1. Introduction
Plants survive with a variety of strategies to defend themselves against
pathogen. Plants are major target of microbe seeking a source of nutrition a
complex array of interactions between plants and microbes evolved that reflects
both the nutrient a acquisition strategies of microbes and defense strategies of
plants. Part of plant defense strategies includes an active offense against invading
microbes using an array of antimicrobial gene products within the context of the
overall plant microbe interaction, we attempt here to emphasize the role of
antimicrobial proteins and peptides in plant defense.
The majority of plant microbe encounters don’t results in disease. Preformed
factors including constitutively expressed waxes cell wall components.,
antimicrobial peptides, proteins and non proteinaceous secondary metabolites that
deter invasion have been proposed to contribute significantly to the host range of
pathogens. The importance of preformed defenses has been inferred from the
observation that plants can be rendered susceptible by a deficiency in the
production of these secondary metabolites or by the abilities of pathogens to
degrade them.
Inducible Defenses Require detection of pathogens by lost surveillance. The
sentinels

2. Plants defense responses are induced by microbial products in non host and
host resistance plant defense systems are also in induced by microbial products in
compatible and incompatible plant microbe interaction.
Specific host- pathogen interaction models describing induced defense responses in
plants have been greatly influenced in recent years gene for gene interaction
originally reported by floor (1956) in these specific host pathogen interactions
resistance to a particular pathogen is conditional on the presence of specific Avr
(avirulence )gene of the pathogen and specific (R) resistance gene in plant host
wide spread interest in gone for gene interaction resulted for recognizing the
resistance was usually controlled by single dominant genes making genetic
analysis very detectable.
Antimicrobial proteins and peptides are important components of innate
immunity
A common feature of the innate immune system of taxonomically diverse
organism i.e. plant is the ability to marshal the accumulation of antimicrobial
proteins and peptides in response to an invasive challenge by foreign organism
more than 500 different antimicrobial proteins and peptides encoded within the
genomes of many organism including plants. Genes encoding these protein
peptides have an important role in host-pathogen interaction. Much less certain is
specific function of each in individual pathogen-plant interaction. In view of this
knowledge gap we emphasize that full susceptibility of any given pathogen to only

3. host-protein toxin should result immunity. Thus it becomes clear that resistance
much have of pathogens against host defense toxins must be widespread and
important to disease development.
Chilly (Capsicum annuum) is an important vegetable crop grown throughout
the world an alkaloid, capsaicin is extracted from chilly, which has medicinal
value. Plants are able to defend themselves successfully with a complex set of
preformed structures and inducible reactions. The inducible reactions require the
perception of either plant derived (endogenous) or pathogen derived (exogenous)
signal molecule.
Chitin and glucan oligomers released during degradation of fungal cell wall
act as elicitors that elicit various defense mechanisms in the plants.High expression
of these defense enzymes in crop plants can enhance resistance to a pathogen.
When a pathogen lands on a host surface, it activates the hosts defense
mechanisms probably by releasing elicitors from its cell walls plants are endowed
with defense genes, which are quiescent in healthy plants, but when activated by
various factors they induce systematic diseases resistance. Defense enzymes that
are induced in response to invasion by a pathogen have greater antifungal activity
to the pathogen in suppressing symptoms induction of defence enzymes makes the
plant resistant to pathogen invasion. Expression of these protein is correlated with
the development of systemic acquired resistance in plants.Thus if mechanisms are
triggered quickly and maintained enhanced level due to infection by a plant
pathogen disease severity can be reduced. In the present study an attempt was

4. made to compare and quantitatively analyze the induction of defense compound
and enzyme in green and ripe chilli fruits inoculated with colletotrichum capsci
.ANTHRACNOSE disease is one of the most serious fungal pathogens of chilli in
Asia. Anthracnose deriving from a Greek word meaning coal is the common name
for plant diseases characterized by very dark sunken lesion containing spores. The
disease appears as small circular spots that coalesce to form large elliptical spots
on fruits and leaves. Under severe conditions ,defoliation of affected plant occurs.
Typical fruit symptoms are circular or angular sunken lesions with
concentric rings of acervuli that are often wet and produce pink to orange conidial
mass. This disease produces symptoms on leaves, stem pre & post harvest fruits
and causes severe damage to mature fruits in the field. More over during transit
and storage this disease are also causes serve damage to chilli fruits. The virulence
degree of disease symptoms host plants reported the existence of 15 pathogens of
C. capsici based on disease system development on inoculated fruit of capsicum
annuum.
There was a significant amount of variation at physiological and
biochemical levels present in chilli gene types for resistance to fruit rot/die back
colletotrichum. The resistance chilli genes types exhibited more physiological
characteristics as compared to susceptible genotypes.

5. A various biochemical characters also affect die back/fruit rot intensively in
chilli such as total phenol, total sugar, capsaicin, ascorbic acid and protein content
that impart resistance fruit.
Red ripe chilli fruits were more susceptible to fruit rot as compared to semi
riped ones. A various biochemical and physiological characters play specific role
in imparting resistance against disease. The resistant chilli genotypes were high in
capsaicin and orthohydroxys phenol and exhibited more plant length days to
picking as compared to susceptible ones.
The other characters which had a positive and direct effect on fruit rot, width
fruit number per plants, stem, thickness, fruit weight per plant, leaf area and, caro
tenes where as total flavonols, ascorbic acid, total sugar content had a direct and
nagative effect on dieback/ fruit rot. A various physiological and biochemical traits
that varied significantly among resistance and susceptible genes type can be
deployed in resistant breeding or as physiological and biochemical.
Materials and Methods
Plant material and Collection of seeds
The plant material was taken chilli plants .The collection of seeds were done
from the OUAT( Orissa University of Agriculture and Technology ) Bhubaneswar
The plant materials were obtained by germinating the seeds of chilly. The
healthy and non diseased seeds were taken.

6. Sterilization of Seeds
The seeds were sterilized with sterile distilled water and then kept in 0.1%
Hgcl2 for 30 seconds washed repeatedly with sterile distilled water and kept in
petriplates layered with moist cotton and also sterilized seeds were sown on soil in
pots.
DETECTION OF PATHOGEN FROM SEEDS
Colletotrichum capsici is both internally and externally seed borne such
effected seed and infected seeding act as primary sources of inoculum.
Blotter methods
Seed borne colletotrichum capsci can be detected by subjecting chilli seed
samples to the standard blotter test. Individual samples of ten seeds are plated in
petriplates.. Plates were placed in petriplates. Plates were placed on three layered
moist blotter paper plates. On the fifth day of the seeds were observed for the
presence of fungal growth with the help of microscope. Colletotrichum capsici is
identified based on morphological characters of acervuli and conidia growing on
the blotter paper.
Casual Organism

7. Colletotrichum capsici
Scientific classification
Kingdom Fungi
Phylum Ascomycota
Class Sordariomycetes
Order Glomerellalels
Family Glomerellaceae
Genus Colletotrichum
Species capsici
Colletotrichum is one of the most important plant pathogens world wide
causing the economically important disease anthracnose in a wide range of hosts
including cereals, legumes, vegetables, perennial crops and tree fruit. Among these
hosts, chilli, an important economic crop worldwide is severely infected by
anthracnose which may cause yield losses of up to 50%.
In the colletotrichum pathosystem, different colletotrichum species can be
associated anthracnose of the same host. Different species cause disease of
different organs of the chilli plant.
For example – C. acutatum and C. gloeosporides infect chilli fruits at all
developmental stages but usually not leaves or stems. Stems or leaves are damaged

8. by C. coccodes and C. dematium. Leaf anthracnose of chilli seeding caused by
C.coccodes was first reported in chilli growing in a field in chungam province of
Korea in 1988. Different colletotrichum species may also play an important role in
different disease of mature stages of chilli fruit.
For example- C. capsici is wide spread in red chilli fruits whereas
C.acutatum , C. gloeosporioides have been reported to be more prevalent both
young and mature green fruits.
Pathogen isolation, culture, maintenance
The anthracnose disease causing Pathogen colletotrichum capsici was
isolated from a diseased seed of chilly which was provided by Department of Plant
pathology, OUAT, Bhubaneswar.
For isolation, culture and maintenance of desired pathogen, Potato Dextrose Agar
(PDA) media was used and the media was prepared as per as the indication given
on the containers, in aseptic conditions with sterilized glass waves & inoculation
loops.
POTATO DEXTROSE AGAR Medium
Component Amount
Peeled potato 250gm
Glucose 20.00gm
Agar 15gm

9. Distilled water 1000ml
pH 6.0-6.6
The seeds were first washed with sterile distilled water and then surface sterilized
with 0.1% Hgdl2 for 30 seconds followed by washing sterile distilled water in three
times. Then the leaves were placed on the PDA plated with aseptic condition in
laminar airflow and incubated at 280C for 48 hours. After 48 hours of incubation
the plates were observed to be containing the colonies of fungal mycelia on the
PDA plates showing the typical colony morphology & growth symmetry. All the
process of isolation, culture and maintenance were done in aseptic condition.
4. Sub culturing of the Organism
The well grown culture plates were sub cultured on PDA and Potato Dextrose
Broth & maintained in incubator at 280C. Two of well grown subcultures plates
were stored at 40C as master plates & were revived monthly. These processes of
sub culturing and maintenance were done in aseptic condition.
Preparation Of Spore Suspension
From the well grown culture plates the development of spore was seen with
the help of electron microscope. The typical structure spore can been seen and
identified. The Potato Dextrose broth with the help of a brush & inoculation loop
under aseptic conditions in laminar air flow & incubated at 280C.

10. Experimental Design
The seeds were sown on the soil in pots. Three types of seeds i.e. sterilized,
unsterilized and seeds soaked with spore suspension were taken.
In each pot there were 8 no. of seeds sown on the pots with the help of forcep &
gloves. First the pots were cleaned & the hole on the pots was closed with the help
of chips of pots. Soil was mixed with biofertilizer and vermicompost and mixed
well.
Then seeds were sown in respective pots. Regular watering and observation was
done and also sterilized, unsterilized & spore suspension seeds were kept in
petriplates with layered moist cotton to know the seed germination and affect of
growth of causal organism.
Germination Percentage
Germination Percentage of chilly was determined by counting the no. of
germinated seeds out of total inoculated seeds & following formula was used to
calculate the germination percentage.
% of Germination = Total no. of germinated seeds/ Total no. of seeds inoculated
Germination Percentage of sterilized, unsterilized and spore inoculated seeds were
observed.

11. Study of Growth Parameters of Plants
The growth of plant was observed from times of seedling to onward, Growth of
plant in terms of its height, leaves was measured.
Measurement of Physiological & Biochemical Parameters
Plant height, stem thickness, no. of leaves & leaf area. At maturity, the height was
measured from the ground level to the highest bud point.
Branches per plant:- The no. of primary branches was counted.
No. of leaves – No. of leaves was counted days to first picking leaf area. Leaf area
of plant was also calculated with graph paper.
Biochemical Parameters
Extraction & estimation of chlorophyll and carotenoids
Estimation of Pigment
The leaf samples from each pot experiment of healthy and unhealthy were taken
for chlorophyll & carotenoid estimation. 500mg of leaf sample was taken &
grinded in motor and pestle by adding 10ml of cold 80% acetone. The mixture was
centrifuged at 2000rpm for 10-15 minutes. The supernatant was taken for
measuring absorbance at 480,663 & 645nm. Chlorophyll a, chlorophyll b and total
chlorophyll content was calculated by using Arnon’s formula (1949).
Chlorophyll a = (12.7xA663-2.69xA645)xV/1000w

12. Chlorophyll b = (22.9xA645-4.68xA645)xV/1000w
Total Chlorophyll = (8.02xA663+20.2xA645)V/1000w
Estimation of carotenoid was done using the formula of Kirk and Allen (1965).
Acar /480per leaf segment = (A480+0.114A663-0.638xA645)x V/1000w
Where A480, A663 & A645 are the absorbance of extract at 480nm, 645nm & 663nm
respectively.
V= volume of acetone extract
w= weight of leaf (gram fresh weight)
Extraction and Estimation of Protein
1gm of plant samples (leaf) were homogenized with cold phosphate buffer (pH
7.4) in a mortar & pestle, the volume of the homogenate was made up to 5ml &
grinded with PVPP. The homogenate was then centrifuged at 14,000rpm for 20
minutes. The supernatant used for estimation of protein.
Soluble Protein was estimated by the method of Lowry etal (1951).
The following reagents were prepared for protein estimation.
Reagent A- 2% (w/v) sodium carbonate in 0.1 N NAOH.
Reagent B- 0.5% (w/v) copper sulphate in 1% (w/v) sodium potassium tartarate

13. Reagent C- 50ml of reagent A mixed with 1ml of reagent B (always freshly
prepared).
5. Reagent D:- Folin phenol Reagent prepared in a ratio of 3:1 (i.e. 3ml of water
1ml of Folin phenol reagent). Always freshly prepared.
The assay mixture for protein estimation was comprised of 0.1 ml of protein
extract, 0.4 ml of distilled water. To the above mixture 5ml of reagent was added
& mixed rapidly & allowed to stand for 10 minutes at room temperature. Then 0.5
ml of reagent D to was added. These were mixed and allowed to stand at room
temperature for 10 minutes. A blank was prepared by taking 0.5 ml of distilled
water instead of plant sample. The absorbance of the developed colour was read at
750 nm. Protein content was calculated by comparing the absorbance of the protein
extract with standard curve calibrated with 10-100µg solution of Bovine serum
albumin (taken as standard).
E Extraction and estimation of total sugar
One gram of leaf sample was taken & thoroughly washed in 80% alcohol using
mortar pestle to make volume up to 10ml. then it was transferred to test tube and
incubated at 600C in a water bath for 15 minutes and cooled and centrifuge at
5000rpm for 10 minutes. Supernatant decanted in to test tube. Process repeated 2-3
times to extracting soluble sugar completely. Supernatant used for total sugar
estimation.

14. The total soluble sugar estimation was done following the anthrone reagent
method of Archwell (1957).
The anthrone reagent was prepared as follows :
Anthrone reagent:- 2 gm of anthrone was dissolved in 1litre of concentrated
H2SO4. The solution was prepared freshly.
The assay of the total sugar with anthrone reagent was performed as follows:
2 ml of anthrone reagent was taken in a wide mouth tube & was chilled in ice bath.
Then 1ml of sugar extract solution was layered carefully over the reagent & was
allowed to cool by keeping in ice bath. Tubes were then shaken vigorously while
still immersed in ice bath. The reaction mixture in tubes were brought to room
temperature and boiled for 20 minutes. After cooling, absorbance was taken at
625nm. Total soluble sugar was calculated by comparing the absorbance of total
soluble sugar extract from the plant with standard curve which was prepared with
10-50µg of D glucose.
Literature Review
T ANAND*, R.BHASKARAN, T.RAGUCHANDER, R.SAMIYAPPAN,
V.PRAKASAM and C.GOPAL KRISHNAN from department of plant pathology,
centre for plant protection studies, Tamil Nadu, Agriculture University Coimbatore
India conducted an experiment defence responses of chilli fruits to Colletotrichum
capsici and Alternaria alternata .They found that, total phenols and activity of

15. phenylalanine ammonia lyase (PAL), peroxidase(PO), polyphenol oxidase (PPO)
and catalase (CAT) increased in the inoculated ripe and green chilli fruits
compared to corresponding healthy chilli. Total phenols and activities of the
enzymes were at maximum 2-3day after inoculation and thereafter declined
sharply in ripe chilli fruits, whereas slowly in green chilli fruits. In comparison
with ripe chilli fruits, green chilli fruits were more resistant as they showed higher
accumulation of total phenols & also higher activities of enzymes.
Eui Hwan Jungal, Ho Won Jungal, Sung chul leea, Sang wook Hana,
Sunggi Heub, Byung Kook, Hwanga*
a. Laboratory of molecular plant pathology, college of life and environmental
sciences, Korea University, Anam-dong, South Korea.
b. Division of plant pathology, National Institute of Agricultural Science and
Technology Rural Development Administration, Suwon, South Korea
identified a novel pathogen-induced gene encoding a leucine-rich repeat
protein expressed in phloem cells of Capsicum annum.
The CALRR1 gene, expressed in pepper leaves following infection by
Xanthomonas campestris pv Vesicatoria , encodes a secreted luecine-rich repeat
(LLR) with five tandem repeats of a 24 amino acid LRR motif. Northern blot
analysis revealed that CALRRI is not constitutively expressed in pepper plant, but
is strongly induced upon the infection by X.campestris pv. Vesicatoria,

16. Phytophthora capsici,Colletotrichum coccodes and Colletotrichum
gloeosporioides leaves.
Morphological, pathological and molecular variability of Colletotrichum
capsici causing anthracnose of chilli in the north-east of Thailand was studied by
Aphidech Sangdee*, Sarawut Sachan and Surasak Khankhum Department of
Biology, Mahasarakham University, Thailand. Anthracnose disease is one the
major economic constraints to chilli production in tropical and subtropical regions.
Ten isolates of Colletotrichum capsici causing chilli anthracnose were collected
from 10 provinces in the north-east of Thailand. The isolates were evaluated for
their morphological and cultural characteristics, pathogenic variability on chilli
fruits and genetic characterization using random amplified polymorphic DNA.
Based on the morphological fruits and cultural characteristics of the C. capsici
population, 10 isolates were categorized in to six groups.
These are designated as cc-I, cc-II, cc-III, cc-III, cc-IV and cc-V respectively. In
Potato Dextrose Agar culture, most of the isolates produced cottony colonies.
However, differences were obtained in colony colour, shape and size of conidia.
Based on the effect of carbendazim, 10 isolates were classified in to two groups
designated as highly resistant group and highly sensitive group. Three virulence
degrees of 10 isolates on chilli fruits were evaluated.
Chilli anthracnose disease caused by colletotrichum species studied by Po Po
Than, Haqryudian prihastuti, Sitthisack phoulivong. Anthracnose disease is one of

17. the major economic constraints to chilli production worldwide, especially in
tropical and subtropical regions. In the colletotrichum patho-system, different
colletotrichum species can be associated with anthracnose of the same host.
Although the management and control of anthracnose disease are still being
extensively researched, commercial cultivars of Capsicum annum that are resistant
to the pathogens that cause chilli anthracnose have not yet been developed. This
paper reviews the causal agents of chilli anthracnose, the disease cycle,
conventional methods in identification of the pathogens and molecular approaches
that have been used for the identification of colletotrichum species. Pathogentic
variation and population structure of the causal agents of chilli anthracnose along
with the current taxonomic status of Colletotrichum species are discussed.