Molecular diagnosis of Citrus tristeza virus in mandarin (Citrus reticulata) orchards of Darjeeling hills of West Bengal

Citrus tristeza virus (CTV) and its vector, brown citrus aphid (BrCA) Toxoptera citricidus are widely distributed in the mandarin (Citrus reticulata) orchards of Darjeeling hills of India. The BrCA incidence upto 32.5% and severity upto 27.2% were recorded. Under field conditions, diagnosis of CTV in Darjeeling mandarin trees is rendered very difficult as no visible symptoms develop. About 16% to 90% of mandarin samples collected from 15 areas were found to be infected by CTV using ELISA with polyclonal antisera to CTV. The mandarin trees of all ages from 10 to 25 yrs at varying altitudes from 500 to 1540 m were found to be similarly infected by CTV. Infections of CTV in kagzi lime (Citrus aurantifolia), sweet orange (C. sinensis) and pumello (C. grandis) in this region were also detected. The citrus species, Rangpur lime (C. limonia), rough lemon (C. jambhiri), trifoliate orange (Poncirus trifoliata) and hybrid trifoliate (Citrange) were found to be free from CTV. Kagzi lime indicator plants when graft inoculated with test samples develop mild to severe vein clearing and vein flecking symptoms characteristic to CTV infection. Electron microscopy (EM) showed long flexuous particles of CTV from inoculated symptomatic kagzi lime plant. The DAC-ELISA and RT-PCR effectively detected CTV in infected trees; the details of which are presented.