DEVELOPMENT OF POLYCLONAL ANTIBODY-BASED IMMUNOLOGICAL FORMATS FOR PATHOGEN DETECTION, DIAGNOSIS AND ESTIMATION IN TEA LEAF TISSUES
DEVELOPMENT OF POLYCLONAL ANTIBODY-BASED IMMUNOLOGICAL FORMATS FOR PATHOGEN DETECTION, DIAGNOSIS AND ESTIMATION IN TEA LEAF TISSUES BN CHAKRABORTY, U CHAKRABORTY, A SAHA, S GUHA, R DAS and P BASU Immuno-Phytopathology Laboratory, Department of Botany, North Bengal University-734430, India Background and objective Tea is one of the important cultivated perennial crops in India, which is the most widely consumed hot beverages in the world. Darjeeling produces the world's finest quality tea in the steep hill slopes of Eastern Himalayas. Some of the most important foliar fungal diseases of tea such as blister blight, black rot, brown blight and grey blight caused by Exobasidium vexans, Corticium invisum,Glomerella cingulataand Pestalotiopsis theae respectively are responsible for considerable economic losses. Early detection and proper diagnosis of these diseases become necessary for their management. Attempts have been made to develop diagnostic kits for four foliar fungal pathogens of tea using various formats of immunoassays and immunofluorescence. Materials and methods Serology - Antigens were prepared from healthy and artificially inoculated (separately with E.vexans,C.invisum,G.cingulata,P.theae) tea leaves of thirty seven clones available at Tea Germplasm Bank, Department of Botany, North Bengal University and fungal mycelia. Purification of antigen was done by saturated ammonium sulphate fractionation and ion exchange chromatography. Polyclonal antisera were raised in separate male white rabbits against mycelial antigens of C.invisum,G.cingulata and P.theae while polyspecific antisera was prepared for E.vexans.immunoglobulin fractions of antisera were purified [ 1 ]. ELISA - Three ELISA formats such as DAC, DAS and Competition were set up for pathogen detection, disease diagnosis and estimation of fungal biomass. lmmunofluorescence - Indirect fluorescence staining of mycelia, conidia and sclerotia of foliar pathogens of tea were done using FITC labelled goat antirabbit IgG [ 2 ]. Results and conclusion Polyclonal antibodies raised against four foliar fungal pathogens of tea have been packaged into ELISA formats for the quick and accurate detection of specific tea pathogens. Differences in ELISA readings between healthy and infected ( artificially or naturally infected ) tea leaf antigens indicates the measure and extent of infection.Concentration of leaf antigens and dilution of pathogen antisera were 40 µg/ml and 1:125 respectively. Absorbance values for artificially inoculated (separately with E.vexans, C.invisum,G.cingulataand P.theae)leaf antigen preparations ( 72h after inoculation ) of all varieties were higher than their respective healthy leaf extracts in both the ELISA-formats ( DAC and DAS ).Pathogens in infected tissues could be detected before appearance of symptoms - as early as 6h after inoculation using DAC-ELISA formats. When antigen dilution ranging from 40 - 1 µg/ml were tested in ELISA, pathogen could be detected in infected leaf extract at a concentration as low as 1 µg/ml. Since very often, brown blight and grey blight occur as part of mixed infection, detection of specific pathogen becomes difficult. Using the format of competition ELISA , major pathogen in the natural infection could be confirmed. There were about 80% cross reactivity between nine isolates of G.cingulata,85-90% cross reactivity between seven isolates of P.theae as measured by DAC-ELISA. The utility of the ELISA was also demonstrated by measuring the growth of G.cingulata,P.theae,C.invisumand E.vexansin tea leaf tissues. Based on an immunofluorescence assay presence of fungal hyphae or conidia on leaf tissues were also detected. The advantages of this assay are its sensitivity and specificity. These methods have enabled the screening and ascertaining of the disease status of the tea clones, and to select the resistant clones with considerable certainty. Disease prediction well in advance has also made it possible to take remedial measures in time. References 1.Chakraborty BN, Basu P,Das R, Saha A,Chakraborty U,1995.Annals of Applied Biologyl27,11-21. 2.Chakraborty BN, Saha A, 1994. Physiological and Molecular Plant Pathology 44, 403-416.