Investigation of Potato Virus Y Strains in Guilan and Mazandaran Provinces Using Biological, Serological and Molecular Methods

Maghsoudi Reza 1, Jafarpoor Behrouz 2, Rastegar Mahrokh 2

1Department of Agriculture, Ramsar, Iran 2Ferdowsi University, Department of Plant Pathology

Received: October 1, 2015 Accepted: December 31, 2015 ABSTRACT

Potato virus Y (PVY) can cause severe crop losses and consequently economic damage. Therefore, the detection of PVY strains in tuber is very important for undertaking appropriate measures to prevent them. Considering that potato cultivation is growing in Guilan and Mazandaran provinces, the goal of the current study is to investigate Potato Virus Y Strains in Guilan and Mazandaran Provinces in Iran. Consequently, 588 tuber samples were collected randomly in summer 2013. After the dormancy period at 7°C, the samples were transferred to a greenhouse to germinate at 25°C. Then, polyclonal antibodies were used to examine buds. Afterwards, samples with the highest absorption in the wavelength were selected. Finally, it was found that 73 samples were infected by PVY. The extracts taken from infected plants were inoculated. To study symptoms necrotic spots were extracted and inoculated into Tobacco (Nicotiana tabaccum var samsun), Physalis Commun (Physalis floridana) and Potato (Solanum tuberosum) after 7 days. Simultaneously, leaf extracts were examined for potato virus y strains using monoclonal antibodies. The results of biological, serological and molecular tests indicated that 17 isolates belonged to PVYO, 30 isolates belonged to PVYN, 12 isolates belonged to PVYC and 14 isolates belonged to PVYNTN. KEY WORDS: Potato, Virus, Leaves, Necrosis, Mosaic,

INTRODUCTION

Potato virus Y, characterized by a broad range of hosts including monocotyledonous and dicotyledonous plants, is Potyvirus, a genus of viruses, and belongs to the family Potyviridae [19, 4, 23, 17, 29]. PVY is among the five most economically harmful viruses [26], with a host range covering major crops, such as Pepper (Capsicum annuum L.), Potato (Solanum tuberosum L.), Tobacco (Nicotiana spp), Tomato (Lycopersicon esculentum Mill.) , as well as less important plants and several species of weed, mainly in the Solanaceae family [33, 21, 28, 13, 17, 7, 15]. PVY has non-enveloped filamentous, flexuous rods and positive- sense single-stranded linear RNA genome with the size of 10kb. At the end of 3' the genome has a Poly A sequence and at the 5' end, it is covalently linked to protein VPg. The RNA of this virus codes a big polypeptide broken down into three areas by three protease codes and creates three protein products [22, 11, 1, 2, 30, 27, 9]. Therefore, the virus can cause heavy yield losses and is a serious threat to potato production [14]. An important factor worth considering is that PVY is easily transmitted by several species of aphids, such as common pests of potatoes [32, 34]. Green peach aphid (Myzus persicae) is the most common in this regard. The best method to prevent the virus infection is to use resistant cultivars [9]. Symptoms are variable depending on viral strain, host cultivar genotype, plant growing step, climatic conditions, and whether it is a primary infection (inoculation by aphid vectors) or secondary infection (when mother tuber is infected). As Potato virus Y can cause necrosis, it is urgent to detect virus Y infection [32]. According to some biological and serological tests PVY is divided into three strains: PVY O ,PVY N and PVY C [30, 21, 33, 5, 27]. However, the new studies reveal that PVY has five strains: PVY N, PVY O, PVY C , PVY Z and PVY E. This virus has strains with recombinant genome including PVY NTN , PVY N-WI , PVY N-NTN and PVY NTN- NWI [7]. The strains PVY N and PVY O more than the other strains were reported worldwide [5]. In tobacco, PVY N causes mild symptoms varying from necrosis, mottle, vein fading to distortion, while in potato, it causes leaf crinkling and foliage necrosis. PVY C causes the symptoms similar to PVYO in tobacco, while in potato and Physalis floridana it causes severe general necrosis and fast plant death [3, 22, 20, 32, 16, 17, 25, 29]. PVY N causes ring spot in potato tubers [14]. This strain can be transmitted not only by aphids but also by infected seed tubers. PVY NTN and PVY NW were first reported in Europe. Since 1980, molecular method has been applied for exact differentiation of PVY strain. Accordingly, two recombinant strains named PVY NTN and PVY NW (PVY N:O ) were differentiated from other PVY strains [21, 13, 2,16,17, 27] . PVY NW strain causes systemic necrosis in tobacco and mild leaf symptoms in potato, similar the ones caused by PVY N strain. The symptoms caused by

*Corresponding Author: Maghsoudi Reza , Department of Agriculture, Ramsar, Iran

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PVY NTN strain includes mosaic, mottle on the leaves and colorless rings on tubers that spread and become necrotic. These symptoms are often undetectable when harvesting and appear gradually during the storage period causing heavy crop losses, decrease in quality and market supply [7]. Two PVY NTN and PVY NWI viruses cause vein necrosis in tobacco and ring chlorosis in potato. PVY Z does not cause vein necrosis in tobacco but creates mosaic symptoms. This strain along with PVY E creates hyper sensitive reaction in several potato and tobacco cultivars. Some strains such as PVY O and PVY N were detected by indicator plants. PVY NTN was differentiated from PVY N using monoclonal antibody [29]. Sing, et al found one isolate of PVY NTN generated via mutation in Northern America in 1998 but recombinant did not interfere with its so called NA-PVY NTN. [21, 7]. Potato virus Y is widespread in almost all potato growing regions in Iran. Pourrahim in 1998 reported the necrotic strain of PVY (PVY N) in Hamedan [24]. Maghsoudi in 2004 reported the existence of three strains PVY N, PNY O and PVY C in Khorasan province. Tousi in 2005 reported the presence of this strain as sub strain of PVY in 5 samples in Iran [31]. Hosseini in 2008 categorized isolates of PVY and placed them into a separate category related to PVYNTN [12]. Majdabadi in 2010 reported PVY NTN in Khorasan e Razavi [18]. The certified seed program of potato tubers is very important to decrease potato virus infection. Since this virus strains can cause severe crop losses, it is necessary to identify them in different regions of the country and undertake appropriate measures to control and prevent them. Considering that potato cultivation is growing in Guilan and Mazandaran provinces, the on time detection of PVY strains in tuber, its identification and control can lead to more production.

MATERIALS AND METHODS

To identify PVY strains, several samples were collected from commercial potato growing areas in Guilan and Mazandaran provinces. Tubers suspected to be infected were collected randomly in harvest period. Then, the collected samples were transferred to the laboratory and were kept at 4 oC to pass the dormancy period. As they started to germinate the infected buds were identified using serological ELISA test and the tubers were separately planted in plastic pots. To study the symptoms of the virus, isolates were inoculated into indicator plants. To increase the efficiency of the virus transfer, the plants were kept in shadow for 24 hours before the inoculation. Then, to identify the presence of PVY in inoculated cultivars, their leaves were separately extracted and used in ELISA test. This test was done according to the test of Clark and Adams [8] and Dr. Burns from Scotland Agricultural Research Center. The LEAF Sap was attenuated by Extraction buffer in proportion of 3 to 1. Virus Immunoglobulin was used in proportion of 3 µl in each milliliter with Coating buffer to cover microplate. Further, the combination of Alkalin phosphate and globulin conjugate in proportion of 1 in each thousand and P-nitro phenol phosphate in proportion of 0.6 milligram in each milliliter were used. In the processes of research 100 µl suspension was added to each cavity. Each microliter was regarded as repeat and each sample included at least 2 repeats. One negative control and one positive control were considered for each plate. Color appearance in microplate was investigated by ELISA reader. All the materials used in this test were provided from International Potato Center and Scottish Agricultural Investigation Center. Further, RNA was extracted from potato leaves according to a slightly modified method of Boonham et al and Mekuria [5]. The solution RNX TM (-Plus) was add to total RNA extraction. The tissues of the potato leaves or tubers (50 mg to100 mg) were frozen in liquid nitrogen and ground to a fine powder to produce a leaf homogenate. 3 mL of the extraction buffer (EDTA 0.01M, Lich 0.1 M, Tris-HCl 0, 1 M, SDS 1%, LiCl 4M, PVP 5%, [w/v]) and Sodium Meta Bisulfate 2% (w/v) were added and mixed. 700 μL of the sample was decanted into a 1.5 mL microfuge tube and 800 μL of phenol-chloroform-iso amyl alcohol was added and mixed. The tube was incubated in ice for 15 minutes before centrifugation. Then, 400 μL of the supernatant was transferred to a fresh microfuge tube and an equal volume of 4 M LiCl was added. All the samples were kept at - 20 ºC for 4 hours following the centrifugation for 15 minites at 14000 rpm and 4 ºC. The supernatant was extruded. 400 μL of distillated water, 800 μL of cold ethanol, and 40 μL of sodium acetate 3 M were added into each tube, all of which were again centrifuged for 15 min at 14000 rpm and 4 ºC. The supernatant was discarded. Then, the microfuge tube was washed with 100 μL of cold ethanol (70%) and centrifuged for 5 min at 4000 rpm and 4 ºC. The supernatant was excreted and the tube was air-dried. Finally, the tube was re-suspended in 30 μL of RNase-free water and stored at -20 ºC. The RNA concentration was calculated by measuring its absorbance at 260 nm. After the extraction of RNA virus, 3 µl of RNA was mixed with 1 µl of MOR2 primer (5' ATA AGC CCA TTC ATC CCA ACC 3') and the resultant mixture was incubated at 70 oC for 5 minutes and then transferred to RT tubes. The cDNA transcription reaction was performed in a thermocycler at 42 oC for an hour and 94 oC for 5 minutes (Boonham, 2002).

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The replication of cDNA fragment made by RT test was performed using Accu Power TM RT-Premix kit. Five µl of cDNA was separately mixed to 1 µl of MOR1 primer (AGG TAA AGG CAC AGG GAA CTG) and MOR3 (GCA CCA ATT CAG GAG ATT CTA). Following, cDNA mixed with 1 µl MOR2 primer and MOR1 primer and cDNA mixed with 1 µl MOR2 primer and 1 µl MOR3 primer were mixed in PCR tubes. Afterwards, PCR, with the total volume of 20 µl, reaction was accomplished in the thermocycler at 94 oC for 4 minutes, followed by 30 cycles including 94 oC for 30 second, 50 oC for 30 second, 72 oC for a minute and a cycle at 72 oC for 10 minutes to complete polymerization. PCR product was kept at -20 oC or was separated by electrophoresis through an agarose gel, and then dyed with Ethidium Bromide. The results were observed using UV transilluminator. Finally, ELISA test was applied to identify the samples infected by PVY considering the average and standard deviation of negative control as infection border.

RESULTS AND DISCUSSION

The results indicated high tuber infection percentage. Out of the total 588 isolates, 481 tubers were infected by PVY and other viruses. 73 tubers were infected only by PVY. About 10 days after inoculation different symptoms appeared. PVY NTN strain created necrotic spots on Nicotiana tabaccum var samsun leading to plant death at the end of growing season. Mosaic spots created in Physalis floridana resulted to chlorosis. To observe the symptoms, DAS-ELISA test was performed with the use of monoclonal antibody related to each PVY strains. The results of DAS-ELISA test, performed with the use of monoclonal antibody, indicated that infected isolates belonged to PVY O, PVY N and PVY C. Hence, PVY isolates existing in Guilan and Mazandaran provinces include 4 types of strains: PVY N , PVY O , PVY C and PVY NTN . The infection ratio in potato fields of both provinces was similar. Biological, serological and molecular tests revealed 17 PVY O isolates, 30 PVY N isolates, 12 PVY C isolates and 14 PVY NTN isolates. The presence of PVY infection can be a potential danger for potato growing in these two provinces. This study is the first to confirm the presence of PVY NTN strain in these regions.

Fig.1.Reaction of PVY in ELISA Plate. Fig.2 Clear veins in tobacco leaves related to PVY C

Fig.3.Necrosis leaf spot related to PVY O Fig.4. Potato tubers infected with virus y.

Figure 5. electrophoresis of RT-PCR product related to PVY on dyed horizontal agarose gel 1 percent Column: 1 DNA marker Column: 2 to 5 PCR products of PVY Column: 6 negative control (distilled water)

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Figure 6. electrophoresis of RT-PCR product related to PVY NTN on dyed horizontal agarose gel 1 percent Column: 1 to 6 PCR products of PVY NTN Column: 7 DNA marker

Table 1: PVY strains reaction with indicator plants and monoclonal antibody Mob Mob Mob Chenepodium quinoa Nicotiana Solanum Nicotiana Physalis PVY N PVY O PVY C glotinosa tubrusum tabaccum floridana PVY O - + - N NS ML MM NC PVY C - - + N NS MV MM-MV ND PVY N + - - N NS NC MV MM-MCL

Table2: The number of infected samples and infection ratio in sampling areas Sampling areas Sampling fileds Total samples Infected samples Infection ratio

Ramsar Javaherde,Filikdom, 153 23 15.03 Salmal, Jirkooh Tonekabon Soleiman abad, Goleijan, 67 11 16.41 Lashtoo Chaboksar Gohar sara, Sorkhani 94 10 10.64 Kelachay Rezamahale, Bibalan 55 17 30.90 Roodsar Aroos mahale, Shahraj, 221 12 5.5 orkom, Vishki, Yasoor

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Table 3: PVY strains abundance in Guilan and Mazandaran provinces PVY O PVY C PVY N PVY NTN Guilan 39 8 5 17 9

Mazandaran 34 9 7 13 5

Total infected samples 74 17 12 30 14

In addition, RT-PCR test proved the presence of virus Y in all samples in accordance with ELISA test. At the meantime, the use of MOR2 primer and MOR1 primer used to identify PVY NTN strain , indicated the presence of PVY NTN in 14 out of 50 samples, causing potato tuber necrotic ring spot disease in potato. The results of ELISA test are in accordance with the researches of Ellis, Cerovska, Nie & Singh and Smith. [10, 21, 22] However, in this study it was not possible to differentiate PVY NTN from PVY N using monoclonal antibody. Similar research has been conducted in Khorasan Razavi province with the same results [18]. This test results are also similar to the results of Boonham [5]. However, this study contradicts to the study conducted by Lorenzen [17]. According to his study, the symptoms appear during storage time in the sensitive cultivars infected by PVY NTN strain; while in this study tubers infected by PVY NTN strain showed no symptoms.

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