Field Evaluation and Enzyme-Linked Immunosorbent Assay Detection of Potato Leaf Roll Virus , Potato Virus X and Potato Virus Y in Potato Germplasm

Polerovirus: potato leaf roll virus (PLRV), Potyvirus: potato virus Y (PVY) and Potexvirus: potato virus X (PVX) is more destructive and well distributed throughout the Pakistan. Incidence has been reported to be as high as 90%, 25%, and ≥ 15%, respectively in the potato growing regions. To find out the source of resistance, twenty-nine virus free potato varieties were grown under field conditions with good agricultural practices. The disease severity of PLRV, PVY and PVX was recorded to determine the level of resistance of the potato varieties according to the disease rating scale. Infectivity and biological assay of all twenty-nine varieties were done in green house on potato, Datura stramonium, Nicotiana glutinosa and Physalis floridana. Non-inoculated plants were served as control. Leaf samples from potato varieties were collected for serological detection of PLRV, PVY and PVX by Double antibody sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA). Out of twenty nine varieties, none of the variety was resistant to PLRV although three varieties; Mirrato, 394021-120 and Orla were moderately resistant. Only FD 48-4 and TPS 9813 showed resistance to PVX and PVY. While FD 3-10, FD 9616 and FD 37-13 were moderately to PVX and PVY. Rest of the varieties was found susceptible to all three viruses.


Introduction
Potato (Solanum tuberosum L.) is the world's third most important food crop and widely distributed (Bhaskar et al., 2010) due to its high production and good nutritional value.Several diseases hamper its production (Ahmed & Bhutta, 1989).Among these most important and extensively widespread are the aphid-transmissible RNA viruses (Robert, 2000;Kogovsek et al., 2010), i.e.Polerovirus: potato leaf roll virus (PLRV), Potyvirus: potato virus Y (PVY) and non aphid-transmissible RNA virus (Adams et al., 2004) Potexvirus: potato virus X (PVX).Potato leaf roll virus is more destructive and well distributed throughout the Pakistan (Mughal et al., 1988).Its incidence has been reported to be as high as 90% in the producing regions (Bhutta & Bhatti, 2002).PLRV is transmitted in a circulative, persistent and non-propagative manner by its most important vector aphid; Myzus persicae Sulz (Salazar, 2003).During the primary infection, the seed tubers are asymptomatic, but symptoms become visible at secondary stage (Chatzivassiliou, 2008).PLRV causes a prominent upright characteristic rolling, chlorosis or reddening, and roughness in the texture of potato plant leaves (Khurana, 2004).The PLRV infected plant remains stunted in growth producing reduced number and size of tubers (Nawres, 2013).On the other hand, PVY is a highly variable virus with new emerging strains PVY O , PVY N , PVY C , PVY NTN , PVY N:O , PVY NW (Dabijev et al., 2005;Kerlan, 2009;Kostiw, 2011).PVY is transmitted mechanically and by aphid (Myzus persicae) in a non-persistent manner (Jones et al., 2009;Kerlan & Moury, 2009).PVY disease losses are up to 25% (Mughal et al., 1988) but it can destroy whole crop if it occurs along with PVX (Loebensstein, 2009).Most prominent symptom is the 'leaf-drop streak' or necrosis along the veins of the underside of leaflets and leaf

Serological Assay
Leaf samples from potato varieties were collected for serological detection of PLRV, PVY and PVX.Double antibody sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA) was used as described by Clark and Adams (1977).Antibody Coating Buffer (Distilled water 1 liter, Sodium carbonate (Na 2 CO 3 ) 1.59 g, Sodium bicarbonate (NaHCO 3 ) 2.93 g); Virus (Antigen) extraction Buffer (Sodium chloride (NaCl) 40 g, Potassium Phosphate (KH 2 PO 4 ) 2 g, Sodium phosphate (Na 2 HPO 4 ) 11.5 g, Potassium chloride (KCl) 2 g, Sodium azide (NaN 3 ) 2 g, Polyvinyl pyrrolidone (PVP.MW.4000) 2 g, Tween-20 (Polyoxyethylenesorbitan monolaurate) 0.5 ml/L, Egg-ova albumin 2 g, Distilled water 800 ml); Washing Buffer (5 × PBS 200 ml, Distilled water 800 ml, Tween-20 1 ml); Substrate Buffer (Diethanolamine 97 ml, Distilled water 800 ml); Enzyme Conjugate Buffer (Bovine serum albumin (BSA) 2 g and Polyvinylpyrrolidone (PVP) Mol.Wt. 40,000 20 g) were prepared by mixing and grinding with a mortar and pestle.Tissue samples were weighed into phosphate-buffered saline containing 0.5 ml/L Tween-20 (PBS-Tween) and 20 g/L PVP and homogenized.Extracts usually were transferred immediately to the test plates.ELISA microtiter plates 96 wells were coated with PLRV, PVY and PVX antibodies (Agdia Company, Elkhart, USA) in three different microtiter plates, each diluted in coating buffer at 1:200.The coated plates were incubated at 4 °C for overnight.After incubation the plates were washed with PBS-Tween 3 times at 5 min intervals.These wells were filled with the sap of PLRV, PVY, and PVX infected tissue extracted in extraction Three and four wells filled with each of buffer and healthy samples, respectively.The plates were incubated for 4 °C and washed 3-4 times with PBST.Enzyme conjugate of 200 μl was diluted at 1:200 and added.Incubated for overnight at 4 °C followed by washing 3-4 times with PBST.200 μl of substrate buffer containing p-nitro phenyl phosphate (75 μg/ml) was freshly prepared and added to each well.Incubation at room temperature for 30 minutes was done and reaction for development of yellow colour was visually observed.The reaction was stopped by adding 50 μl 3M NaOH to each well.Colour intensity is proportional to the conc. of virus.Test results were scored visually as positive or negative based on the colour intensity of the reaction mixture.
In confirmation of the ELISA results, disease incidence data was observed as per internationally accepted disease rating scale (Table 1) for PLRV, PVX and PVY (Mughal & Khan, 2001).

Results
The twenty nine potato varieties tested against PLRV, PVY and PVX showed a range of symptoms from mild to severe mosaic and rugosity.
Attack on the twenty nine potato varieties by PLRV was most severe of the three viruses.Notwithstanding, three of the varieties; Mirrato, 394021-120 and Orla were moderately resistant to PLRV.FD 48-4, FD 3-10, TPS 9802, FD 37-13, TPS 9813and Hermes were moderately susceptible.The remaining twenty one varieties were very susceptible to PLRV (Table 4).
ELISA reaction tested positive in all the twenty nine varieties tested against the three viruses.The test results were observed visually and recorded as positive or negative (Tables 2-4).

Discussion
The field observation of the potato varieties showed that PVY, PVX and PLRV are very much distributed in the field.The widespread of these viruses has been reported previously (Jan & Khan, 1995;Muhammad, 1990;Anwar & Mirza, 1984).The presence of the potato viruses was detected through graft inoculation and mechanical transmission.These methods have been reported severally (Khan et al., 2002;Mayo et al., 2000;Muhammad, 1990;Mughal et al., 1988;Arif, 1988).The ELISA further corroborates the finding.Other studies have also shown the efficacy of ELISA in detecting viruses (Jarjees, 2000;Abou-Jawdah, 2001;Khan & Saif, 2001;Abdullah, 1992).The potato varieties showed variable responses against the viruses.Hossain et al. ( 2002) also found similar responses to PLRV and PVY in different varieties.Most of the potato varieties evaluated in this experiment were moderately resistant and moderately susceptible.This pattern of resistant response was also observed for most potato varieties by Ahmad and Ahmad (1995).However, such type of varieties exhibiting tolerant responses have been found to be usually high yielding and might be a good source for the breeders to produce virus free seed through tissue culture techniques.

Conclusion
Further screening of potato germplasm is necessary for identification of resistant source.

Table 2 .
Potato germplasm screening against PVX