High Throughput Molecular Screening of Citrus Tristeza Virus from trhe Citrus Germplasm of Punjab and Production of Virus-free Citrus Plants via Shoot-Tip Grafting.

Abstract

Citrus is the prominent tree fruit crop consumed world wide because of its wide nutritional and health values. Pakistan is also famous for its citrus production that plays an important role in establishing the country’s economy. Tactlessly, various diseases including many viruses, virus-like, bacterial and fungal diseases are damaging the citrus crop in the world including Pakistan. Citrus tristeza virus (CTV) is the causal agent of the most deadly virus disease of citrus leaving a disastrous impact on citrus production. It is a phloem-limited and graft-transmissible virus that belongs to Closteroviridae family transferred by using the already infected propagative material or by vector, causing a wide range of symptoms depending on the isolate and the host. The current research was designed to study the prevalence of CTV in Pakistan screening the two major regions of Punjab i.e. Sargodha and Silanwali using PCR techniques and the ultimate goal was the establishment of virus-free citrus plants via in vitro shoot-tip grafting (STG). A total of 400 citrus samples representing major citrus groups including sweet oranges, mandarins, grapefruits, kumquats, tangelos, lemons and limes, rootstocks and others were collected. Symptoms in the field ranged from asymptomatic or the symptomatic plants bearing pin holing on the bark, seedling yellow, vein clearing, leaf and bark flecking, necrotic lesions on the leaf edges and twigs, stunted growth and quick decline like symptoms were noticed. RNA extraction was done using two methods i.e. Trizol and SDS-Triton. The quality of RNA from both the extraction methods was checked using 37% formaldehyde denaturing agarose gel electrophoresis as well as using UV-VIS Nanodrop spectrophotometer. Molecular screening of CTV using PCR techniques was used to confirm the presence or absence of CTV. Initial detection was done using conventional PCR with cDNA as a template in which 12 samples were positive for CTV with an infection rate of only 3%. A total of 400 RNA samples were checked for their integrity, out of which 392 samples were positive for NAD5 and COX assay and were used for further CTV screening. Cq values for NAD5 ranged from 15.40 to 28.50 with an average of 19.84 for 392 samples. COX Cq values ranged from 12.33 to 27.00 with an average of 16.00 for 392 samples. The detection of CTV using highthroughput qRT-PCR was done using reported primers and TaqMan probe. Twenty-five samples were found positive for the CTV using both the multiplex (CTV, CPsV and CLBV) as well as singleplex (CTV) assay. The Cq values for the CTV positive samples in a multiplex and singleplex assay ranged from 11.4-32.0 and 11.413-31.749 respectively. The estimated infection rate for CTV was 7% in both the regions ix of Punjab. The most infected group found was sweet oranges (22/252), followed by mandarins (2/52) and lemons and limes (1/25). The infection rate between the two regions Sargodha and Silanwali was 6.7% and 2.6% respectively. A one-step RT-PCR was used to further characterizes the CTV positive samples. As one-step RT-PCR is far less sensitive from qPCR, out of 25, only 15 samples reacted with universal coat protein primers for the detection of CTV at the desired amplicon size of 672bp. The fifteen CTV positive samples from one-step RT-PCR for the conserved generic coat protein unit (CPU) were characterized for the presence of common CTV genotypes as VT, T30. T36, T3, RB and a newly found S1 using a multiple molecular marker (MMM) method based on the one-step RT-PCR amplification of sequence specific PCR products with sets of primers derived from the most conserved sites within the genomes of VT, T30, T36, T3, RB and a newly discovered S1 isolates. Out 15 CTV-CPU positive samples 10 samples reacted with VT marker, 2 reacted with T30 and RB marker and 1 reacted for T36, T3 and S1 marker respectively. Many of the genotypes were found in mix infection such as VT+T30, VT+T36 and VT+T30+S1 etc. The CTV coat protein gene, VT, T30, T36, T3, RB and S1 isolates were sequenced for the phylogenetic analysis with isolates present worldwide. The results of the nucleotide sequence comparison of individual isolate were done using MEGA 7.0. phylogenetically. The Pakistani isolates showed highly similar homology with American, Italian, Indian and Chinese isolates. The control of CTV and many other graft-transmissible diseases is now possible by using the disease free nursery plants, controlling the vectors and the eradication of diseased plants. Availability of virus free planting material is the utmost necessity of the citrus industry in Pakistan for which in vitro shoot-tip grafting has already proved successful in establishing the clean plant material. In vitro shoot-tip grafting technique was introduced for the production of pathogen tested citrus plants. Nine commercially important citrus cultivars were STGed and were indexed at various steps. The most significant aspect of the present study was to learn and develop the procedure for in vitro shoot-tip grafting for establishing the virus-free citrus plants that will help the citrus growers of Pakistan in providing the clean plant material. The procedure involved rootstock preparation, scion preparation, in vitro shoot-tip grafting, acclimatization, pre- and post- indexing of the successful grafts.