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Researcher: Sharma, Ms Meenakshi
Guide(s): Kulshreshtha, Dr Saurabh
Keywords: C. gloeosporioides
Inverse PCR
University: Shoolini University of Biotechnology and Management Sciences
Completed Date: 06-05-2015
Abstract: newline ABSTRACT newlineColletotrichum gloeosporioides is an ubiquitous pathogen and widely distributed in tropical and newlinesubtropical regions than temperate regions. The various infected leaves samples were collected newlinefrom different places of H.P and causative agent was isolated from five different samples, collec- newline-ted from Dhaula kuan, Shimla, Solan, Chandigarh. The causative agent of anthracnose disease newlinewas found to be C. gloeosporioides by morphological characterization. DNA of all isolates was newlineisolated and all isolates were confirmed by amplification of their ITS region with ITS region newlinespecific primers of C. gloeosporioides. The product of 450bp was obtained and sequenced, which newlinefurther confirms the identity of fungus as C. gloeosporioides. The morphological study of newlinepathogen was also done using different growth media such as PDA, lima bean agar, oat meal newlineagar, malt extract agar. Out of all tested media, the oatmeal agar media was found to be best newlinemedia for growth and sporulation of pathogen. Random hygromycin resistant mutants were gen- newline-erated by using REMI method. Generated mutants were selected on PDA plate containing newline50and#956;g/ml hygromycin and mutants were single spored to get pure colony. Confirmation of newlinemutants was done by ITS region specific primers of C. gloeosporioides and by hygromycin gene newlinespecific primers which confirms that all the mutants were C. gloeosporioides and were true newlinetransformants. Southern hybridization was also performed to check gene integration in mutants. newlineThe effects of mutation on morphology of mutants were also detected by two sets of experiment newlinein triplet such as: spore germination and spore penetration by using 104/spore /ml suspension. newlineExcept mutants 2.3, B27, C80 and H6 which showed more spore germination than wild type, all newlineother mutants showed less spore germination. Spore penetration was less in all mutants as newlinecompared to the wild type. Pathogenesis assay was done by inoculating 106spores/ml suspension newlineof mutants and wild type on different detached leaves/fruits/vegetables. Mutants showed reduced newlineinfection as compared to wild type. But mutant 2.2 showed more infection as compared to other newlinemutants but less in comparison to wild type. Inverse PCR was performed with three set of nested newlineprimers in order to check tagged gene in mutant s genome. In case of mutant H4, site of newlinehygromycin gene integration occur at 1095 bp in diacylglycerol acyltransferase. This gene is newlineinvolved in triglycerolacyl glycerol and size of this gene is 1986bp.
Pagination: vii,137
Appears in Departments:Faculty Of Biotechnology

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