Biology and Molecular Biology of Major Viruses Infecting Potato Crop in Northern Parts of Khyber Pakhtunkhwa, Pakistan.

Abstract

The hilly area of Khyber Pakhtunkhwa (KP) province is a leading potato seed producing region of Pakistan. Field symptoms and laboratory analysis revealed that Potato Leaf Roll Virus, Potato Virus Y (PVY) and Potato mop virus (PMTV) were the major viral diseases of potato in the areas surveyed. On the basis of characteristic symptoms the average incidence of PLRV ranged from 3.89 - 46.66 % with maximum incidence in Kalam North (46.66 %) at Ushu (56.66%) and minimum incidence in Miandam (3.89 %) at Seney locality (3.33%). The incidence of PVY ranged from 67.04% (Shangla) to 78.95% (Swat). The infestation of aphid ranged from 2.21 (Kalam East, Swat) to 5.50 (Pir Khana, Shangla) in summer crop while in spring crop it is ranged from 7.00 (Abbotabad) to 10.66 (Mansehra). The incidence of PMTV ranged from 2.44 - 9.07% with maximum incidence in district Swat and minimum incidence in district Shangla. The incidence of PMTV in tubers showing spraing symptoms are ranged from 7.33 - 4.99% with maximum incidence in district Swat and minimum incidence in district Abbotabad. Symptomatologcal studies also showed that mostly necrosis for PVY, and leaf rolling and yellowing of leaflets for PLRV and no exact symptoms in case of PMTV and TRV were appeared in the fields surveyed. The ELISA results revealed that average incidence of PLRV was ranged from 8.66 - 44.33 % and PVY ranged from 67.04 - 78.95 %. The average incidence of PMTV ranged from 2.44 - 9.07% and it is ranged from 7.33 - 24.99% in tubers showing spraing symptoms. The ELISA test in case of TRV was negative and showed zero incidences. Serological results were confirmed on the basis of molecular properties during six month exposure visit to USA funded by HEC, Islamabad, Pakistan where the molecular detection and characterization was carried out in the Department of Plant Pathology (Washington State University, Pullman) under the Supervision of Prof. Dr. Hanu R. Pappu and his team. RNA was extracted from ELISA positive samples and C DNA was synthesized and was used as a template in RT-PCR amplification. In aphid borne viruses only PVY was detected and its CP gene of 801bp was amplified using CP gene specific primers. Plasmid DNA having CP gene was isolated, purified and sequenced by ELIM Biopharmaceutical, Inc. 25495 Whitesell St, Hayward, CA 94545, USA after successful cloning, transformation and confirmation by colony PCR. All samples (two from each zone) of plasmid DNA having CP gene of PVY showed similar sequence. The nucleotide sequence of 801bp on translation resulted in 267 amino acids and sequence analysis showed high variability of the Pakistani isolates with isolates reported from other countries. Nucleotide identity of 99% was observed with Poland N-W, Germanyw, Saudi Arabia, South Africa and Cuba and amino acid identity level of 100% was observed with isolates reported from Poland, Saudi Arabia, India and Pakistan. Phylogenetic analysis revealed Pakistani isolate clustered with India, Pakistan, Poland and Saudi Arabia isolates. Similar process was performed for soil borne viruses and only PMTV was detected and amplified using PMTV- CP gene specific primers. The expected size (~ 553bp) of the PCR product was achieved in all samples tested. Plasmid DNA having CP gene was isolated and sequenced. All the samples two from each zone of plasmid DNA having CP gene of PMTV showed same sequence similarly as in PVY. Sequence analysis revealed that coat protein region of 540 bp has 176 deduced amino acids. Nucleotide identity of 99% was shown by Pakistani PMTV Isolate with Canada and USA isolates. The amino acids level was as high 99% when compared with rest of the isolates except few isolates reported from other countries which showed no significant similarity. Phylogenetic analysis revealed that Pakistani isolate clustered with Canada and USA isolates. Regarding the screening of varieties the PVY showed the maximum disease severity in Racco (80.33%) and minimum in Raja (41.13%. PLRV showed maximum disease severity in Paramont (69.50%) and minimum in Desiree (41.80%). No typical symptoms was appeared in case of PMTV infection, therefore, data were recorded on powdery scab disease and the maximum disease severity was recorded in Diamont (70.00%) with minimum in Karuda (11.11%) and these results were confirmed through DAS-ELISA