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Subject: Plant Pathology × Author: Baruah, Borsha Rani × End date: 2019 × Start date: 2016 × Type: Thesis × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    Evaluation of protective strains for cross-protection against Citrus tristeza virus disease
    (2019-07) Baruah, Borsha Rani; Nath, P. D.
    For Citrus tristeza virus strains differentiation and identification, leaf samples were collected from Khasi Mandarin (Citrus reticulata) 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, viz. low range, medium range and high range based on ELISA OD405 values. Biological indexing with CTV positive samples from these three serological categories on Mexican lime or Kaghzi 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 some plants, no visible symptom development took place within this period. Based on the results, the plants were grouped into two groups- mild and severe, and were confirmed through Bi-directional PCR with mild and severe strain specific primers. PCR products for both mild and severe isolates were sequenced. Consensus sequences showed a single nucleotide difference at position 371 for mild and severe isolates, thereby confirming the identity. Two mild isolates and two severe isolates were selected for the cross protection experiment on Khasi mandarin seedlings. Cross protection experiment was carried out in seven treatments with five replications. Real time-PCR analysis of the grafted plants was carried out six months post grafting to determine the virus titre values through absolute quantification by developing an external standard curve with the equation ‗y = -3.0543x + 37.018‘ (R2 = 0.9981), using 6 serial dilutions of eluted DNA (10-1 to 10-6). The average estimated gRNA copies were 1.02 x 105 + 8.33 x 103 mol/ng and 9.55 x 104 + 5.89 x 103 mol/ng of total RNA in mild inoculated plants, 3.80 x 105 + 3.05 x 104 mol/ng and 3.94 x 105 + 3.53 x 104 mol/ng in severe inoculated plants and, 2.20 x 104 + 2.14 x 103 mol/ng and 3.44 x 104 + 3.20 x 103 mol/ng in mild inoculated plants, later challenged by severe, with no observed amplification in the un-inoculated control. It was observed that the mild inoculated plants, challenged by severe i.e. cross protected plants exhibited the lowest virus copy numbers. Melting curve analysis revealed that the Tm of the mild CTV isolates (80.37–80.820C) was lower than that of the severe isolates (81.57–81.970C). In the cross protected plants, Tm in the range of 80.73-80.880C was recorded, indicating the presence of mild isolates.