Introduction      Plants survive with a variety of strategies to defend themselves againstpathogen. Plants are major targe...
Plants defense responses are induced by microbial products in non host andhost resistance plant defense systems are also i...
host-protein toxin should result immunity. Thus it becomes clear that resistancemuch have of pathogens against host defens...
made to compare and quantitatively analyze the induction of defense compoundand enzyme in green and ripe chilli fruits ino...
A various biochemical characters also affect die back/fruit rot intensively inchilli such as total phenol, total sugar, ca...
Sterilization of Seeds        The seeds were sterilized with sterile distilled water and then kept in 0.1%Hgcl2 for 30 sec...
Colletotrichum capsiciScientific classificationKingdom      FungiPhylum      AscomycotaClass       SordariomycetesOrder   ...
by C. coccodes and C. dematium. Leaf anthracnose of chilli seeding caused byC.coccodes was first reported in chilli growin...
Distilled water                     1000ml      pH                                  6.0-6.6The seeds were first washed wit...
Experimental Design The seeds were sown on the soil in pots. Three types of seeds i.e. sterilized,unsterilized and seeds s...
Study of Growth Parameters of PlantsThe growth of plant was observed from times of seedling to onward, Growth ofplant in t...
Chlorophyll b = (22.9xA645-4.68xA645)xV/1000wTotal Chlorophyll = (8.02xA663+20.2xA645)V/1000wEstimation of carotenoid was ...
Reagent C- 50ml of reagent A mixed with 1ml of reagent B (always freshlyprepared).5. Reagent D:- Folin phenol Reagent prep...
The total soluble sugar estimation was done following the anthrone reagentmethod of Archwell (1957).The anthrone reagent w...
phenylalanine ammonia lyase (PAL), peroxidase(PO), polyphenol oxidase (PPO)and catalase (CAT) increased in the inoculated ...
Phytophthora       capsici,Colletotrichum       coccodes     and      Colletotrichumgloeosporioides leaves.     Morphologi...
the major economic constraints to chilli production worldwide, especially intropical and subtropical regions. In the colle...
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Niharika

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Niharika

  1. 1. Introduction Plants survive with a variety of strategies to defend themselves againstpathogen. Plants are major target of microbe seeking a source of nutrition acomplex array of interactions between plants and microbes evolved that reflectsboth the nutrient a acquisition strategies of microbes and defense strategies ofplants. Part of plant defense strategies includes an active offense against invadingmicrobes using an array of antimicrobial gene products within the context of theoverall plant microbe interaction, we attempt here to emphasize the role ofantimicrobial proteins and peptides in plant defense. The majority of plant microbe encounters don’t results in disease. Preformedfactors including constitutively expressed waxes cell wall components.,antimicrobial peptides, proteins and non proteinaceous secondary metabolites thatdeter invasion have been proposed to contribute significantly to the host range ofpathogens. The importance of preformed defenses has been inferred from theobservation that plants can be rendered susceptible by a deficiency in theproduction of these secondary metabolites or by the abilities of pathogens todegrade them.Inducible Defenses Require detection of pathogens by lost surveillance. Thesentinels
  2. 2. Plants defense responses are induced by microbial products in non host andhost resistance plant defense systems are also in induced by microbial products incompatible and incompatible plant microbe interaction.Specific host- pathogen interaction models describing induced defense responses inplants have been greatly influenced in recent years gene for gene interactionoriginally reported by floor (1956) in these specific host pathogen interactionsresistance to a particular pathogen is conditional on the presence of specific Avr(avirulence )gene of the pathogen and specific (R) resistance gene in plant hostwide spread interest in gone for gene interaction resulted for recognizing theresistance was usually controlled by single dominant genes making geneticanalysis very detectable.Antimicrobial proteins and peptides are important components of innateimmunity A common feature of the innate immune system of taxonomically diverseorganism i.e. plant is the ability to marshal the accumulation of antimicrobialproteins and peptides in response to an invasive challenge by foreign organismmore than 500 different antimicrobial proteins and peptides encoded within thegenomes of many organism including plants. Genes encoding these proteinpeptides have an important role in host-pathogen interaction. Much less certain isspecific function of each in individual pathogen-plant interaction. In view of thisknowledge gap we emphasize that full susceptibility of any given pathogen to only
  3. 3. host-protein toxin should result immunity. Thus it becomes clear that resistancemuch have of pathogens against host defense toxins must be widespread andimportant to disease development. Chilly (Capsicum annuum) is an important vegetable crop grown throughoutthe world an alkaloid, capsaicin is extracted from chilly, which has medicinalvalue. Plants are able to defend themselves successfully with a complex set ofpreformed structures and inducible reactions. The inducible reactions require theperception of either plant derived (endogenous) or pathogen derived (exogenous)signal molecule. Chitin and glucan oligomers released during degradation of fungal cell wallact as elicitors that elicit various defense mechanisms in the plants.High expressionof these defense enzymes in crop plants can enhance resistance to a pathogen.When a pathogen lands on a host surface, it activates the hosts defensemechanisms probably by releasing elicitors from its cell walls plants are endowedwith defense genes, which are quiescent in healthy plants, but when activated byvarious factors they induce systematic diseases resistance. Defense enzymes thatare induced in response to invasion by a pathogen have greater antifungal activityto the pathogen in suppressing symptoms induction of defence enzymes makes theplant resistant to pathogen invasion. Expression of these protein is correlated withthe development of systemic acquired resistance in plants.Thus if mechanisms aretriggered quickly and maintained enhanced level due to infection by a plantpathogen disease severity can be reduced. In the present study an attempt was
  4. 4. made to compare and quantitatively analyze the induction of defense compoundand enzyme in green and ripe chilli fruits inoculated with colletotrichum capsci.ANTHRACNOSE disease is one of the most serious fungal pathogens of chilli inAsia. Anthracnose deriving from a Greek word meaning coal is the common namefor plant diseases characterized by very dark sunken lesion containing spores. Thedisease appears as small circular spots that coalesce to form large elliptical spotson fruits and leaves. Under severe conditions ,defoliation of affected plant occurs. Typical fruit symptoms are circular or angular sunken lesions withconcentric rings of acervuli that are often wet and produce pink to orange conidialmass. This disease produces symptoms on leaves, stem pre & post harvest fruitsand causes severe damage to mature fruits in the field. More over during transitand storage this disease are also causes serve damage to chilli fruits. The virulencedegree of disease symptoms host plants reported the existence of 15 pathogens ofC. capsici based on disease system development on inoculated fruit of capsicumannuum. There was a significant amount of variation at physiological andbiochemical levels present in chilli gene types for resistance to fruit rot/die backcolletotrichum. The resistance chilli genes types exhibited more physiologicalcharacteristics as compared to susceptible genotypes.
  5. 5. A various biochemical characters also affect die back/fruit rot intensively inchilli such as total phenol, total sugar, capsaicin, ascorbic acid and protein contentthat impart resistance fruit. Red ripe chilli fruits were more susceptible to fruit rot as compared to semiriped ones. A various biochemical and physiological characters play specific rolein imparting resistance against disease. The resistant chilli genotypes were high incapsaicin and orthohydroxys phenol and exhibited more plant length days topicking as compared to susceptible ones. The other characters which had a positive and direct effect on fruit rot, widthfruit number per plants, stem, thickness, fruit weight per plant, leaf area and, carotenes where as total flavonols, ascorbic acid, total sugar content had a direct andnagative effect on dieback/ fruit rot. A various physiological and biochemical traitsthat varied significantly among resistance and susceptible genes type can bedeployed in resistant breeding or as physiological and biochemical.Materials and MethodsPlant material and Collection of seeds The plant material was taken chilli plants .The collection of seeds were donefrom the OUAT( Orissa University of Agriculture and Technology ) Bhubaneswar The plant materials were obtained by germinating the seeds of chilly. Thehealthy and non diseased seeds were taken.
  6. 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 inpetriplates layered with moist cotton and also sterilized seeds were sown on soil inpots.DETECTION OF PATHOGEN FROM SEEDS Colletotrichum capsici is both internally and externally seed borne sucheffected seed and infected seeding act as primary sources of inoculum.Blotter methods Seed borne colletotrichum capsci can be detected by subjecting chilli seedsamples to the standard blotter test. Individual samples of ten seeds are plated inpetriplates.. Plates were placed in petriplates. Plates were placed on three layeredmoist blotter paper plates. On the fifth day of the seeds were observed for thepresence of fungal growth with the help of microscope. Colletotrichum capsici isidentified based on morphological characters of acervuli and conidia growing onthe blotter paper.Casual Organism
  7. 7. Colletotrichum capsiciScientific classificationKingdom FungiPhylum AscomycotaClass SordariomycetesOrder GlomerellalelsFamily GlomerellaceaeGenus ColletotrichumSpecies capsici Colletotrichum is one of the most important plant pathogens world widecausing the economically important disease anthracnose in a wide range of hostsincluding cereals, legumes, vegetables, perennial crops and tree fruit. Among thesehosts, chilli, an important economic crop worldwide is severely infected byanthracnose which may cause yield losses of up to 50%. In the colletotrichum pathosystem, different colletotrichum species can beassociated anthracnose of the same host. Different species cause disease ofdifferent organs of the chilli plant. For example – C. acutatum and C. gloeosporides infect chilli fruits at alldevelopmental stages but usually not leaves or stems. Stems or leaves are damaged
  8. 8. by C. coccodes and C. dematium. Leaf anthracnose of chilli seeding caused byC.coccodes was first reported in chilli growing in a field in chungam province ofKorea in 1988. Different colletotrichum species may also play an important role indifferent disease of mature stages of chilli fruit. For example- C. capsici is wide spread in red chilli fruits whereasC.acutatum , C. gloeosporioides have been reported to be more prevalent bothyoung and mature green fruits.Pathogen isolation, culture, maintenance The anthracnose disease causing Pathogen colletotrichum capsici wasisolated from a diseased seed of chilly which was provided by Department of Plantpathology, 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 givenon the containers, in aseptic conditions with sterilized glass waves & inoculationloops.POTATO DEXTROSE AGAR MediumComponent Amount Peeled potato 250gm Glucose 20.00gm Agar 15gm
  9. 9. Distilled water 1000ml pH 6.0-6.6The seeds were first washed with sterile distilled water and then surface sterilizedwith 0.1% Hgdl2 for 30 seconds followed by washing sterile distilled water in threetimes. Then the leaves were placed on the PDA plated with aseptic condition inlaminar airflow and incubated at 280C for 48 hours. After 48 hours of incubationthe plates were observed to be containing the colonies of fungal mycelia on thePDA plates showing the typical colony morphology & growth symmetry. All theprocess 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 DextroseBroth & maintained in incubator at 280C. Two of well grown subcultures plateswere stored at 40C as master plates & were revived monthly. These processes ofsub culturing and maintenance were done in aseptic condition.Preparation Of Spore Suspension From the well grown culture plates the development of spore was seen withthe help of electron microscope. The typical structure spore can been seen andidentified. The Potato Dextrose broth with the help of a brush & inoculation loopunder aseptic conditions in laminar air flow & incubated at 280C.
  10. 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 helpof chips of pots. Soil was mixed with biofertilizer and vermicompost and mixedwell.Then seeds were sown in respective pots. Regular watering and observation wasdone and also sterilized, unsterilized & spore suspension seeds were kept inpetriplates with layered moist cotton to know the seed germination and affect ofgrowth of causal organism.Germination PercentageGermination Percentage of chilly was determined by counting the no. ofgerminated seeds out of total inoculated seeds & following formula was used tocalculate the germination percentage. % of Germination = Total no. of germinated seeds/ Total no. of seeds inoculatedGermination Percentage of sterilized, unsterilized and spore inoculated seeds wereobserved.
  11. 11. Study of Growth Parameters of PlantsThe growth of plant was observed from times of seedling to onward, Growth ofplant in terms of its height, leaves was measured.Measurement of Physiological & Biochemical ParametersPlant height, stem thickness, no. of leaves & leaf area. At maturity, the height wasmeasured 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 areaof plant was also calculated with graph paper.Biochemical ParametersExtraction & estimation of chlorophyll and carotenoidsEstimation of PigmentThe leaf samples from each pot experiment of healthy and unhealthy were takenfor chlorophyll & carotenoid estimation. 500mg of leaf sample was taken &grinded in motor and pestle by adding 10ml of cold 80% acetone. The mixture wascentrifuged at 2000rpm for 10-15 minutes. The supernatant was taken formeasuring absorbance at 480,663 & 645nm. Chlorophyll a, chlorophyll b and totalchlorophyll content was calculated by using Arnon’s formula (1949).Chlorophyll a = (12.7xA663-2.69xA645)xV/1000w
  12. 12. Chlorophyll b = (22.9xA645-4.68xA645)xV/1000wTotal Chlorophyll = (8.02xA663+20.2xA645)V/1000wEstimation of carotenoid was done using the formula of Kirk and Allen (1965).Acar /480per leaf segment = (A480+0.114A663-0.638xA645)x V/1000wWhere A480, A663 & A645 are the absorbance of extract at 480nm, 645nm & 663nmrespectively.V= volume of acetone extractw= weight of leaf (gram fresh weight)Extraction and Estimation of Protein1gm of plant samples (leaf) were homogenized with cold phosphate buffer (pH7.4) 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 20minutes. 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. 13. Reagent C- 50ml of reagent A mixed with 1ml of reagent B (always freshlyprepared).5. Reagent D:- Folin phenol Reagent prepared in a ratio of 3:1 (i.e. 3ml of water1ml of Folin phenol reagent). Always freshly prepared.The assay mixture for protein estimation was comprised of 0.1 ml of proteinextract, 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 0.5ml of reagent D to was added. These were mixed and allowed to stand at roomtemperature for 10 minutes. A blank was prepared by taking 0.5 ml of distilledwater instead of plant sample. The absorbance of the developed colour was read at750 nm. Protein content was calculated by comparing the absorbance of the proteinextract with standard curve calibrated with 10-100µg solution of Bovine serumalbumin (taken as standard).E Extraction and estimation of total sugarOne gram of leaf sample was taken & thoroughly washed in 80% alcohol usingmortar pestle to make volume up to 10ml. then it was transferred to test tube andincubated at 600C in a water bath for 15 minutes and cooled and centrifuge at5000rpm for 10 minutes. Supernatant decanted in to test tube. Process repeated 2-3times to extracting soluble sugar completely. Supernatant used for total sugarestimation.
  14. 14. The total soluble sugar estimation was done following the anthrone reagentmethod of Archwell (1957).The anthrone reagent was prepared as follows :Anthrone reagent:- 2 gm of anthrone was dissolved in 1litre of concentratedH2SO4. 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 & wasallowed to cool by keeping in ice bath. Tubes were then shaken vigorously whilestill immersed in ice bath. The reaction mixture in tubes were brought to roomtemperature and boiled for 20 minutes. After cooling, absorbance was taken at625nm. Total soluble sugar was calculated by comparing the absorbance of totalsoluble sugar extract from the plant with standard curve which was prepared with10-50µg of D glucose.Literature ReviewT 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 CoimbatoreIndia conducted an experiment defence responses of chilli fruits to Colletotrichumcapsici and Alternaria alternata .They found that, total phenols and activity of
  15. 15. phenylalanine ammonia lyase (PAL), peroxidase(PO), polyphenol oxidase (PPO)and catalase (CAT) increased in the inoculated ripe and green chilli fruitscompared to corresponding healthy chilli. Total phenols and activities of theenzymes were at maximum 2-3day after inoculation and thereafter declinedsharply in ripe chilli fruits, whereas slowly in green chilli fruits. In comparisonwith ripe chilli fruits, green chilli fruits were more resistant as they showed higheraccumulation 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 byXanthomonas campestris pv Vesicatoria , encodes a secreted luecine-rich repeat(LLR) with five tandem repeats of a 24 amino acid LRR motif. Northern blotanalysis revealed that CALRRI is not constitutively expressed in pepper plant, butis strongly induced upon the infection by X.campestris pv. Vesicatoria,
  16. 16. Phytophthora capsici,Colletotrichum coccodes and Colletotrichumgloeosporioides leaves. Morphological, pathological and molecular variability of Colletotrichumcapsici causing anthracnose of chilli in the north-east of Thailand was studied byAphidech Sangdee*, Sarawut Sachan and Surasak Khankhum Department ofBiology, Mahasarakham University, Thailand. Anthracnose disease is one themajor economic constraints to chilli production in tropical and subtropical regions.Ten isolates of Colletotrichum capsici causing chilli anthracnose were collectedfrom 10 provinces in the north-east of Thailand. The isolates were evaluated fortheir morphological and cultural characteristics, pathogenic variability on chillifruits and genetic characterization using random amplified polymorphic DNA.Based on the morphological fruits and cultural characteristics of the C. capsicipopulation, 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. InPotato 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 groupsdesignated as highly resistant group and highly sensitive group. Three virulencedegrees of 10 isolates on chilli fruits were evaluated. Chilli anthracnose disease caused by colletotrichum species studied by Po PoThan, Haqryudian prihastuti, Sitthisack phoulivong. Anthracnose disease is one of
  17. 17. the major economic constraints to chilli production worldwide, especially intropical and subtropical regions. In the colletotrichum patho-system, differentcolletotrichum species can be associated with anthracnose of the same host.Although the management and control of anthracnose disease are still beingextensively researched, commercial cultivars of Capsicum annum that are resistantto the pathogens that cause chilli anthracnose have not yet been developed. Thispaper reviews the causal agents of chilli anthracnose, the disease cycle,conventional methods in identification of the pathogens and molecular approachesthat have been used for the identification of colletotrichum species. Pathogenticvariation and population structure of the causal agents of chilli anthracnose alongwith the current taxonomic status of Colletotrichum species are discussed.

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