Acta Physiologiae Plantarum (2021) 43:155

https://doi.org/10.1007/s11738-021-03334-x

ORIGINAL ARTICLE

Elucidation of the biochemical and molecular basis of the differential

disease expression in two cultivars of chili (Capsicum annuum)
in response to Colletotrichum capsici infection
Jayeeta Bijali1 · Tanmoy Halder2 · Krishnendu Acharya1

Received: 10 November 2020 / Revised: 22 January 2021 / Accepted: 2 November 2021 / Published online: 10 November 2021
© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2021

Abstract
Chili plants are affected by the hemibiotrophic ascomycota fungus Colletotrichum capsici causing Anthracnose. Infection
results in yield and marketability loss due to a decrease in the quality of fruits. The study of morphological symptom devel-
opment in two cultivars, Bullet, and Beldanga, showed very different disease expression pattern. To understand the reasons
behind such differential response, we investigated, in a time-dependent manner, biochemical activities of important defense
enzymes, PR proteins, like peroxidase, polyphenol-oxidase, phenylalanine ammonia lyase, β-glucanase, chitinase, catalase,
as well as phenols, flavonoids, chlorophyll and the key signaling molecule nitric oxide in their leaves. We further performed
real-time nitric oxide (NO) detection studies. The results showed striking differences in the activity profile of these defense
molecules through the course of the study. We monitored the gene expression levels of 12 important defense-related genes
under in vivo condition. The transcription levels were mostly increased in the tolerant cultivar till 7 days post-infection (DPI),
while downregulation of some of the genes were observed in the susceptible one. These data indicated that disease manifesta-
tion is a simulated response of these defense molecules which can nullify the effect of the pathogen and its products, when
resistance occurs. Alternatively, the pathogen suppresses the host defense when the disease develops.

Keywords Pathogenesis · Disease development · Anthracnose · Chili · Colletotrichum · Tolerant · Susceptible · Enzyme
assay

Abbreviations SBM Standard blotter method

PR Pathogenesis related BAP 6-Benzylaminopurine
PO Peroxidase PDA Potato dextrose agar
PPO Polyphenol oxidase DSI Disease severity index
PAL Phenylalanine ammonia lyase PVP Polyvinylpyrrolidone
DPI Days post-inoculation PMSF Phenylmethylsulfonyl fluoride
NO Nitric oxide DNS Dinitrosalicylic acid
ROS Reactive oxygen species EA Enzyme activity
SDW Sterile distilled water BSA Bovine serum albumin
DAF-2DA 4,5-Diaminofluorescein diacetate

Communicated by V. P. Singh.

* Krishnendu Acharya
Introduction
krish_paper@yahoo.com
Chili, Capsicum annum, is an important cash crop of the
1
Molecular and Applied Mycology and Plant world and through centuries, it has made its place, as an
Pathology Laboratory Centre of Advanced Study, important condiment, in the cuisines of many cultures of
Department of Botany, University of Calcutta, Kolkata,
West Bengal 700019, India the world, especially because of its tangy-pungent spicy
2 taste of capsaicinoids (Mishra et al., 2017). Neverthe-
Plant Functional Genomics Lab, Department of Botany,
Centre of Advanced Study, University of Calcutta, Kolkata, less, it has been found to have rich nutritional status,
West Bengal 700019, India particularly in its content of vitamin A and C, phenolic

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compounds, carotenoids, and flavonoids (Mateos et al., transmitted to distal parts of the plant via phloem resulting
2013; Mishra et al., 2017). However huge economic loss is in systemic acquired resistance or SAR (Saxena et al., 2019).
incurred globally with respect to its production and yield, The disease-causing virulent pathogen releases plant cell
due to profound damage caused by anthracnose disease wall-degrading enzymes and toxins, and can silence or even
(Sharma et al., 2005). The pathogen has been widely iden- evade off the defense response (Anand et al., 2009; Mbengue
tified as the ascomycota fungus, Colletotrichum species et al., 2016). Each successful disease establishment involves
(Than et al., 2008), Colletotrichum capsici (Sydow) But- a plethora of molecular and biochemical events between the
ler, and Bisby, being the most devastating species (Taylor host plant and its pathogen, which involves defense genes
et al., 2007). The disease appears as small sunken necrotic and enzymes, viz., PO, PPO, PAL, β-glucanase, chitinase,
lesions (Isaac 1992), in leaves, stems, fruits during pre and catalase, etc. (Ramamoorthy and Samiyappan, 2001). This
post-harvest stage, and also within seeds during storage particular arena is well documented in the literature and has
(Than et al. 2008). been found in a wide range of crops such as tomato and
The hemibiotrophic pathogen infects nearly all plant rice (Chandrasekhar and Umesha, 2012; Suthin et al. 2019).
parts- leaves, stem, fruits, nodes, flowers in all stages of crop Higher activity of these enzymes has also been reported in
development (Isaac 1992) and can even propagate in affected resistant green chili fruits against Alternaria alternata as
plant debris lying in the field and gets propagated with fresh well as C. capsici (Anand et al., 2009) as well as in conjunc-
plantations, thus amplifying the damage (Saxena et al., tion with induced systemic resistance (Banu et al., 2019).
2019). It can also remain quiescent in the seeds of shelved For a clearer understanding of chili defense mechanisms,
fruits during storage (Than et al., 2008). A more threatening we monitored the activity levels of the defense enzymes and
reason is anthracnose infected chili fruits become an easy PR proteins in two cultivars of chili, Bullet and Beldanga, in
target for secondary infection due to Aspergillus sp. which response to Colletotrichum capsici infection from 0 to 3 days
produces aflatoxin (Roy et al. 2013). Thus studies regard- post-infection (DPI). We report changes in the activity levels
ing its pathogenicity become necessary not only to control of the defense molecules with time and striking differences
yield and marketability loss but also in the broader picture in the two cultivars which are commensurate with the differ-
of global food security. ences in their morphological disease symptom expression.
The rapidity and promptness with which a plant fights The comparative study of disease resistance and suscepti-
back the pathogen depends on a number of factors and takes bility in the two cultivars against the pathogen was carried
place along multiple lines of action. Presence of pathogen or out by wound inoculation with the pathogen. This approach
pathogen sensing on plants is aided by pathogen-associated might surpass some of the initial resistance mechanisms of
molecular pattern (PAMP) (Monaghan and Zipfel, 2012) the plant, yet it results in more discrete disease development
which are recognized by host cell surface receptors called and thus helps in studies. Our studies focused on morpho-
pathogen recognition receptors or PRRs, (Du et al., 2015), logical symptom development after pathogen inoculation,
resulting in PAMP triggered immunity (PTI) (Monaghan and assayed the defense enzymes and PR proteins biochemically,
Zipfel, 2012). This triggers signal transduction cascades via and concomitantly studied the gene expression levels in the
several downstream effecter molecules. PTI is followed by two cultivars. Based on the morphological differences of
effecter triggered immunity (ETI) that occurs in the cyto- symptom development on leaves of these two varieties, we
plasm (Singla et al., 2019). The immediate host response chose a specific time frame, 0, 1, 2, 3 DPI, to assess the dif-
involves the generation of reactive oxygen species or ROS ferences in host response. We evaluated the activity levels
(Heller et al., 2011), and initiates a localized hypersensitive of important molecules of plant defense—PO, PPO, PAL,
response (HR), wherein the plant chooses to kill its own β-glucanase, chitinase, catalase, chlorophylls a and b, total
cells, i.e., necrosis to contain the pathogen within the infec- phenol and flavonoids, and NO levels, as well as studied the
tion spot by a series of controlled cellular events resulting gene expression profile. This is the first time course report
in PCD or programmed cell death (Mbengue et al., 2016; on the comparative response of disease resistance and dis-
Prasad et al. 2020). A more potent response is achieved ease susceptibility in chili leaves against anthracnose patho-
through increased expression and activation of antioxida- gen to the best of our knowledge.
tive enzymes and defense enzymes, such as catalase, per-
oxidase, polyphenol oxidase, phenylalanine ammonia lyase;
pathogenesis-related proteins such as β,1–3, glucanase, and Materials and method
chitinase; and phenolics and other antimicrobial compounds
such as defensing and phytoalexins. (Yadav et al., 2020; Plant material
Malik et al. 2020). Nitric oxide (NO) (Mur et al., 2013;
Nasir et al., 2020) has also been reported as a key regulator Seeds of two cultivars of chilli were obtained from Bala-
in these signaling pathways. Eventually, the signal may be gar in West Bengal, India, and seedlings raised in natural

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conditions in the experimental garden in a completely rand- Disease severity index was deduced according to Song
omized pattern. Young leaves from 45 days old healthy chili et al., (2004) using the formula:
plants were surface sterilized with 0.1% sodium hypochlo-
Disease severity index (DSI ) %
rite followed by several washes with sterile distilled water
disease severity scale no. of samples in each scale
(SDW). The detached leaves were subjected to pathogen = Σ
highest numerical scale index × total no. of samples
× 100.
challenge by standard blotter method or SBM (ISTA, 2005).
The leaves were kept on three layered sterile moist blotting
paper bed supplemented with 0.1% 6-benzylaminopurine
(BAP) in 20 cm Petri plates, thus maintaining moist cham-
Biochemical assay of defense enzymes and PR
bers. In these conditions, biochemical experiments were car-
proteins
ried out from 0 to 3 DPI.
Control water-inoculated and infected leaves were assayed
for different defense enzymes and PR proteins, from 0 to
Fungal Isolate and Inoculation
3 DPI. For each sample preparation, collected leaves were
wiped with sterile blotting paper, weighed and leaf tissue
Pure cultures of virulent Colletotrichum capsici purchased
was extracted in the chilling condition in different enzyme-
from the culture collection unit of Agharkar Research Insti-
specific extraction buffers each containing 0.1% polyvi-
tute, Pune (NFCCI No. 1660), were maintained in the labo-
nylpyrrolidone (PVP) and 20 µl of 1 mM phenylmethyl-
ratory in potato dextrose agar or PDA slants by subculturing.
sulfonyl fluoride (PMSF). The resultant solutions were
From 8-day-old PDA slant cultures, spore suspension was
centrifuged at 4 °C, 1200 × g for 20 min. The supernatant
made with SDW (Sharma et al., 2005) and diluted with SDW
was collected and used as the enzyme solution.
to a concentration of 2 × ­107cells/ml using a hemocytometer.
From this spore suspension, 10 μl was used for each infec-
tion spot made in between two veins by wound pricking. Peroxidase (PO) assay
10 μl of SDW was added at the wound pricked spot on the
control sets. PO was assayed following the protocol given by Hameda and
Klein (1990). The control and infected leaves were weighed
Morphological disease progression studies and each sample of 150 mg of leaf tissue was extracted in
3 ml of 0.1 M of sodium phosphate buffer (pH 7.0). Change
Seeds were isolated from ripe mature red chili fruits and in absorbance due to guiacol oxidation at 470 nm was moni-
seedlings raised therefrom. Fully expanded leaves from tored for 3 min in the reaction mixture.
45 days old seedlings were excised and surface sterilized
with 0.1% NaOCl, followed by repeated SDW wash. Leaves
in humid chambers (prepared as mentioned above) were Polyphenol oxidase (PPO) assay
observed for disease symptom development. The percent
disease area was calculated and disease scoring was done PPO activity was assayed following the method outlined by
following Inglis et al. (1988) with little modifications in Kumar and Khan (1982) with slight modifications. 150 mg
scale for chili leaves in ex vivo condition. A disease scale leaf tissue was extracted in 3 ml of 0.1 M of sodium phos-
from 0 to7 was assigned (Table 1). phate buffer (pH 7.0). The absorbance of purpurogallin

Table 1  Disease scoring scale of infected excised chili leaves

Score Affected area (% of whole leaf Symptom details
area)

0 0 No infection
1 Up to 5 With a light colored water-soaked lesion
2 > 5–10 Larger light brownish water-soaked lesion
3 > 10–20 Necrotic lesions with limited chlorosis surrounding the lesions
4 > 20–30 Necrotic lesions turn dark brown with chlorosis along the midrib
5 > 30–40 Larger necrotic lesions with extensive chlorosis
6 > 40–50 Raised pustule-like appearances, possibly acervuli, at wound-prick points within the
necrotic lesions, extensive chlorosis and browning at the margins
7 > 50 Fungal mycelium mats over the lesions, complete chlorosis and loss of tissue integrity

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formed was measured at 495 nm against the enzyme blank. gallic acid, the phenol quantity was expressed as μg of gallic
PPO activity was measured as a change in absorbance (U) acid produced ­g−1 of tissue.
­min−1 ­mg−1 of protein.
Estimation of Flavonoids content
Phenylalanine ammonia lyase (PAL) assay
Total flavonoids content was estimated following the pro-
tocol outlined by Chang et al., (2002), with little modifica-
Quantitative estimation of PAL was carried out following
tions. 250 mg of leaf tissue was homogenized in 3 ml of
the protocol given by Dickerson, (1984) with minor modifi-
80% aqueous ethanol, followed by dark incubation for half
cations. 150 mg of leaf tissue was extracted in 3 ml of 0.1 M
an hour at room temperature. To 1 ml of the supernatant
sodium borate buffer (pH 8.7). Formation of trans-cinnamic
collected after centrifugation, 4.3 ml of 80% aqueous etha-
from L-phenylalanine was determined from ­OD290.
nol was added along with 100 μL of 10% aluminum nitrate
and 100 μL of 1 M sodium acetate solution, followed by
β ‑1,3‑ glucanase assay 30 min of dark incubation, and absorbance was measured
at 495 nm. Total flavonoids content was expressed as mg
β-Glucanase activity was assayed as described by Pan et al., of quercitin ­g−1 of the tissue sample.
(1991). The absorbance of the red-colored solution formed
due to reaction with laminarin was measured at 525 nm Estimation of chlorophyll content
against a zero minute incubation blank set.
For chlorophyll content estimation, 250 mg of leaf tis-
Chitinase sue was ground in 4 ml of 80% alkaline acetone solution
(in 0.1 N NaOH solution). Following protocol outlined by
Chitinase activity was assayed following the protocol from Kamble et al., (2015), absorbance was recorded at 663 and
Bansode and Bajekals, (2006) with little modifications. 645 nm. Chlorophyll content was calculated according to
100 mg of leaf tissue was crushed in 3 ml of 0.1 M of sodium the following formula:
acetate buffer (pH 5.0). The reaction mixture comprised 1 ml
Chlorophyll a = 11.75 A663 − 2.35 A645 ,
of 1% colloidal chitin, 2 ml of buffer, and 500 μL of enzyme
solution and absorbance measured at 540 nm. Chlorophyll b = 18.61 A645 − 3.96 A663 .

Catalase Estimation of Total protein

Catalase activity was estimated following the protocol out- The total protein content in each sample extract was esti-
lined by Anderson et al. 1995 with slight changes. 250 mg mated with Bradford assay (1976) using bovine serum
of leaf tissue was extracted in 5 ml of 0.067 M sodium phos- albumin (BSA) as standard.
phate buffer, pH 7. The reaction mixture comprised 3 ml of
0.1% of ­H2O2 solution, 3 ml of 0.067 M phosphate buffer Nitric oxide (NO) estimation
(ph 7), and 60 μL of the enzyme extract. The decrease in
absorbance at 240 nm was recorded for 1 min. The enzyme Quantitative estimation of nitric oxide production was
activity was calculated with € = 36 ­M−1 ­cm−1 and expressed done by the hemoglobin assay method (Delledonne 2005).
as μmol ­min−1 ­g−1 of protein. Each leaf sample was dipped in hemoglobin buffer com-
prising 10 mM l -arginine and 10 mM hemoglobin in a
Estimation of total phenol 0.1 M phosphate buffer solution. The OD of the reac-
tion mixture was recorded every 2 h from 0 to 4 h. NO
250 mg of leaf tissue from each sample was homogenized content in the reaction mixture was calculated using
with 3 ml of 80% methanol, and the homogenate was kept at E = 38,600 ­M−1 ­cm−1 and expressed as moles ­g−1 of tis-
65 ˚C in a dry incubator for 15 min. It was centrifuged and sue ­h−1 (Salter and Knowles, 1998).
the supernatant collected for phenol estimation (Zeislin and
Ben Zaken, 1993). To 5 ml of double-distilled water, 1 ml Real‑time NO detection
of the supernatant and 250 μL of Folin–Ciocalteu reagent
were added, incubated for 30 min at room temperature, and The lower epidermal layer of leaves was peeled off and
OD value recorded at 725 nm. Using the standard curve of subjected to real-time NO detection with DAF-2DA

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(4,5-diaminofluorescein diacetate) (Bartha et al., 2005). software version 8.4.3. Comparison has been performed for
Leaf peels from each sample were dark incubated for the infected plants with respect to the corresponding con-
20 min in a loading buffer comprising 10 mM KCl and trol of their respective varieties. *p < 0.05, **p < 0.01 and
10 mM Tris– HCl (pH 7.2) with 10 μM final concentra- ***p < 0.001 show statistically significant differences with
tion of DAF-2DA. Real-time NO imaging in epidermal control.
cells was observed owing to the cell-permeable fluores-
cent probe DAF-2DA, using Leica DMLS microscope at
excitation and emission maxima of 490 nm and 515 nm, Results
respectively.
Disease progression studies
Gene expression studies
Chili leaves challenge inoculated with Colletotrichum cap-
Pathogen challenge to chili plants sici were studied for morphological expression of disease
symptoms. Contrasting features were exhibited by leaves of
45 days old plants of both the cultivars grown under natural two chili cultivars. The Bullet cultivar showed severe disease
greenhouse conditions were inoculated with 10 μl of spore symptoms with sunken necrotic lesions with leaf discolora-
suspension by wound pricking. Wounded spot mock inocu- tion surrounding each lesion and also along the midrib. Leaf
lated with an equal volume of SDW served as control sets. integrity was lost somewhat near the margins after 3 DPI.
Each infected leaf was covered with sterile plastic cups for However, Beldanga cultivar retained leaf color and integrity
24 h to maintain aseptic isolation conditions. Leaf samples after 3 DPI. Localized deep brown-colored necrotic spots
were collected at 0, 1, 3, and 7 DPI, immediately dipped in developed with raised margins on infection spots without
liquid nitrogen and stored at − 80 °C. much effect on the rest of the leaf (Fig. 1). The transverse
section from near the lesions at 3 DPI revealed compara-
RNA isolation and semi‑quantitative RT‑PCR analysis tively more localized fungal infestation in Beldanga. In Bul-
let, massive tissue damage and relatively more abundance of
Total RNA was isolated from 100 mg leaf tissue (collected mycelium were observed at 3DPI.
at different time points post-inoculation) using the RNeasy The disease scoring data revealed a 100% disease severity
Plant Mini Kit (Qiagen, Germany) following the manufac- index (DSI) in Bullet at 5 DPI with a corresponding mean
turer’s protocol. First-strand cDNA synthesis was done from percent disease area of 66.29% (Supplementary Fig. 1). The
1 µg of total RNA using RevertAid First-strand cDNA syn- DSI of Beldanga was calculated to be 28.58% with a cor-
thesizing kit [Thermo Scientific, (EU) Lithuania] according responding mean percent disease area of 7.66%. The percent
to the manufacturer’s instruction using an oligo-dT primer. disease area in Bullet at 3 DPI was 33.06% with 71.42% DSI
The genes targeted for the semi-quantitative RT-PCR analy- and that in Beldanga was 2.89% with 14.28% DSI.
sis and the primers used for the amplification of those genes
are enlisted in the Supplementary Table S1. Actin was used
as an internal control to normalize the sample amounts. Biochemical studies
The semi-qRT-PCR was carried out in MJ Mini Personal
Thermo Cycler (Bio-Rad, USA). The PCR cycling condi- Peroxidase (PO) assay
tions were as follows: 95 °C for 2 min, followed by 35 cycles
of 95 °C for 20 s, 55 °C for 20 s and 72 °C for 20 s; a final The PO activity levels in the pathogen challenged and
extension for 5 min at 72 °C was given at the end of the unchallenged sets of Bullet and Beldanga through a course
PCR reaction. of 0 to 3 DPI differed considerably. PO activity in Bul-
let increased sharply at 1 DPI, thereafter it decreased and
Statistical analysis at 3 DPI the activity fell significantly. In the pathogen-
infected Beldanga sets, a gradual increase in PO activity
The plants were raised in a completely randomized design, was observed. At 3 DPI, activity was maximum with an
with one plant per experimental pot. In each experiment, increase of about 1.86-fold compared to the control set of
three replicates for each set were used. The experiments Beldanga. (Fig. 2a, b).
were repeated at least thrice. All values were means of three
individual experiments each in triplicate. The individual Polyphenyl oxidase (PPO) assay
bar represents the mean value of three replicates. All results
obtained were subjected to one-way variance ANOVA using PPO activity differed in the two cultivars. In the pathogen-
Dunnett’s multiple comparison test with GraphPad Prism infected sets of Bullet, there was a sharp increase in PPO

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Fig. 1  Disease progression stud-

ies of the two cultivars. Images
showing symptom development
at different time intervals, i.e.,
0 DPI, 1 DPI, 2 DPI, 3DPI, 4
DPI, 5 DPI in a Bullet cultivar
(upper panel) and b Beldanga
cultivar (lower panel),along
with their corresponding
controls; c and d magnified
image of infection site at 3
DPI in Bullet and in Beldanga,
respectively, where the infected
site is highlighted with white
dotted circle. e Microscopic
view of a section through the
infection lesion in Bullet and f
in Beldanga. Identified infection
sites are marked with black
arrowheads

Fig. 2  Specific activity of three enzymes in pathogen-challenged represent mean ± SD of three independent experiments each in tripli-
sets of Bullet and Beldanga cultivars at different time intervals, i.e., cate. Comparison was performed for the infected plants with respect
0 DPI, 1 DPI, 2 DPI, and 3 DPI. a Peroxidase (PO), b polyphenol to the corresponding control of their respective varieties. ns = not sig-
oxidase (PPO) and (c) phenylalanine ammonia lyase (PAL). Non- nificant, *p < 0.05, **p < 0.01 and ***p < 0.001 representing statisti-
infected plants were used as a control for those experiments. Values cally significant differences with control

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activity at 1 DPI (1.87 fold), followed by a gradual decrease there was a considerable increase at 3 DPI when it peaked
and at 3 DPI, PPO activity was lesser than the control set. (1.27 times) Fig. 3c and d).
PPO level did not increase much at 1 DPI in the pathogen-
infected sets of Beldanga, at 2 DPI it was highest (1.1 fold), Catalase assay
and thereafter declined at 3 DPI (Fig. 2c, d).
Catalase activity in Bullet cultivar rapidly increased at 1 DPI
Phenylalanine ammonia lyase (PAL) assay (2.2 fold) and then sharply decreased at 2 DPI compared to
that of the corresponding control. Another sharp decline was
The activity level of PAL in Bullet cultivar was highest at 1 observed at 3 DPI. Contrastingly, progressive enhancement
DPI, when its increase was 1.16-fold, and then decreased. in catalase activity was observed in Beldanga. From 0 to 1
However, the decrease from 2 to 3 DPI was very little. In DPI, the catalase activity showed a remarkable increase. At
Beldanga cultivar, there was a gradual and steady increase 3 DPI it was highest (2.6 fold) (Fig. 3e and f).
in PAL activity from 0 to 3 DPI. It peaked at 3 DPI with a
1.2-fold increases (Fig. 2e, f).
Phenol estimation
β‑Glucanase assay
Phenol was estimated as mg of gallic acid/g of fresh tissue.
The phenol profile of the two cultivars showed a complete
Bullet cultivar showed a decrease in the activity levels of
contrasting pattern in the duration of the experiment. While
β-glucanase post-inoculation. At 1 DPI the activity level fell
the total phenol level in the Bullet gradually decreased
and the decreased level was maintained at 2 DPI. At 3 DPI
from 0 to 1 DPI, the same in Beldanga showed progres-
another significant drop in the activity level was noted. In
sive enhancement. After an initial significant dip in the phe-
contrast, the enzyme activity in Beldanga cultivar showed a
nol levels of Bullet from 0 to 1 DPI, the level continued to
progressive increase from 0 DPI. The initial level of β- glu-
decrease slowly from 1 to 3 DPI. On the other hand, a sharp
canase activity in Beldanga was slightly higher than that in
increase was observed from 0 to 1 DPI in the Beldanga cul-
Bullet (1.19 times). At 2 DPI, it increased, with the highest
tivar. A further increase was observed from 1 to 3 DPI in this
level being at 3 DPI (1.3 fold) (Fig. 3a, b).
cultivar (Figs. 4a and b). The increase in Beldanga was 1.52
times the initial level when it peaked at 3 DPI.
Chitinase assay

The initial level of chitinase activity was found to be 1.21 Flavonoids estimation
times more in Beldanga than in Bullet. Its levels in Bul-
let decreased steadily post-infection, and the decrease was While Bullet showed a slow decrease in flavonoid levels
significant at 2 DPI and further decreased at 3 DPI. In the from 1 to 3 DPI, the corresponding levels were increased
Beldanga cultivar, chitinase activity initially increased very in Beldanga, sharply at 1 DPI, and maintained with slight
slowly and a slight increase was observed at 2 DPI. However, enhancement at each day (Fig. 4c, d). The decrease in Bullet

Fig. 3  Specific activity of three enzymes in pathogen-challenged each in triplicate. Comparison was performed for the infected plants
sets of Bullet and Beldanga cultivars at different time intervals, i.e., with respect to the corresponding control of their respective varieties.
0 DPI, 1 DPI, 2 DPI, and 3 DPI. a β-Glucanase, b chitinase, and c ns = not significant, *p < 0.05, **p < 0.01 and ***p < 0.001 represent-
catalase. Non-infected plants were used as a control for those experi- ing statistically significant differences with the control
ments. Values represent mean ± SD of three independent experiments

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Fig. 4  Phenols and flavonoids estimation in pathogen-challenged iments each in triplicate. Comparison was performed for the infected
sets of Bullet and Beldanga cultivars at different time intervals, i.e., plants with respect to the corresponding control of their respective
0 DPI, 1 DPI, 2 DPI, and 3 DPI. a Total phenol content and b flavo- varieties. ns = not significant, *p < 0.05, **p < 0.01 and ***p < 0.001
noids content. Non-infected plants were used as a control for those representing statistically significant differences with control
experiments. Values represent mean ± SD of three independent exper-

was more pronounced at 3 DPI. In the pathogen-challenged at 3 DPI (Fig. 5b). The initial chlorophyll a and b lev-
sets of Beldanga, flavonoid level peaked at 3 DPI (1.58 els were observed to be higher in the Bullet cultivar. The
times). decrease in infected sets of Bullet and Beldanga chloro-
phyll a levels at 3 DPI was 1.21 and 1.06 times of their
Chlorophyll initial levels, respectively.
Chlorophyll b level in infected sets of Bullet cultivar
In the Bullet cultivar, chlorophyll a decreased post-inoc- was steady till 1 DPI with a slight decrease at 2 and 3 DPI
ulation, and the decrease was significant at 2 DPI. From (Fig. 5c). In Beldanga cultivar too there was a slight decrease
there it again dropped at 3 DPI (Fig. 5a). In the Beldanga in chlorophyll b levels from 0 to 3 DPI (Fig. 5d).
cultivar, the chlorophyll level decreased from 0 to 1 DPI, Overall, there were significant differences in the activ-
maintained the same levels at 2 DPI, and further dropped ity levels of the defense enzymes and PR proteins in the

Fig. 5  Chlorophyll estimation in pathogen-challenged sets of Bul- each in triplicate. Comparison was performed for the infected plants
let and Beldanga cultivars at different time intervals, i.e., 0 DPI, 1 with respect to the corresponding control of their respective varieties.
DPI, 2 DPI, and 3 DPI. a Chlorophyll a content and b chlorophyll b ns = not significant, *p < 0.05, **p < 0.01 and ***p < 0.001 represent-
content. Non-infected plants were used as a control for those experi- ing statistically significant differences with control
ments. Values represent mean ± SD of three independent experiments

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of the study. An increase in their activity levels was also

observed in Bullet after pathogen challenge. However, their
higher activities were not maintained consistently and a
sharp decline in the levels was observed.

Nitric oxide estimation

NO levels in the pathogen-challenged sets of Bullet cultivar

peaked at 1 DPI after which the levels started decreasing and
at 3 DPI the levels were lower than that of the control set.
NO levels in the pathogen-challenged sets of Beldanga cul-
tivar started increasing from 1 DPI and continued to increase
till 3 DPI (Fig. 6).
A similar pattern of a greater increase in NO levels was
observed in pathogen-challenged sets of Beldanga cultivar
than that of Bullet sets in real-time imaging of NO levels in
the two cultivars (Fig. 7).
Fig. 6  Nitric oxide (NO) estimation in pathogen-challenged sets of
Bullet and Beldanga cultivars at different time intervals, i.e., 0 DPI, Gene expression studies
1 DPI, 2 DPI, and 3 DPI. Non-infected plants were used as a control
for the experiment. Values represent mean ± SD of three independ-
ent experiments each in triplicate. Comparison was performed for The semi-qRT-PCR results for 12 genes following infec-
the infected plants with respect to the corresponding control of their tion in the chili cultivars Beldanga and Bullet depicted a
respective varieties. ns = not significant, *p < 0.05, **p < 0.01 and disease resistance response in Beldanga cultivar. Gene
***p < 0.001 representing statistically significant differences with expression analysis of important transcription factors such
control
as WRKY33, CaMYB, CaNAC, and bZIP were carried
two cultivars throughout the experiment. In the Beldanga out. The expression of WRKY33 increased post-infection,
cultivar, in general, the levels were higher after pathogen expression dropped at 3DPI in both the Bullet and Beldanga
challenge and never fell below the initial level in the course

Fig. 7  Real-time NO detection

in control and pathogen-chal-
lenged sets of the two cultivars.
1–3 DPI of Bullet control sets
(a–c) and pathogen-challenged
sets (d–f); 1–3 DPI of Beldanga
control sets (i–k) and pathogen-
challenged sets (l–n)

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155 Page 10 of 16 Acta Physiologiae Plantarum (2021) 43:155

cultivars, and then rose again at 7DPI. The expression levels Zn SOD. MnSOD showed an increase in gene expression
of CaMYB also increase post-infection in both. The basal at 3DPI for Beldanga, whereas at 7 DPI for Bullet. PR 5
expression of CaNAC is higher in Beldanga than in Bullet. showed several-fold upregulation in expression from 3DPI
Gene expression levels drop post-infection and rise again at in Beldanga, whereas its expression dropped post-3 DPI in
7 DPI being similar to the basal level in both the cultivars. Bullet (Fig. 8).
Expression levels of bZIP 10 showed a steady rise in the
cultivars of Beldanga and Bullet. However, expression fold
dropped at 7 DPI for the susceptible cultivar of Bullet. Discussion
The sweet sugar transporter gene expression levels
showed a sharp increase in fold expression post-3 DPI in Our results showed that, among the two cultivars of Cap-
Beldanga and at 7 DPI in Bullet. The basal level expression sicum annuum, the overall defense response was more
of CAT gene is lower in Beldanga as compared to Bullet. pronounced and consistent in the Beldanga cultivar. The
It shows increased gene expression at 7 DPI for both. The phenotypic disease expression in this cultivar showed dis-
expression profile of ascorbate peroxidase increased several crete necrotic spots in the infection site without much effect
fold from 3DPI in Beldanga, whereas in Bullet, it increased on the whole leaf area. These necrotic spots pointed to an
and remained more or less constant till 7 DPI. Glutathione acute hypersensitive response in this cultivar leading to pro-
reductase showed increased gene expression profile post-3 grammed cell death at the infection site containing the path-
DPI in both the cultivars. SODs such as Cu/Zn SODs and ogen within and severing its connections with the rest of the
FeSOD showed higher expression at 7 DPI in Beldanga, leaf tissue. To destroy the invading pathogen, immediately
whereas in Bullet, FeSOD expression declined at 7 DPI and after detection, the host resorts to ROS production (Heller
there is no significant change in expression levels of Cu/ et al., 2011; Prasad et al., 2020); the resultant oxidative burst

Fig. 8  Relative expression

level analysis of several genes
in Colletotrichum capsici-
challenged sets of Bullet and
Beldanga cultivars through
semi-quantitative real-time
PCR. Gene expression was ana-
lyzed at different time intervals,
i.e., 0 DPI, 1 DPI, 3 DPI, and
7 DPI. Actin was used as an
internal control

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Acta Physiologiae Plantarum (2021) 43:155 Page 11 of 16 155

causes damage in the cell membranes and membrane-bound activity of PO, PPO, and PAL was observed in the patho-
structures, eventually aiding the process of programmed cell gen-challenged sets of both Bullet and Beldanga cultivar
death (PCD) (Agrios, 2005; Prasad et al., 2020). Thus, the post-inoculation. The activities peaked off and decreased in
percent disease area and corresponding disease severity Bullet cultivar which clearly explains the extensive disease
index (DSI) were much lower in the Beldanga cultivar which manifestation in this cultivar, while in the pathogen-chal-
clearly points to the tolerance nature of Beldanga cultivar lenged sets of Beldanga cultivar, PO, PPO, and PAL activi-
against anthracnose. In the Bullet cultivar, a rapid and mas- ties increased post-inoculation and maintained the enhanced
sive pathogen spread caused yellowing of leaves and loss of levels till 3 DPI. The differences in the activity patterns of
tissue integrity, as evidenced by heavy pathogen localization the enzymes PO, PPO, and PAL in the two cultivars further
in the leaf tissue. The might of this cultivar was not sufficient substantiates their differential response toward the pathogen.
enough to completely restrict the infection within the infec- Studies with chili fruits infected with C. capsici have previ-
tion site only, which points to more susceptible nature of ously reported higher total content of orthodideoxy phenols
this cultivar. Hence, greater percent disease area and disease as well as PO, PPO, and PAL levels in resistant genotypes
severity indices (DSI) were recorded in Bullet. and moderately resistant hybrids (Prasath and Ponnuswami,
Our study was intended to explain the differences in 2008). The researchers screened a total of 17 genotypes of
morphological disease symptoms of the two chili cultivars chili against anthracnose pathogen and recorded the activ-
Bullet and Beldanga and to better characterize the underly- ity levels of the enzymes PO, PPO, PAL, total phenol, and
ing host–pathogen interaction. Evidence till date supports orthodihydroxyphenol. The researchers further demonstrated
elevated levels of defense molecules which include defense that the least disease incidence in the resistant genotype
enzymes, antioxidative enzymes, PR proteins as well as phe- Capsicum baccatum was concomitant with the highest lev-
nolics behind an overall improved disease resistance (Majid, els of these enzymes and the highest accumulation of total
2017). Previous studies have reported enhanced activities of phenol and orthodihydroxyphenol in this genotype. Our
these defense molecules in a number of crop plants such as results thus are seeded well into the existing understanding
rice (Suthin et al. 2019), tomato (Anand et al. 2007), and tea of the role of these defense molecules in eliciting an immune
(Chandra et al., 2015) resistant to specific diseases. In the response.
present study, we found higher levels of all defense mole- Phenol is the precursor of lignin and is an important
cules post-inoculation in both the cultivars, the enhancement signal molecule that activates a number of defense-related
being more pronounced in Beldanga than in Bullet. Higher genes and pathways including systemic acquired resistance
PO, PPO, and catalase activities were reported in chili or SARS (Madhavan et al., 2010). Enhanced level of phenols
fruits upon pathogen inoculation with C. capsici (Koppad in response to pathogen invasion has been reported owing
and Mesta, 2017). The researchers further reported higher to its role in the deposition of secondary cell wall materi-
levels of these enzymes in infected resistant genotypes than als around the infection spot (Saxena et al., 2019). In the
in susceptible ones. These are antioxidant enzymes that present study, phenol levels were found to be elevated in
are required to maintain the delicate balance between ROS both the cultivars post-inoculation. Previous reports have
production and scavenging (Singla et al. 2019). In the pre- shown an improved level of phenolics in chili, upon patho-
sent study, both PO and catalase activity increased in Bullet gen ingress by C. truncatum (Madhavan et al., 2010; Sax-
as well as Beldanga cultivar following inoculation, which ena et al., 2019). Flavonoids are important phytoalexins
certainly testifies to ROS-mediated triggering of the down- that have antimicrobial properties (Facchini et al., 2002). In
stream defense signaling pathways. complete agreement with the previous reports, our results
PO also plays important role in cell wall fortification have shown enhanced levels of total phenol and flavonoids
(Saxena et al. 2019) which occurs to provide a mechanical after pathogen invasion. The effect was more pronounced
barrier to the spread of the pathogen from the infection spot in Beldanga, owing to its severe fight-back mechanism that
to the adjacent cells as well as to prevent additional invasion contributes to its resistance response. Thus total phenol and
from the cell surface. Deposition of wall thickening materi- flavonoid levels were observed to be continuously increasing
als such as lignin, suberin, and callose takes place with the till 3 DPI in this cultivar. The levels dropped after 1 DPI in
aid of PO, PPO, PAL as well as phenolics (Saxena et al., Bullet cultivar due to its eventual surrender to the disease as
2019). Increased lignin deposition along with enhanced PO was substantiated by the disease progress studies. Our results
activity has been reported in C. annuum against C. cap- are further validated by previous reports on an increased
sici (Abhyashree et al. 2017; Saxena et al. 2019). In chili level of phenol against pathogen ingress in C. annum (Anand
fruits, previous reports have shown induction of PO, PPO, et al., 2009; Madhavan et al., 2010).
PAL, and other enzymes in response to Alternaria alternata Another way of combating is to secrete extracellu-
(Anand et al., 2009) and Pseudomonas (Ramamoorthy and lar enzymes that degrade fungal cell wall components.
Samiyappan, 2001). In the present study, an increase in the β-Glucanase and chitinase are two important PR proteins

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that are bestowed with this function. The former hydro- study could contribute to the current understanding of suc-
lyzes glucan and the latter degrades chitin, thus diluting cessful disease establishment. To get a more complete pic-
important cell wall constituents of the pathogen (Tian ture of the differential disease response in the two cultivars,
et al., 2006). Moreover, the interaction of these enzymes we studied the gene expression levels in whole plants.
with the fungal cell wall induces the release of elicitor The gene expression profile of 12 important genes
molecules that interact with the plant cell receptors and remarkably substantiated the differences in morphological
initiates downstream signal transduction (Ham et al., disease expression of the two cultivars. Till now, a total of 17
1991). Accumulation of these PR proteins eventually leads PR proteins have been identified in plants (Soh et al., 2012).
to SAR (Ryals et al., 1996). Increased β-glucanase activity Previous studies have linked increased expression levels of
was reported in cucumber and tobacco against powdery PR 2, 5, 9 to improved disease resistance in plants (Hong
mildew and in pear fruits against Alternaria alternata, et al., 2018). Semi-q-RTPCR results showed a several-fold
in response to elicitor-mediated resistance (Herger and increase in the expression of pathogenesis responsive pro-
Klingauf, 1990; Tian 2006). Separate reports on increased tein-5 in Beldanga cultivar from 3 DPI, whereas its expres-
activity of β-glucanase and chitinase have been correlated sion dropped post-3DPI in the susceptible cultivar Bullet.
with improved resistance response in both tomato (Chak- Transcription factors (TFs) play a key role in transcriptional
taborty et al., 2017), and chili (Banu et al., 2019). In the reprogramming, triggered by pathogen perception in plants,
current study, we report enhanced β-glucanase and chi- resulting in the differential regulation of downstream defense
tinase activity in Beldanga cultivar in lieu of pathogen responsive genes and signal transduction (Peng et al., 2018).
invasion, and in Bullet the activity levels have shown a Transcription factor families of WRKY, NAC, MYB and
gradual decrease, which is commensurate with the pheno- bZIP are instrumental in plant immune response (Yuan et al.,
typic characteristics. Thus a simulated defense response 2019). A large repertoire of the transcription factor family
is touched up by close coordination of these enzymes PO, comprises WRKY, bZIP, bHLH, NAC, and AP2-ERF with
PPO, PAL, β-glucanase, chitinase, catalase as well as phe- a role in plant defense response (Seo et al., 2015). bZIPs
nols and flavonoids to shape into a composite and more play an important role in hormone and sugar signaling, oxi-
coordinated defense response. dative stress and pathogen defense (Thurow et al., 2005).
Various signaling molecules have been identified as medi- bZIP10 has been reported to play a role in the activation
ators in between the defense signaling cascades, of which of pathogenesis-related genes and induce basal defense
NO has been recognized as an important modulator (Nasir responses (Kaminaka et. al., 2006). Several bZIP genes are
et al., 2020). The role of NO in ROS scavenging (Mur et al., known to be involved upon biotic stimuli such as salicylic
2013) has been well elucidated. In the present study, NO acid, methyl jasmonic acid, ethylene and pathogen defense
levels as found in the quantitative estimation, and real time (Gai et al., 2020).The gene expression analysis from semi-
NO detection are in congruence with these findings. Both qRT-PCR results showed a steady rise in the expression of
the cultivars showed an upsurge of NO levels post-pathogen bZIP 10 in the resistant Beldanga cultivar post-infection.
inoculation and a prolonged elevated level was maintained An increase in expression was also observed in the Bullet
in the Beldanga cultivar. Studies with both NO-enriching cultivar; however, its expression dropped below the basal
systems and NO-scavenging systems have shown a direct level after 3DPI. Plant NAC TFs have also been known to
correlation of NO levels with plant defense (Chandra et al., play an important role in the development and biotic stress
2015; Chakraborty et al., 2017). (Puranik et al., 2012). These TFs contain binding sites for
Thus, the fundamental differences in disease response of W boxes, GCC boxes and MYB that mediates its interaction
the two cultivars of the same chili plant to the same pathogen with other TFs such as WRKY and MYB in signal trans-
could be explained due to differences in the activity levels duction (Pascual et al., 2015). An NAC domain containing
of their important defense molecules. Enhanced levels of transcription factor CaNAC1 was found to be involved in
the defense molecules have been positively correlated to interactions between chili pepper and pathogens (Oh et al.,
an enhanced degree of tolerance toward successful disease 2005). Here, the expression levels of CaNAC in the resistant
establishment (Ramamoorthy and Vellaiswamy, 2001; Banu Beldanga cultivar were higher as compared to the Bullet cul-
et al., 2019). Previous reports have also suggested the role of tivar. The expression dropped at 1 DP1 and 3 DPI following
sesquiterpenoids such as capsidiol and related genes active which it increased at 7 DPI and was the same as 0 DPI, sug-
in pepper fruit in defense against Colletotrichum scovil- gesting an establishment of defense response after pathogen
lei; however, their mechanism of biosynthesis remain to be infection. The R2R3 type MYB transcription factors play
understood (Baba et al., 2020). In our study, the elevated a crucial role in plant defense, also regulating the expres-
response of the defense molecules was maintained in Bel- sion of pathogen responsive proteins (Hong et al., 2018).
danga cultivar, but not in the Bullet cultivar which explains Semi-qRT-PCR results show a steady rise in the expression
the susceptibility of the latter. In this context, the present level of CaMYB from 3DPI suggesting a basal response.

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Acta Physiologiae Plantarum (2021) 43:155 Page 13 of 16 155

WRKY33 is implicated in pathogen response in PTI (Hong studies carried out. A previous study reported a new gene
et al., 2018) and via interactions with MAP kinase cascades RCt1 for resistance against anthracnose and the same report
(Mao et al., 2011). Earlier reports of a WRKY transcription identified few ISSR and AFLP markers showing polymor-
factor, CaWRKY1 from chilli, have been shown to act as a phism between resistant and susceptible cultivars (Mishra
negative regulator of defense for virus-induced gene silenc- et al., 2019). Future studies related to such marker-assisted
ing of the gene that decreased the growth of Xanthomonas, analysis can be helpful for the identification and breeding of
whereas overexpression resulted in a hypersensitive response disease-resistant varieties.
and cell death in response to P. syringae and tobacco mosaic
virus (Oh et al., 2008). Expression levels of WRKY33 in
both the cultivars dropped at 3DPI and then increased again Conclusion
at 7 DPI suggesting a hypersensitive immune response.
The expression of sweet sugar transporters is often modi- Anthracnose is a devastating disease of chili which causes
fied by extracellular pathogens so they can direct the sugars huge economic loss. Thus studies contributing to the rec-
for their growth (Chen et al., 2010). AtSTP4 from Arabidop- ognition of the basis of resistance in the tolerant cultivars
sis thaliana is overexpressed upon pathogen attack and inva- can facilitate identification of genes responsible for the trait
sion (Truernit et al., 1996). Sugar transporter proteins have and, in turn, accelerate breeding programs to develop resist-
been identified from Capsicum and implicated in growth, ant cultivars. With this view, our study was aimed at better
development and abiotic stress resistance (Wei et al., 2020). characterizing the defense response in the two host culti-
In the present study, upon pathogen attack, the semi qRT- vars. While our disease progression and histological stud-
PCR results showed a sharp rise in the expression level of ies have shown discrete patterns of C. capsici colonization,
the gene in both Beldanga cultivar (from 3 DPI) and Bullet the biochemical assay of defense enzymes and PR proteins
cultivar (at 7 DPI) suggesting a faster response to pathogen have clearly pointed the differences in their activity levels.
attack in the resistant cultivar. These results clearly explain their differential response. The
SODs play an important role in converting ROS, pro- results are further substantiated by the amount of total phe-
duced as a result of oxidative burst, into hydrogen peroxide nols, flavonoids and chlorophyll. Our characterization of the
and oxygen. The hydrogen peroxide is then broken down differentially expressed gene and proteins could facilitate the
by enzymes such as catalase (CAT) and ascorbate peroxi- identification of important genetic markers in future studies
dase, thereby neutralizing the toxicity of ROS (Cox et al., and their application in successful breeding programs.
2003). Earlier studies have reported the induced expression
of ascorbate peroxidase and thioredoxin peroxidase upon Author contribution statement KA designed the experi-
infection of Xanthomonas campestris triggering a hypersen- ments. JB and TH performed the experiments and analyzed
sitive response (Do et al., 2003) High expression of the SOD the data. JB wrote the manuscript. All the authors have read
metallo-enzymes at 7 DPI in Beldanga suggested a pro- and approved the manuscript.
nounced defense response and mitigation of oxidative stress.
In the Bullet cultivar, there was a decline in the expression of
FeSOD at 7 DPI, whereas the CuSOD expression profile did Supplementary Information The online version contains supplemen-
not show a significant change. MnSOD showed an increased tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11738-0​ 21-0​ 3334-x.
expression post 3 DPI in Beldanga cultivar but at 7DPI in
the Bullet cultivar. The CAT gene expression also showed Acknowledgements The authors thank DST (WOS-A), UGC-UPE II,
CAS Phase VII and DST (FIST), Govt. of India, for providing instru-
an increase at 7 DPI in both. The expression profile of ascor- mental facility.
bate peroxidase increased post-infection in the Bullet culti-
var and remained more or less constant till 7 DPI. However, Funding This research project was funded by Women Scientist
comparably in the Beldanga cultivar, several-fold increase Scheme–A (WOSA), Department of Science and Technology (DST),
from 3DPI was observed suggesting a pronounced defense Government of India [Project sanction no. SR/WOS-A/LS-272/2017].
response to infection. Flavoproteins such as glutathione
reductase (GR) play an important role in the conversion of Declarations
glutathione disulfide to glutathione (GSSG/GSH cycle) and
Conflict of interest The authors declare that they have no conflict of
in the process eliminates excessive burst of ­H2O2 in the cell. interest.
The expression profile of glutathione reductase showed an
increase in expression post-3 DPI in both the cultivars.
High-throughput phenotyping and gene expression analy-
sis can also be complemented with the study of disease-asso-
ciated marker analysis. There are very few marker-associated

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155 Page 14 of 16 Acta Physiologiae Plantarum (2021) 43:155

References Dickerson DP, Pascholati SF, Hagerman AE et al (1984) Phenyla-

lanine ammonia-lyase and hydroxycinnamate: CoA ligase in
maize mesocotyls inoculated with Helminthosporium maydis or
Abhayashree MS, Murali M, Thriveni MC, Sindhu GM, Amruthesh
Helminthosporium carbonum. Physiol Plant Pathol 25:111–123.
KN (2017) Crude oligosaccharides mediated resistance and histo-
https://​doi.​org/​10.​1016/​0048-​4059(84)​90050-X
chemical changes in Capsicum annuum against anthracnose dis-
Do HM, Hong JK, Jung HW, Kim SH, Ham JH, Hwang BK (2003)
ease caused by Colletotrichum capsici. Plant Biosyst 151:221–233
Expression of peroxidase-like genes, ­H 2O 2 production, and
Agrios GN (2005) Plant pathology, 5th edn. Academic Press, San
peroxidase activity during the hypersensitive response to Xan-
Diego, p 922
thomonas campestris pv. vesicatoria in Capsicum annuum. Mol
Anand T, Chandrasekaran A, Kuttalam S, Raguchander T, Prakasam V,
Plant Microbe Interact 16(3):196–205. https://​doi.​org/​10.​1094/​
Samiyappan T (2007) Association of some plant defense enzyme
MPMI.​2003.​16.3.​196 (PMID: 12650451)
activities with systemic resistance to early leaf blight and leaf
Du J, Verzaux E, Chaparro-Garcia A, Bijsterbosch G, Keizer LP,
spot induced in tomato plants by azoxystrobin and Pseudomonas
Zhou J, Liebrand TW, Xie C, Govers F, Robatzek S (2015)
fluorescens. J Plant Interact 2(4):233–244. https://d​ oi.o​ rg/1​ 0.1​ 080/​
Elicitin recognition confers enhanced resistance to phytophthora
17429​14070​17089​85
infestans in potato. Nat Plants 1:15034
Anand T, Bhaskaran R, Raguchander T, Samiyappan R, Prakasham
Facchini PJ, Hagel J, Zulak KG (2002) Hydroxycinnamic acid
V, Gopalakrihnan C (2009) Defence responses of chilli fruits
amide metabolism: physiology and biochemistry. Can J Bot
to Colletotrichum capsici and Alternaria alternata. Biol Plant
80:577–589
53:553. https://​doi.​org/​10.​1007/​s10535-​009-​0100-5
Gai WX, Ma X, Qiao YM et al (2020) Characterization of the bZIP
Anderson MD, Prasad TK, Stewart CR (1995) Changes in Isozyme
transcription factor family in pepper (Capsicum annuum L.):
Profiles of catalase, peroxidase, and glutathione reductase during
CabZIP25 positively modulates the salt tolerance. Front Plant
acclimation to chilling in mesocotyls of maize seedlings. Plant
Sci 11:139. https://​doi.​org/​10.​3389/​fpls.​2020.​00139
Physiol 109(4):1247–1257. https://d​ oi.o​ rg/1​ 0.1​ 104/p​ p.1​ 09.4.1​ 247
Ham K, Kauffmann SS, Albersheim P, Darvill AG (1991) Host-path-
Baba VY, Powell AF, Ivamoto-Suzuki ST et al (2020) Capsidiol-related
ogen interactions. A soybean pathogenesis-related protein with
genes are highly expressed in response to Colletotrichum scovil-
b-1,3- glucanase activity releases phytoalexin elicitor-active heat-
lei during Capsicum annuum fruit development stages. Sci Rep
stable fragments from fungal cell walls. Mol Pl Microbe Interact
10:12048. https://​doi.​org/​10.​1038/​s41598-​020-​68949-5
4:545–552
Bansode Y, Bajekals S (2006) Characterization of chitinase from
Hameda HM, Klein BP (1990) Effects of naturally occurring anti-
microorganisms isolated from Lonar Lake. Indian J Biotechnol
oxidants on peroxidase activity of vegetable extracts. J Food Sci
5:357–363
55:184–185
Banu N, Mahadevamurthy M, Amruthesh K (2019) Plant growth-pro-
Heller J, Tudzynski P (2011) Reactive oxygen speciesin phytopath-
moting fungi (PGPF) instigate plant growth and induce disease
ogenic fungi: signaling, development, and dis-ease. Annu Rev
resistance in Capsicum annuum L. upon Infection with Colle-
Phytopathol 49(1):369–390. https:// ​ d oi. ​ o rg/ ​ 1 0. ​ 1 146/ ​ a nnur​
totrichum capsici (Syd.) Butler and Bisby. Biomolecules 10:41.
ev-​phyto-​072910-​095355
https://​doi.​org/​10.​3390/​biom1​00100​41
Herger G, Klingauf F (1990) Control of powdery mildew fungi
Bartha B, Kolbert Z, Erdei L (2005) Nitric oxide production induced
with extracts of the giant knotweed, Reynoutria sachalinensis
by heavy metals in Brassica juncea L. Czern and Pisum sativum
(Polygonaceae). Mededelingen Faculteit Landbouwwetenschap-
L. Acta Biol Szeged 49:9–12
pen Rijksuniversiteit Gent 55:1007–1014
Bradford M (1976) A rapid and sensitive method for the quantitation of
Hong Z, Shikai L, Changyou W, Wanquan J (2018) The role of tran-
microgram quantities of protein utilizing the principle of protein
scription factor in wheat defense against pathogen and its prospect
dye binding. Anal Biochem 72:248–254
in breeding. J Plant Biol Crop Res 1:1005
Chakraborty N, Chandra S, Acharya K (2017) Biochemical basis of
Inglis DA, Haedorn DJ, Rand RE (1988) Use of dry inoculum to evalu-
improvement of defense in tomato plant against Fusarium wilt by
ate beans for resistance to anthracnose and angular leaf spot. Plant
­CaCl2. Physiol Mol Biol Plants Int J Funct Plant Biol 23(3):581–
Dis 72:771–774
596. https://​doi.​org/​10.​1007/​s12298-​017-​0450-y
Isaac S (1992) Fungal plant interaction. Chapman and Hall Press, Lon-
Chandra S, Chakraborty N, Dasgupta A, Sarkar J, Panda K, Acharya
don, p 115
K (2015) Chitosan nanoparticles: a positive modulator of innate
ISTA (2005) International seed testing association. In: Proceedings of
immune responses in plants. Sci Rep 5:15195. https://​doi.​org/​10.​
the international seed testing association. International rules of
1038/​srep1​5195
seed testing. Seed Sci Technol 15: 1–9.
Chandrashekar S, Umesha S (2012) Induction of antioxidant enzymes
Kamble P, Giri S, Mane R, Tiwana A (2015) Estimation of chlorophyll
associated with bacterial spot pathogenesis in tomato. Int J Food
content in young and adult leaves of some selected plants. Univ J
Agric Veterin Sci 2:22–34
Environ Technol 5:306–310
Chang CC, Yang MH, Wen HM, Chern JC (2002) Estimation of total
Kaminaka H et al (2006) bZIP10-LSD1 antagonism modulates basal
flavonoid content in propolis by two complementary colorimetric
defense and cell death in Arabidopsis following infection. EMBO
methods. J Food Drug Anal 10:178–182
J 25:4400–4411
Chen LQ, Hou BH, Lalonde S, Takanaga H, Hartung ML, Qu XQ et al
Koppad S, Mesta RK (2017) Role of enzymes in resistance against fruit
(2010) Sugar transporters for intercellular exchange and nutri-
rot of chilli caused by Colletotrichum capsici (Syd.) Butler and
tion of pathogens. Nature 468:527–532. https://​doi.​org/​10.​1038/​
Bisby. Int J Chem Stud 5(4):536–540
natur​e09606
Kumar K, Khan P (1982) Peroxidase and polyphenol oxidase in excised
Cox GM, Harrison TS, McDade HC et al (2003) Superoxide dismutase
ragi (Eleusine corocana cv PR 202) leaves during senescence.
influences the virulence of Cryptococcus neoformans by affecting
Indian J Exp Biol 20:412–416
growth within macrophages. Infect Immun 71(1):173–180. https://​
Madhavan S, Paranidharan V, Velazhahan R (2010) Foliar application
doi.​org/​10.​1128/​iai.​71.1.​173-​180.​2003
of Burkholderia sp. strain TNAU-1 leads to activation of defense
Delledonne M (2005) NO news is good news for plants. Curr Opin
responses in chili (Capsicum annuum L.). Braz J Plant Physiol
Plant Biol 8:390–396
23:261–266

13
Acta Physiologiae Plantarum (2021) 43:155 Page 15 of 16 155

Majid MU, Awan MF, Fatima K et al (2017) Genetic resources of chili of biochemical and enzymatic activities in inducing resistance.
pepper (Capsicum annuum L.) against Phytophthora capsici and Indian J Genet Plant Breed 68:344–346
their induction through various biotic and abiotic factors. Cytol Puranik S et al (2012) NAC proteins: regulation and role in stress toler-
Genet 51:296–304. https://​doi.​org/​10.​3103/​S0095​45271​70400​3X ance. Trends Plant Sci 17:369–381
Malik NA, Kumar IS, Nadarajah K (2020) Elicitor and receptor mol- Ramamoorthy V, Samiyappan R (2001) Induction of defense-related
ecules: orchestrators of plant defense and immunity. Int J Mol Sci genes in Pseudomonas fluorescens treated chilli plants in response
21(3):963. https://​doi.​org/​10.​3390/​ijms2​10309​63 to infection by Colletotrichum capsici. J Mycol Plant Pathol
Mao G et al (2011) Phosphorylation of a WRKY transcription factor by 31:146–155
two pathogen-responsive MAPKs drives phytoalexin biosynthesis Roy M, Harris J, Afreen S, Deak E, Gade L, Balajee SA, Park B,
in Arabidopsis. Plant Cell 23:1639–1653 Chiller T, Luby S (2013) Aflatoxin contamination in food com-
Mateos RM, Jimenez A, Roman P, Romojaro F, Bacaeizo S, Leterrier modities in Bangladesh. Food Addit Contam B6(1):17–23
M, Gomez M, Sevilla F, del Rio LA, Corpas FJ, Palma JM (2013) Ryals JA, Neuenschwander UH, Willits MG, Molina A, Steiner
Antioxidant systems from pepper (Capsicum annuum L.): involve- HY, Hunt MD (1996) Systemic acquired resistance. Plant Cell
ment in the response to temperature changes in ripe fruits. Int 8:1809–1819
J Mol Sci 14:9556–9580. https://​doi.​org/​10.​3390/​ijms1​40595​56 Salter M, Knowles GR (1998) Assay of NOS activity by the measure-
Mbengue M, Navaud O, Peyraud R, Barascud M, Badet T, Vincent ment of conversion of oxyhemoglobin to methemoglobin by NO.
R, Barbacci A, Raffael S (2016) Emerging trends in molecular In: Titheradge MA (ed) Nitric oxide protocols. Humana Press,
interactions between plants and the broad host range fungal patho- Totowa, pp 61–65
gens Botrytis cinerea and Sclerotinia sclerotiorum. Front Plant Sci Saxena A, Mishra S, Ray S, Raghuwanshi R, Singh H (2019) Dif-
7:422. https://​doi.​org/​10.​3389/​fpls.​2016.​00422 ferential reprogramming of defense network in Capsicum annum
Mishra R, Nanda S, Rout E, Chand SK, Mohanty JN, Joshi RK (2017) L. plants against colletotrichum truncatum infection by phyl-
Differential expression of defense-related genes in chilli pepper lospheric and rhizospheric trichoderma strains. J Plant Growth
infected with anthracnose pathogen Colletotrichum truncatum. Regul. https://​doi.​org/​10.​1007/​s00344-​019-​10017-y
Physiol Mol Plant Pathol 97:1–10 Seo E, Choi D (2015) Functional studies of transcription factors
Mishra R, Rout E, Mohanty JN, Joshi RK (2019) Sequence-tagged involved in plant defenses in the genomics era. Brief Funct Genom
site-based diagnostic markers linked to a novel anthracnose resist- 14(4):260-267@@
ance gene RCt1 in chili pepper (Capsicum annuum L). 3 Biotech Sharma PN, Kaur M, Sharma OP, Sharma P, Pathania A (2005) Mor-
9(1):9. https://​doi.​org/​10.​1007/​s13205-​018-​1552-0 (10.1007/ phological, pathological and molecular variability in Colletotri-
s13205-018-1552-0. Epub 2019 Jan 2. PMID: 30622847; chum capsici, the cause of fruit rot of chillies in the subtropical
PMCID: PMC6312824) region of north-western India. J Phytopathol 153:232–237
Monaghan J, Zipfel C (2012) Plant pattern recognition receptor com- Singla P, Devi R, Kaur S, Kaur J (2019) Stripe rust induced defence
plexes at the plasma membrane. Curr Opin Plant Biol 15(4):349– mechanisms in the leaves of contrasting barley genotypes (Hor-
357. https://​doi.​org/​10.​1016/j.​pbi.​2012.​05.​006 deum vulgare L.) at the seedling stage. Protoplasma. https://​doi.​
Mur LAJ, Mandon J, Persijn S, Cristescu SM, Moshkov IE, Novikova org/​10.​1007/​s00709-​019-​01428-5
GV, Hall MA, Harren FJM, Hebelstrup KH, Gupta KJ (2013) Soh HC, Park AR, Park S, Back K, Yoon JB, Park HG, Kim YS (2012)
Nitric oxide in plants: an assessment of the current state of knowl- Comparative analysis of pathogenesis-related protein 10 (PR10)
edge. AoB Plants 5:52. https://​doi.​org/​10.​1093/​aobpla/​pls052 genes between fungal resistant and susceptible peppers. Eur J
Nasir NNM, Ho CL, Lamasudin DU, Saidi NB (2020) Nitric oxide Plant Pathol 132(1):37–48
improves tolerance to Fusarium oxysporum f. sp. cubense tropi- Suthin T, Sudhagar GB, Rao S, Ann RS, Muthukumar A, Muthuku-
cal race 4 in banana. Physiol Mol Plant Pathol. https://​doi.​org/​10.​ maran N, Rao S (2019) Induction of defence enzymes activities
1016/J.​PMPP.​2020.​101503 in rice plant treated by seaweed algae against Rhizoctonia solani
Oh SK, Lee S, Yu SH, Choi D (2005) Expression of a novel NAC Kuhn causing sheath blight of rice. 210–218.
domain-containing transcription factor (CaNAC1) is preferentially Taylor PWJ, Mongkolporn O, Than PP, et al (2007) Pathotypes of
associated with incompatible interactions between chili pepper Colletotrichum spp. infecting chili peppers and mechanisms of
and pathogens. Planta 222(5):876–887. https://​doi.​org/​10.​1007/​ resistance. In: Oh DG, Kim KT (eds) First International Sym-
s00425-​005-​0030-1 (Epub 2005 Aug 3 PMID: 16078072) posium on Chilli Anthracnose, Abstracts, Seoul, South Korea.
Oh SK, Baek KH, Park JM, Yi SY, Yu SH, Kamoun S, Choi D (2008) National Horticultural Research Institute. p. 29.
Capsicum annuum WRKY protein CaWRKY1 is a negative regu- Than PP, Jeewon R, Hyde KD, Pongsupasamit S, Mongkolporn O,
lator of pathogen defense. New Phytol 177(4):977–989 Taylor PWJ (2008) Characterization and pathogenicity of Colle-
Pan SQ, Ye XS, Kuć J (1991) Association of β-1,3-glucanase activity totrichum species associated with anthracnose disease on chili
and isoform pattern with systemic resistance to blue mould in (Capsicum spp.) in Thailand. Plant Pathol 57:562–572
tobacco induced by stem injection with Peronospora tabacina Thurow C et al (2005) Tobacco bZIP transcription factor TGA2.2
or leaf inoculation with tobacco mosaic virus. Physiol Mol Plant and related factor TGA2.1 have distinct roles in plant defense
Pathol 39:25–39. https://d​ oi.o​ rg/1​ 0.1​ 016/0​ 885-​5765(91)​90029-H responses and plant development. Plant J 44:100–113
Pascual MB, Canovas FM, Avila C (2015) The NAC transcription fac- Tian S, Wan Y, Qin G, Xu Y (2006) Induction of defense responses
tor family in maritime pine (Pinus pinaster): molecular regulation against Alternaria rot by different elicitors in harvested pear fruit.
of two genes involved in stress responses. BMC Plant Biol 15:254 Appl Microbiol Biotechnol 70:729–734
Peng Y, van Wersch R, Zhang Y (2018) Convergent and divergent Truernit E, Schmid J, Epple P, Illig J, Sauer N (1996) The sink-specific
signaling in PAMP-triggered immunity and effector-triggered and stress regulated Arabidopsis gene: enhanced expression of a
immunity. Mol Plant Microbe Interact 31:403–409 gene encoding a monosaccharide transporter by wounding, elici-
Prasad A, Sedlářová M, Balukova A, Rác M, Pospíšil P (2020) Reac- tors, and pathogen challenge. Plant Cell 8:2169–2182
tive oxygen species as a response to wounding. Vivo imaging in Wei H, Liu J, Zheng J et al (2020) Sugar transporter proteins in Cap-
Arabidopsis thaliana. Front Plant Sci 10:1660 sicum: identification, characterization, evolution and expression
Prasath D, Ponnuswami V (2008) Screening of chilli (Capsicum ann- patterns. Biotechnol Biotechnol Equip 34(1):341–353
uum L.) genotypes against Colletotrichum capsici and analysis

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155 Page 16 of 16 Acta Physiologiae Plantarum (2021) 43:155

Yadav V, Wang Z, Wei C, Amo A, Ahmed B, Yang X, Zhang X (2020) Publisher's Note Springer Nature remains neutral with regard to
Phenylpropanoid pathway engineering: an emerging approach jurisdictional claims in published maps and institutional affiliations.
towards plant defense. Pathogens 9(4):312. https://​ doi.​org/​10.​
3390/​patho​gens9​040312
Yuan X, Wang H, Cai J et al (2019) NAC transcription factors in
plant immunity. Phytopathol Res 1:3. https://​doi.​org/​10.​1186/​
s42483-​018-​0008-0
Zieslin N, Ben Zaken R (1993) Peroxidase activity and presence of
phenolic substances in peduncles of rose flowers. Plant Physiol
Biochem 31:333–339

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