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Citrus tristeza virus (CTV) is a plant pathogenic virus belonging to the genus Closterovirus and family Closteroviridae. It is transmitted by vegetative propagation and by several aphid species. It has been reported that CTV has killed millions of citrus trees worldwide. CTV has previously been reported in Pakistan. A survey was carried out in the year 2014 and 1260 random samples were tested from six districts of Punjab and seven districts of Khyber Pakhtunkhwa (KP). Results from DAS-ELISA revealed incidence of 28.3 percent in Punjab and 30.8 percent in KP. Seventy symptomatic samples from both the provinces were collected and tested through DAS-ELISA. The major coat protein coding gene of CTV from forty-eight ELISA positive samples were amplified, cloned into pGEM®-T Easy vector, sequenced and phylogenetic analysis was carried out. Nucleotide sequence analysis revealed 90-100% similarity within indigenous forty-eight isolates, 91.1 to 100 percent similarity with six isolates previously reported from Pakistan and 97.7 to 99.7 percent similarity with T3 USA, VT USA, VT Israel, RB New Zealand, VT India and an Indian isolate of unknown genotype. A maximum likelihood phylogenetic tree indicated that CTV population is diverse in Pakistan with different isolates consisting of one major isolate, T3, and three minor isolates, VT, RB, and VT IND. The most common group (T3 like) is comprised of 42 Pakistani isolates including 36 of our samples and is dispersed all over the country irrespective of the region and province. The remaining three groups are related to VT Israel, VT India and RB New Zealand confined to specific regions. One coat protein gene sequence from the major clade were selected and expressed in E. coli Expression system. The expressed protein was purified and used to raise antibodies in rabbits. Our own produced antisera was tested by DAC-ELISA with the infected and healthy plant tissues. The ELISA readings showed positive results with the infected tissues from Pakistan and USA and negative results with the healthy tissues which confirmed the authenticity of antisera. Results indicated that our prepared antisera is more specific and sensitive against Pakistani CTV isolates as compare to commercially available kit. ELISA readings showed difference in reactivity between the antiserum from two rabbits. No difference in the ELISA readings were observed among five bleeds at the same dilution factor which revealed no difference in the antibody titer among the bleeds. Antisera was also tested against four different dilutions of plant extracts and results showed the sensitivity of one antiserum up to 1/100 of the plant extract. We can use the antisera for the indexing of trees in Pakistan and certification schemes could be introduced to reduce the spread of the virus in the country.
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