Biological Indexing and Molecular Approaches in Detecting a Mild Strain ‘CRS 4’ against Citrus tristeza Virus in Khasi Mandarin (Citrus reticulata)

Abstract

Citrus tristeza virus, the most important viral disease of citrus, is reported in Assam and other NE states of India to infect Khasi mandarin (Citrus reticulata), the most economically important citrus crop of the region. For effective management, an attempt was made to identify a potential mild isolate against virus. Leaf samples were collected from Khasi Mandarin plants expressing differential symptoms from three different locations viz., Tinsukia, Golaghat and Mariani of Upper Brahmaputra Valley Zone of Assam. These were then grouped into three categories based on ELISA OD 405 values. Biological indexing with CTV positive samples from these three serological categories on Mexican lime (Citrus aurantifolia) seedlings resulted in symptom expression within three months post grafting. Visible symptoms of CTV infection were observed in some of the graft successful indicator plants whereas, in Khasi mandarin selection ‗CRS 4', no visible symptom development took place within this period. Based on the results, the plants were grouped into two groups-symptom producing and nonsymptom producing, and were confirmed through Bi-directional RT-PCR with mild and severe strain primers. PCR products for ‗CRS 4' were sequenced. Consensus sequences showed a single nucleotide difference at position 371 for mild isolates (CRS 4), thereby confirming the identity.