combination
of O•–
with pathogen at different concentrations, led to maximum formation of
blue formazan and thus the highest accumulation of O•– 2 ions after
inoculation. The pathogen expressed accumulation of O•– 2 is high in
comparison to the control but lower in comparison to colletotrichumine
A and the colletotrichumine A-pathogen combination (Fig. 5.1). The bar
diagram (Fig. 5.2) also supports the pattern of accumulation of O•–2 ions,
which was initially 24 h and 48 h after inoculation increased slowly
then, increased gradually after 72 and 96 h as the dose increased in the
colletotrichumine A-pathogen combination and the individual colleto­
trichumine A inoculation. In the case of the individual pathogen inoc­
ulation, the accumulation of O•–2 ions gradually increases after pathogen
infection, but DMSO and unchallenged plants showed insignificant
accumulation of O•– 2 ions over time. These results also supported by the
histochemical analysis of production of blue formazan in leaves of the
chilli plants.
M.K. Chitara et al.
3.6. Production of H2O2 and PPO within host leaf tissues
Accumulation of H2O2 within leaf tissues was determined by histo­
chemical assay using DAB treatment, in which H2O2 reacts with DAB in
presence of peroxidase to produce an alcohol insoluble brown DAB
precipitate on the leaf surface (Fig. 6.1). The leaf tissue treated with
colletotrichumine A both individually and in combination with path­
ogen displayed prominent number of brown spots in contrast to the
untreated plants. Among the infected plants, the colletotrichumine A-
7
Rhizosphere 16 (2020) 100247
Fig. 6. 6.1: Formation of H2O2 in leaves of chilli plants. A
represents production of superoxide ions in untreated
control after 24, 48, 72 and 96 h, respectively. Uninfected
control (A), Pathogen infected (B), DMSO (C), pathogen-
colletotrichumine A @ 40 μg/mL (D),pathogen-colleto­
trichumine A @ 50 μg/mL (E), pathogen-
colletotrichumine A @ 60 μg/mL (F), colletotrichumine
A @ 40 μg/mL (G), colletotrichumine A @ 50 μg/mL (H),
colletotrichumine A @ 60 μg/mL (I), 6.2: Comparison of
the amount of H2O2 production in uninfected control (A),
pathogen infected (B), DMSO (C), pathogen-
colletotrichumine A @ 40 μg/mL (D),pathogen-colleto­
trichumine A @ 50 μg/mL (E), pathogen-
colletotrichumine A @ 60 μg/mL (F), colletotrichumine
A @ 40 μg/mL (G), colletotrichumine A @ 50 μg/mL (H),
colletotrichumine A @ 60 μg/mL (I). 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 Duncan’s multiple
range test at P ≤ 0.05. 6.3: Comparison of amount of PPO
formation in uninfected control (A), pathogen infected
(B), DMSO (C), pathogen-colletotrichumine A @ 40 μg/
mL (D), pathogen-colletotrichumine A @ 50 μg/mL (E),
pathogen-colletotrichumine A @ 60 μg/mL (F), colleto­
trichumine A @ 40 μg/mL (G), colletotrichumine A @ 50
μg/mL (H), colletotrichumine A @ 60 μg/mL (I). 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 Dun­
can’s multiple range test at P ≤ 0.05.
pathogen combination displayed maximum staining in leaves, while the
plants inoculated with colletotrichumine A alone showed lower levels of
staining in their leaves. The production of H2O2 in pathogen challenged
plants was less compared to both the colletotrichumine A-pathogen
combination and colletotrichumine A alone, but more when compared
to DMSO treated and untreated chilli plants.
The bar diagram (Fig. 6.2) indicates that a sharp increase in H2O2
production was observed in the hosts infected with the pathogen-
colletotrichumine A combination @ 40, 50, 60 mg/mL after 72 h and
M.K. Chitara et al.
Fig. 7. Detection of hypersensitivity cell death in leaves of chilli plant. A
represents lipid peroxidation in untreated control after 24, 48, 72, 96 h,
respectively. Uninfected control (A), pathogen infected (B), DMSO (C),
pathogen-colletotrichumine A @ 40 μg/mL (D),pathogen-colletotrichumine A
@ 50 μg/mL (E), pathogen-colletotrichumine A @ 60 μg/mL (F), colleto­
trichumine A @ 40 μg/mL (G), colletotrichumine A @ 50 μg/mL (H), colleto­
trichumine A @ 60 μg/mL (I).
96 h (4.91, 5.31, 5.64 and 7.11, 6.48, 6.66 mM). However, there was
also increase in H2O2 production in the hosts infected with colleto­
trichumine A alone @ 40, 50, 60 μg/mL dose after 72 h and 96 h (3.80,
5.23, 5.41 and 5.41, 6.66, 6.92 mM) followed by pathogen (3.54 and
3.70) infected plants. The maximum accumulation of H2O2 was recor­
ded in the host plants inoculated with colletotrichumine A @ 40 μg/mL
(7.11 mM) and the pathogen after 96 h. On the other hand, the pathogen
inoculated plants accumulated lower levels of H2O2 in comparison to the
aforementioned treatments with colletotrichumine A, but more in
comparison to DMSO treated and untreated plants. The DMSO treated
and untreated plants serving as controls accumulated the minimum
amount of H2O2 in their leaves.
Plant polyphenol oxidases (PPOs) are widely distributed and well-
studied oxidative enzymes that are responsible for discoloration in
damaged and diseased plant tissues. PPOs are ubiquitous copper-
containing enzymes which use molecular oxygen to oxidize common
ortho-diphenols such as caffeic acid and catechol to their corresponding
quinones. PPO-generated quinones are highly reactive and may cross-
link or alkylate proteins, leading to the commonly observed brown
pigments in damaged plant tissues and plant extracts. The histochemical
analysis showed maximum browning in the host treated with the col­
letotrichumine A-pathogen combination after 72 and 96 h, compared to
those treated with colletotrichumine A alone. Pathogen treated plants
showed less browning in comparison to both the colletotrichumine A-
pathogen combination as well as colletotrichumine A alone. The DMSO
treated and untreated plants exhibited minimum amount of browning in
their leaves (Fig. 6.1). This hypothesis was also supported by the
biochemical estimation of PPO in chili plants (Fig. 6.3).
3.7. Induction of cell death due to HR
In vitro cell death was detected by Evan’s blue treatment (Ray et al.,
2016). The highest cell death owing to HR was recorded in the plants
treated with the colletotrichumine A-pathogen combination. The prog­
ress of cell death increased as the dose of colletotrichumine A @ 40, 50,
60 μg/mL was increased. However, the plants treated with colleto­
trichumine A alone @ 40, 50, 60 mg/mL showed less cell death in
comparison to the colletotrichumine A-pathogen combination, but more
in comparison to pathogen alone, DMSO treated and untreated plant
(Fig. 7).
8
Rhizosphere 16 (2020) 100247
3.8. Role of PAL, PO in lignification as a plausible mode of containment
of the pathogen from invasion to other plant parts
PAL and PO are actively involved in the synthesis of lignin in host
tissue, providing cellular strength against pathogen attack (Singh et al.,
2016). Lignification has been suggested to play a role in plant devel­
opment as well as disease resistance owing to the formation of a physical
barrier against pathogen attack. Prominent differences in lignification
were observed between challenged and unchallenged plants in our case
(Fig. 8.1). In the colletotrichumine A-pathogen combination treatment,
less lignification was observed in comparison to individual exposure to
colletotrichumine A and the pathogen. Plants subjected to individual
inoculation with colletotrichumine A and the pathogen displayed high
levels of lignification in the inter-fascicular cambium of the plants.
DMSO and untreated plants showed normal lignification levels. These
results are also supported by the biochemical analysis of PAL and PO
(Figs. 8.2 and 8.3).
3.9. Qualitative and quantitative analysis of phenolics
In order to gain insight into the induced systemic response of the
host, qualitative and quantitative enhancement of phenolic content was
assessed. The plants infected with colletotrichumine A-pathogen com­
bination, colletotrichumine A alone and pathogen alone all exhibited a
sharp elevation in the total phenol content in comparison to the DMSO
treated and untreated control (Fig. 9.1).
Figs. 9.2, 9.3, 9.4 and 9.5 show the HPLC chromatogram of the
phenolics in the leaves after all the different treatments. The plants
treated with pathogen (C. capsici), colletotrichumine A alone @ 40, 50,
60 μg/mL and colletotrichumine A-pathogen combination @ 40, 50, 60
μg/mL, including unchallenged control showed significant accumula­
tion of phenols after inoculation. The significant accumulation of free
phenolics was enumerated in the form of per gram fresh weight basis of
challenged and unchallenged chilli plant (Figs. 9.4 and 9.5).
4. Discussion
The fungal phytopathogen C. capsici causes severe crop losses
worldwide (Dean et al., 2012). Our study suggests that colleto­
trichumine A leads to increased disease severity, when applied on chilli
plants both in combination with the pathogen (C. capsici) and individ­
ually. The in vitro pathogenicity test revealed the ability of
pathogen-colletotrichumine A combination enhanced disease severity in
comparision to pathogen alone, indicating the impact of colleto­
trichumine A which may involve increasing the virulence of the path­
ogen. Colletotrichumine A alone also caused mortality in chilli plants
but relatively lesser in comparison to pathogen-colletotrichumine A
combination.
Colletotrichum spp. generally exhibit three types of infection strategy;
intracellular hemibiotrophic infection, subcuticular intramural infection
and a combination thereof (Bailey et al., 1992). C. capsici proliferation
and differentiation in vivo is characterized by an elevated localized
production of H2O2 and a simultaneous inhibition of the oxidative burst
pathway generated by the host in response to biotic stress (Miles et al.,
2011). Thus, it can be postulated that the high H2O2 production in the
colletotrichumine A-pathogen combination is due to localized produc­
tion and release of H2O2 at the sites of infection throughout the plant
(Figs. 6.1 and 6.2). On the contrary, the comparatively lower levels of
H2O2 in colletotrichumine A treated plants followed by pathogen
(C. capsici) infected plants is likely related to elevated production of
oxalate oxidase and other defense enzymes, such as SOD, PO, etc. H2O2
serves as a signaling molecule in various physiological processes and
plays a significant role in intra- and intercellular processes as the levels
of H2O2 increases during both biotic and abiotic stresses. It primarily
interacts with thiol-containing proteins and triggers different signaling
pathways as well as associated transcription factors which regulate gene
M.K. Chitara et al.
expression, cell-cycle processes and cellular homeostasis of challenged
plants (Slesak et al., 2007). Thus, under stress conditions the highest
levels of H2O2 were observed in the leaves. Here the colletotrichumine
A-pathogen combination caused most severity which produces higher
amounts of H2O2 for the regulation of gene expression, cell-cycle pro­
cesses and cellular homeostasis.
The production of O•– 2 ions was observed by the histochemical
staining of the leaves treated with colletotrichumine A-pathogen com­
bination, colletotrichumine A alone and C. capsici alone. Staining leaf
tissues revealed that the plants treated with colletotrichumine A-path­
ogen combination and colletotrichumine A alone expressed significant
O•–
2 accumulation within 72 and 96 h of treatment. This suggests that
activation of SOD in the plants treated with the colletotrichumine A-
9
Rhizosphere 16 (2020) 100247
Fig. 8. 8.1: Variations in lignification pattern of chilli
plants, represent accumulation of lignin after 24, 48, 72
and 96 h, respectively. Uninfected control (A), pathogen
infected (B), DMSO (C), pathogen-colletotrichumine A @
40 μg/mL (D), pathogen-colletotrichumine A @ 50 μg/mL
(E), pathogen-colletotrichumine A @ 60 μg/mL (F), col­
letotrichumine A @ 40 μg/mL (G), colletotrichumine A @
50 μg/mL (H), colletotrichumine A @ 60 μg/mL (I). 8.2:
Comparison of amount of PAL formation in uninfected
control (A), pathogen infected (B), DMSO (C), pathogen-
colletotrichumine A @ 40 μg/mL (D), pathogen-
colletotrichumine A @ 50 μg/mL (E), pathogen-
colletotrichumine A @ 60 μg/mL (F), colletotrichumine
A @ 40 μg/mL (G), colletotrichumine A @ 50 μg/mL (H),
colletotrichumine A @ 60 μg/mL (I). 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 Duncan’s multiple
range test at P ≤ 0.05. 8.3: Comparison of amount of PPO
formation in uninfected control (A), pathogen infected
(B), DMSO (C), pathogen-colletotrichumine A @ 40 μg/
mL (D), pathogen-colletotrichumine A @ 50 μg/mL (E),
pathogen-colletotrichumine A @ 60 μg/mL (F), colleto­
trichumine A @ 40 μg/mL (G), colletotrichumine A @ 50
μg/mL (H), colletotrichumine A @ 60 μg/mL (I). 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 Dun­
can’s multiple range test at P ≤ 0.05.
pathogen combination and colletotrichumine A alone after 72 h of
infection. On the contrary, the plants infected with only pathogen
showed less expression of SOD but more in comparison to DMSO treated
plants. The untreated plants showed the least accumulation of O•– 2 ions,
suggesting least oxidative stress in this case. SOD catalyzes dismutation
of the quenched O•–2 ions to H2O2. H2O2 not only directly inhibits and
reduces the growth of plant pathogens, but also contributes to the for­
mation of various secondary cell wall defense structures, such as lignin
and papillae formation, cross-linking of hydroxyproline/proline-rich
proteins etc (Lu and Higgins, 1999), (Jain et al., 2015b), (Keswani
et al., 2019). On the contrary, the plants treated with DMSO and un­
treated plants accumulated lower levels of H2O2, probably due to the
enhanced expression of antioxidant enzymes as expressed by augmented
M.K. Chitara et al. Rhizosphere 16 (2020) 100247

Fig. 9. 9.1: Total phenolic content activity at different time intervals in chilli plants. Control (A), pathogen treated (B), DMSO (C). D, E, F correspond to treatment
with pathogen-colletotrichumine A @ 40, 50, 60 μg/mL and G, H, I correspond to treatment with colletotrichumine A @ 40, 50, 60 μg/mL. 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 Duncan’s multiple range test at P ≤ 0.05. 9.2:9.2: HPLC profiles of free phenols in leaves 24 hours after infection. Control (A),
pathogen treated (B), DMSO (C). D, E, F correspond to treatment with pathogen-colletotrichumine A @ 40, 50, 60 μg/mL and G, H, I correspond to treatment with
colletotrichumine A @ 40, 50, 60 μg/mL 1=Catechin, 2=Ferulic acid. 9.3: HPLC profiles of free phenols in leaves 48 hours after infection. Control (A), pathogen
treated (B), DMSO (C). D, E, F correspond to treatment with pathogen-colletotrichumine A @ 40, 50, 60 μg/mL and G, H, I correspond to treatment with colle­
totrichumine A @ 40, 50, 60 μg/mL 1=Catechin, 2=Ferulic acid. 9.4: HPLC profiles of free phenols in leaves 72 hours after infection. Control (A), pathogen treated
(B), DMSO (C). D, E, F correspond to treatment with pathogen-colletotrichumine A @ 40, 50, 60 μg/mL and G, H, I correspond to treatment with colletotrichumine A
@ 40, 50, 60 μg/mL 1=Catechin, 2=Ferulic acid. HPLC profiles of free phenols in leaves 96 hours after infection. Control (A), pathogen treated (B), DMSO (C). D, E, F
correspond to treatment with pathogen-colletotrichumine A @ 40, 50, 60 μg/mL and G, H, I correspond to treatment with colletotrichumine A @ 40, 50, 60 μg/mL
1=Catechin, 2=Ferulic acid. HPLC profiles of free phenols in leaves 24 hours after infection. Control (A), pathogen treated (B), DMSO (C). D, E, F correspond to
treatment with pathogen-colletotrichumine A @ 40, 50, 60 μg/mL and G, H, I correspond to treatment with colletotrichumine A @ 40, 50, 60 μg/mL 1=Catechin,
2=Ferulic acid. 9.3: HPLC profiles of free phenols in leaves 48 hours after infection. Control (A), pathogen treated (B), DMSO (C). D, E, F correspond to treatment
with pathogen-colletotrichumine A @ 40, 50, 60 μg/mL and G, H, I correspond to treatment with colletotrichumine A @ 40, 50, 60 μg/mL 1=Catechin, 2=Ferulic
acid. 9.4: HPLC profiles of free phenols in leaves 72 hours after infection. Control (A), pathogen treated (B), DMSO (C). D, E, F correspond to treatment with
pathogen-colletotrichumine A @ 40, 50, 60 μg/mL and G, H, I correspond to treatment with colletotrichumine A @ 40, 50, 60 μg/mL 1=Catechin, 2=Ferulic acid.
9.5: HPLC profiles of free phenols in leaves 96 hours after infection. Control (A), pathogen treated (B), DMSO (C). D, E, F correspond to treatment with pathogen-
colletotrichumine A @ 40, 50, 60 μg/mL and G, H, I correspond to treatment with colletotrichumine A @ 40, 50, 60 μg/mL 1=Catechin, 2=Ferulic acid.

10
M.K. Chitara et al.
Fig. 9. (continued).
SOD production, because it regulates the invasion of the pathogen by
hypersensitive cell death which may be associated with elicitation of
physiological and biochemical reactions associated with resistance in
the tissue cells neighboring the dead cells (Doke, 1983).
Obstruction of the oxidative network in the colletotrichumine A-
pathogen combination was observed which may be due to the produc­
tion of oxalate and its role in the oxidative reactions catalyzed by lignin-
11
Rhizosphere 16 (2020) 100247
degrading enzymes. In this case the colletotrichumine A-pathogen
combination produced a significantly higher amount of oxalate as
compared to colletotrichumine A, pathogen challenged and unchal­
lenged plants. Accumulation of H2O2 coupled with lipid peroxidation is
a hallmark of cell membrane damage (Airaki et al., 2012). The colleto­
trichumine A-pathogen combination led to the highest level of lipid
peroxidation, possibly due to the collapse oxidative burst pathway.
M.K. Chitara et al.
Fig. 9. (continued).
Plants adopt numerous defense strategies to escape from pathogen
attack (Osbourn, 1996; Keswani, 2015). The primary response of the
host against phytopathogens is to produce pathogen related (PR) pro­
teins viz. PO, PPO, SOD, etc. The activity of the defense enzymes PO,
PAL, PPO, SOD were all elevated, as were the levels of salicylic acid and
phenols after 72 h of inoculation. Antimicrobial free phenols are vital
defense compounds which not only act as signaling molecules for
expression of defense related genes but also polymerize to form the
structural barrier lignin (Madhavan et al., 2011; Dakora and Phillips,
1996). The first line of host defense against phytopathogens is lignin
layer, which if penetrated leads to the activation of a series of
highly-coordinated cytosolic enzymes including PO, SOD, catalase, PPO,
PAL, β-1,3-glucanase, etc. resisting disease progression (Inkha and
Boonyakiat, 2010).
PAL catalyzes a committed reaction in the phenylpropanoid pathway
leading to the conversion of L-phenylalanine to trans-cinnamic acid
(Cheng and Breen, 1991). PAL activity is significantly upregulated in
host tissues under biotic stress or wounding (Ronald and Söderhäll,
1985). Peroxidases are involved in the formation of iso-di-Tyr bridges in
the cross-linking of cell wall proteins, the cross-linking of pectins by
diferulic bridges, and the oxidation of cinnamyl alcohols prior to their
polymerization during lignin formation. Polymerization of lignin has
been considered to be catalyzed by cell wall bound guaiacol-type per­
oxidases (Whetten and Sederoff, 1995). The activity of cell
wall-associated peroxidase correlates with the extent of lignification
(Sattler and Funnell-Harris, 2013). An increase in lignin synthesis is
observed in response to biotic stress (Rogers and Campbell, 2004).
Lignification is a mechanism for disease resistance in plants (Singh et al.,
2013).
In our case, the accumulation of lignin was significantly decreased in
the plants treated with the colletotrichumine A-pathogen combination,
but the colletotrichumine A only treated plants showed comparatively
more lignification in colletotrichumine A-pathogen combination and
pathogen challenged plants than untreated control. An increase in lignin
12
Rhizosphere 16 (2020) 100247
production is often observed in response to attack by the pathogens.
Minimum accumulation of lignin was observed in DMSO-treated and in
the untreated control. Comparatively high lignification suggests a cor­
responding induction of PAL, an important enzyme induced in the
synthesis of lignin and intermediates of phenylpropanoid pathway. The
highest levels of PAL activity were observed in the plants treated with
colletotrichumine A individually, slightly less in the colletotrichumine
A-pathogen combination followed by the inoculation with the pathogen
alone. DMSO treated and control plants showed less PAL activity.
Similar induction patterns in PPO activity were also recorded in
response to the aforementioned treatments.
Phenolic compounds are secondary metabolites which primarily act
as antimicrobial agents, providing the second line of defense against
pathogenic fungi along with the cuticle at the surface of fruits, but also
as phytoalexins formed in plant tissues in response to the infection
process (Lattanzio, 2003). The progression of phytopathogens can be
significantly reduced when free phenols are present at the site of inva­
sion (Maqbool et al., 1012). Although many phenolic compounds pre­
sent in the host tissues lack anti-microbial activity per se, but there are
several cases where the oxidized counterparts of pre-existing phenolics
have demonstrated significantly higher antimicrobial activity (Jain
et al., 2015b; Lattanzio, 2003).
To conclude, anthracnose is one of the major economic constraints to
chilli production worldwide, especially in tropical and subtropical re­
gions including India. The present study was undertaken to investigate
the role of the C. capsici-derived alkaloid colletotrichumine A in
anthracnose disease development in chilli cultivated in north eastern
districts of Uttar Pradesh, India. The effect of colletotrichumine A, both
alone and in combination with the pathogen (C. capsici), on chilli plants
was evaluated. It was found that the colletotrichumine A-pathogen
combination significantly increased the activity of defense related en­
zymes viz. PPO, SOD and TPC, also lowering the activity lignification
enzymes PAL and PO. An increased accumulation of phenols like cate­
chin, ferulic acid, kaempferol, shikimic acid, gentistic acid were
M.K. Chitara et al.
observed after both treatments with the pathogen and colletotrichumine
A individually, as well as in combination, with the highest accumulation
in the latter. Moreover, the highest levels of cell death resulted from the
colletotrichumine A-pathogen combination followed by colleto­
trichumine A alone. This study proves the active role of colleto­
trichumine A in enhancing the disease severity of C. capsici infection in
chili plants. Further investigations are needed to evaluate and ascertain
the exact target(s) of colletotrichumine A at molecular level.
Author contributions
Manoj K. Chitara: did the experiments and wrote the manuscript.
Chetan Keswani: conceived the idea, provided the general concept and
inputs for each specificsection, and drafted part of the manuscript, did
the experiments and wrote the manuscript. Kyriakos G. Varnava: did
the experiments and wrote the manuscript. Hareram Birla: did the
experiments and wrote the manuscript, supervised the whole work. All
authors read and approved it for publication. Hagera Dilnashin: did the
experiments and wrote the manuscript. Surya P. Singh: supervised the
whole work. All authors read and approved it for publication. Viji
Sarojini: conceived the idea, provided the general concept and inputs
for each specificsection, and drafted part of the manuscript, supervised
the whole work. All authors read and approved it for publication.
Jonathan Sperry: conceived the idea, provided the general concept and
inputs for each specificsection, and drafted part of the manuscript, su­
pervised the whole work. All authors read and approved it for publica­
tion. Harikesh B. Singh: conceived the idea, provided the general
concept and inputs for each specific section, and drafted part of the
manuscript.
Ethical statement
The work was done in compliance with the ethical standards.
Ethical approval
This article does not contain any studies with human participants or
animals performed by any of the authors.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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