MOLECULAR DETECTION OF FUNGAL PATHOGENS OF CITRUS RETICULATE GROWN IN DARJEELING HILLS AND THEIR MANAGEMENT

Mandarin orchards in Darjeeling hills are recently showing severe signs of citrus decline caused by Macrophomina phaseolina and Fusarium solani resulting in decreased fruit production. Polyclonal antibodies (PAbs) were raised against mycelial antigens of M. phaseolina and F. solani; IgG were purified and further packaged into immunological formats using PTA-ELISA, dot blot, western blot and immunofluorescence for quick and accurate detection of pathogens from soil and mandarin root tissue. Indirect staining of mycelia and sclerotia of M. phaseolina as well as conidia of F. solani with homologous PAb and labeling with goat antirabbit IgG conjugated with FITC developed strong fluorescence in young hyphal tips, conidia and sclerotia of pathogens. Besides, PCR based molecular detection of pathogens have also been developed. Genomic DNA from M. phaseolina, F. solani and twenty five Trichoderma isolates were prepared, purified and PCR amplification of 18S rDNA were done using ITS region specific primer pair keeping annealing temperature of 59°C with good product yield and minimum nonspecific amplification. The product size was approximately 570 bp for Fusarium, 620 bp for Macrophomina and 600 bp for Trichoderma with the size variation across the isolates. RAPD profile was obtained using six random decamers from Operon technology kit [OPA1, OPA4, OPB2, OPB3, OPB6 and OPD5] for Fusarium and Macrophomina, while A-5, AA-11 and AA-04 random primers specifically for Trichoderma. All reproducible polymorphic bands were scored and analysed following UPGMA cluster analysis protocol and computed into similarity matrix using NTSYS computer programme. A dendogram was made with similarity coefficient ranged from 0.67 to 0.95. In vitro screening of all these Trichoderma isolates were made for evaluation of their antagonistic activity against M. phaseolina and F. solani and further evaluated in vivo on the development of root rot disease of mandarin under green house as well as field conditions.