Microscopic and polyclonal antibody-based detection of yellow leaf disease of arecanut (Areca catechu L.)

Abstract

Phytoplasma, the pathogen of yellow leaf disease (YLD) of arecanut (Areca catechu L.) was detected by transmission and scanning electron microscopy. Tissues of YLD affected palms contained phytoplasmas in the phloem sieve elements, but not in symptomless healthy palm tissues. Phytoplasma was purified from tissues of diseased palms employing percoll density gradient centrifugation and confirmed by transmission electron microscopy. Using the purified phytoplasma preparation, a polyclonal antiserum was raised in rabbits and used for standardisation of agar gel double diffusion (Ouchterlony) test and DAC-ELISA. Clear precipitin line was observed in Ouchterlony test between the antigen from diseased palms and the pathogen-specific antibodies after 48 h incubation and only undiluted antiserum showed best result in the test. However, in ELISA, 1:10 antigen dilution and 1:400 pathogen-specific antibodies dilution produced sensitive detection of the pathogen with a difference of >3.5 times absorption values between healthy and diseased samples. The results thus confirmed the ability of antiserum to distinguish healthy and infected plants and utility of ELISA for effective diagnosis of YLD.