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Molecular characterization of two alfalfa mosaic virus isolates infecting potato crop in Central Region of Saudi Arabia.

Byline: Mohammed A. Al-Saleh Mahmoud A. Amer Ibrahim M. Al-Shahwan Omer A. Abdalla and M.T. Shakeel

Abstract

During the autumn growing season 2012 thirty-two leaf samples of potato (Solanum tuberosum L. cv. Herms) plants showing mottling symptoms suspected to be due to viral infection were collected from Hail and Wadi aldawasser regions in Saudi Arabia. These leaf samples were tested serologically using DAS ELISA against Alfalfa Mosaic Virus (AMV). Twenty-three out of the thirty-two samples were positive for AMV. To determine host range plant sap was extracted from two diseased potato samples representing the two regions and different healthy plants were mechanically inoculated. The disease symptoms induced by the two selected isolates were divided into three groups: systemic and calico symptoms local chlorotic lesions and non-symptomatic ones. Total RNA was extracted from different plants showing positive reaction to AMV. RT-PCR was performed using AMV coat protein specific primers. Approximately 700-bp RT-PCR products were amplified and these PCR products were sequenced in both directions. Nucleotide sequencing analysis showed that these two Saudi Arabian AMV isolates [AMV-PSA-Ha (for the Hail isolate) and AMV-PSA-Wd (for the Wadi Al Dawasser isolate] had a 96.5% identity. Sequence comparison showed that these two isolates of AMV shared 93.2% to 99.7% sequence similarity with the twenty- eight reported isolates of AMV obtained from GenBank. The cDNA probe was prepared and hybridized with RNA extraction from symptomatic potato plants. No hybridization was shown with RNA extracts from asymptomatic plants. This is the first report on the genetic variability of AMV isolates infecting potato crop in Saudi Arabia. Copyright 2014 Friends Science Publishers

Keywords: Potato; AMV; ELISA; RT-PCR; CP sequence

Introduction

Potato (Solanum tuberosum L.) is a very important staple crop in many parts of the world due to its nutritional values. It is used for human consumption animal feed as well as a source of starch carbohydrates alcohol and protein (Al-Saikhan 2000). In Saudi Arabia potato is considered to be one of the most important and potential vegetable crops. It is grown during spring and autumn growing seasons. Seed tubers of different potato cultivars are usually imported from France Holland and USA for planting the spring season whereas the autumn season is planted from seed tubers propagated locally from the imported tubers harvested from the previous spring season. Potato acreage has increased in some areas and this expansion is mainly due to the government policy of diverting food production and reducing wheat acreage. Potato is grown in different regions in Saudi Arabia and the total area reached 15312 ha with a production that reached up to 444138 tons in 2010 (Anonymous 2010). Many viruses viroids and phytoplasmas have been reported to infect potato. More than forty different viruses or virus like-diseases have been reported as pathogenic to potato (Ross 1986; Salazar 1996; Al-Shahwan et al. 1998). These plant viruses are responsible for severe diseases in potato plants resulting in severe losses in potato production (Aranda and Maule1998). In Saudi Arabia twelve potato viruses have been detected (Al-Shahwan et al. 1998). Out of these 12 viruses Alfalfa Mosaic Virus (AMV) was found to be the most important and widely distributed one infecting potato plants in several locations and was considered as one of the mostprevalent viruses in the country (Al-Shahwan et al. 1998;Al-Shahwan 2003). The main objective of this study was to molecularly study the genetic variability of AMV isolates recovered from potato plants from two different regions in Saudi Arabia and assess the DNA homology between the Saudi AMV isolates and other AMV strains reported elsewhere and available in GenBank database.

Materials and Methods

Source of Virus Isolates Serological and BiologicalIdentification

In December 2012 thirty-two symptomatic and asymptomatic leaves of potato plants of cv. Hermes growing under field conditions which exhibited leaf distortion and mottling symptoms suspected to be virusinfections (Fig. 1) were collected from Hail (North of the Riyadh region) and Wadi Aldawasser (South of the Riyadh region) Saudi Arabia. These samples were tested serologically using Direct ELISA against AMV (Agdia Co. France). One sample from each region (Hail and Wadi aldawasser) was chosen for further plant host range characterization. The sap was extracted and ground separately in phosphate buffer (1:3 w/v 0.1 M pH 7.2) passed through a double layer of cheesecloth and mechanically inoculated onto carborandoum (600 mesh)- dusted leaves by gently rubbing the freshly prepared inoculums (Xu and Nie 2006). The selected plant species belonged to different plant families including Chenopodium amaranticolor Cost and Reyn C. quinoa Vigna unguiculata Pisum sativum Vicia faba Nicotiana tabacum N. benthamiana Capsicum annum Cucumis sativus N. rustica and S. tuberosum. For biological purification the single local lesion technique was carried out according to Kahn and Monroe 1963 using C. amaranticolor as a local lesion host. The resulting local lesions were singly back inoculated onto potato plants whereas N. tabacum was used as propagative host for the AMV isolates (Fig. 2). Five seedlings of each host plant were inoculated and observed daily for symptom development. A number of healthy seedlings of the same species and age were inoculated with buffer to serve as a control. The inoculated plants were maintained in an insect-proof greenhouse with naturalillumination and temperatures of 2532oC. The symptoms expressed on each species were described and recorded. The experiment was repeated twice.

Reverse Transcriptase-Polymerase Chain Reaction

Total RNA was extracted from four out of twenty-three samples which tested positive for AMV by ELISA (two samples from each region). These samples and one uninfected potato sample served as a negative control were extracted using the Isolate Plant RNA Mini Kit (Bioline). The oligonucleotide primers designed according to (Xu and Nie 2006) was as follows the upstream primer AMV-coat- F: 5'-GTG GTG GGA AAG CTG GTA AA-3'] and downstream primer AMV-coat-R: 5'-CAC CCA GTG GAG GTC AGC ATT-3' were used. RT-PCR was performed using a thermal cycler (Eppendorf Germany) according to Xu and Nie 2006; Al-Saleh and Amer 2013. DNA was visualized and photographed using DNA documentation gel analysis (IN GENIUS Syngene Bio Imaging UK). HyperLadder IV DNA marker (Bioline) was used to determine the size of RT-PCR amplified cDNA products.

Probe Preparation and Dot Blot Hybridization

The viral fragment was labeled according to the Digoxigenin Non-radioactive Labeling System (Roche Boehringer Mannheim Germany). Total clarified sap preparations from infected plant materials were diluted ten-fold with 6X SSC buffer heated to 95C for 10 min and chilled on ice. A volume of 5 L of total RNA extracted from each of the twenty-three symptomatic and healthy samples that were collected were prepared and directly applied to nitrocellulose membranes according to established methods (Podleckis et al. 1993; Pallas et al.1998). Prehybridization hybridization and colorimetric detection with a single digoxigenin labeled probe were carried out using a hybridization oven (Amersham Biosciences Piscataway NJ USA) following the protocol recommended by Boehringer Mannheim. The results were documented by photography.

Nucleotide Sequence and Phylogenetic Analyses

Virus-specific RT-PCR products from two out of the four samples tested positive for AMV and were designated as AMV-PSA-Ha (for the Hail isolate) and AMV-PSA-Wd (for the Wadi Al Dawasser isolate). The RT-PCR products were sequenced using two directional sequencing with the AMV-coat-F and AMV-coat-R primer specific for AMV-CP (Xu and Nie 2006) using an Applied Biosystem AB3730xI DNA analyzer (Life Technologies USA). The partial nucleotide sequence of the CP gene was subjected to a Blast N search for comparison with published AMV-CP gene sequences retrieved from GenBank. The multiple sequence alignment and phylogenetic relationships for the AMV isolates available in GenBank (Table 1) were analyzed and reconstructed using Lasergene DNASTAR V5-05Software.

Results

Serological and Biological Identification

Twenty-three out of thirty-two tested samples were shown to be positive to AMV by ELISA. Two isolates (AMV-PSA- Ha and AMV-PSA-Wd) were selected and inoculated mechanically onto various plants to determine host range. The induced symptoms were divided into three groups. The first group included the variable systemic symptoms exhibited by V. unguiculata N. tabacum and V. faba. Calico symptoms were induced on inoculated S. tuberosum plants. The second group included chlorotic local lesions exhibited by Ch. quinoa and Ch. amaranticolor. The third group included the other plants that were not infected by these isolates (P. sativum N. benthamiana C. annum C. sativus and N. rustica).

RT-PCR

Figure (3) show electrophoresis analysis of the RT-PCR products of AMV-CP gene generated from leaf samples from symptomatic potato plants collected from Hail (lanes 23) and Wadi aldawasser (lanea 45) regions respectively. No fragments were amplified from the RNA extracted from healthy plants (lane 1).

Dot Blot Hybridization Assay

Dot blot hybridization was used to detect the virus in infected potato plant tissues. Fig. 4 shows a moderate reaction (blue signal) resulting from dot blot hybridization of the DIG labeled probe with nucleic acids extracts from all twenty-three potato samples collected from Hail (Row A: 1-8 and Row B: 1-5) and Wadi aldawasser regions (Row B: 6-8 and Row C: 2-8). However hybridization was not observed between the probe and total nucleic acids from uninfected potato leaf tissue (Row C: 1).

Nucleotide Sequence and Phylogenetic Analyses

Sequence comparisons showed that the percentage of similarity between AMV-PSA-Ha and twenty-eight reported isolates of AMV obtained from GenBank ranged from 96.5 to 99.7% while the identity ranged from 93.2% -97.4%with the AMV-PSA-Wd isolate. The AMV-PSA-Ha isolate was closely related (99.7%) to AMV-Saj Saudi Arabia (KC434083) and Aq Australia (JX112758) isolates also high similarity (99.5%) was shares with Hu (JX112759) in Australia and Brazil (FJ858265) isolates isolated from alfalfa. Also this isolate was shares the high sequence identity (99.1%) with N20 (HM807304) S40 (HM807306) W1( HM807307 ) isolates in Australia one isolate from China ( JQ281522) isolated from alfalfa and Ca175 isolate ( DQ314750) in Canada which isolated from potato. AMV- PSA-Ha shares the lowest sequence homology (94.1%) with the AMV-WD isolate (KC434084) which was isolated from alfalfa in Wadi aldawasser region Saudi ArabiaThe AMV-PSA-Wd isolate shares the highest sequence homology (97.4%) with the AMV-Wd isolate (KC434084) isolated from alfalfa grown in Wadi Aldawasser region Saudi Arabia. The high similarity (96.2 to 96.3%) was shares with the Chinese (JQ281522) Brazilian (FJ858265) Australian isolates of N20 (HM807304) S40 (HM807306) and W1 (HM807307) isolated from alfalfa. The AMV-PSA- Wd isolate shares the lowest sequence homology (93.2%) with a Serbian isolate (FJ527748) isolated from alfalfa (Fig.5 and Table 1).The partial nucleotide sequences containing the CP genes of the AMV-Saudi Arabian isolates were deposited in the GenBank under the accession number no. KC569796 and KC569797 for the AMV-PSA-Wd and AMV-PSA-Ha isolates respectively.

Discussion

Potato plants are infected by many viruses under field conditions (Salazar 1996; Al-Shahwan et al. 1998). Such viruses have been shown to cause great economic losses and are considered to be the major limiting factors of potato production. Among such viral diseases AMV was found to be widely distributed on potato plants. AMV is the type member of the genus Alfamovirus in the Bromoviridae family of plant viruses. AMV is a world-wide distributed virus (Jasper and Bos 1980) with a very wide host range. This virus can naturally infect many herbaceous and some woody plant hosts and causes diseases of many economically important crops including the families Solanaceae and Leguminosae (Hiruki and Miczynski1987) however AMV was considered as economically less important when compared to potato viruses (Salazar1996) but was considered as one of the most prevalent viruses in the Saudi Arabia (Al-Shahwan et al. 1998; Al- Shahwan 2003) and also in other countries (Jung et al.2000; Xu and Nie 2006; Pourrahim et al. 2007; El-Helalyet al. 2012). In this study AMV was mechanically inoculated onto different host plants and the resultsobtained in this report were compatible with the findings oother reports (Xu and Nie 2006; Baldo et al. 2010; Parrella and Acanfora 2010).In this study host range studies and serological reactions with AMV antisera showed that the two isolatesisolated from potato were of the Alfamoviruses previously reported to be pathogenic on several crop species (Paliwal1982; Valkonen et al. 1992). The close relationship between the two isolates was confirmed based on biological assays serological reactions and molecular techniquesincluding RT-PCR molocular hybridization andsequencing.The size of the major RT-PCR product from AMV- infected tissue was identical to that of the 700 bp CP gene of AMV; however the specific primer pair did not amplify viral cDNA from extracts of uninfected potato plants. The primers for detection of AMV (AMV coat-F and AMV coat-R were specific for AMV RNA (Xu and Nie 2006). PCR and RT-PCR assays (Saiki et al. 1988) which involve the enzymatic amplification of a DNA fragment defined by two oligonucleotide primers have been used to diagnose a number of plant viruses belonging to several different groups (Henson and French 1993). These method has been successfully utilized to detect viruses from infected potato tubers and leaves (Lopez-Moya and Loez-Abella 1992; Singh and Singh 1995).

Table 1: Nucleotide identity percentage of the CP gene sequences of the two AMV Saudi Arabian isolates isolated from potato with those of AMV isolates originating in different countries

AMV###Country###Isolate/###Organic###Percentage of Identity

###AMV- AMV-

GenBank###Strain###Host

Accession###PSA-Ha PSA-Wd

Number

AF294432###Korea###KR1###S. tuberosum###96.8%###94.4%

AF294433###Korea###KR2###S. tuberosum###97.1%###94.7%

AMU12509###M. sativa

###New Zealand###NZ1###96.1%###93.2%

AMU12510###New Zealand###NZ2###M. sativa###96.1%###93.2%

DQ314749###Canada###Ca375###S. tuberosum###96.1%###93.7%

DQ314750###Canada###Ca175###S. tuberosum###96.3%###95.1%

DQ314751###Canada###Ca399###S. tuberosum###96.1%###93.5%

###S. tuberosum

DQ314752###Canada###Ca400###96.1%###93.7%

DQ314753###Canada###Ca401###S. tuberosum###96.1%###93.5%

DQ314754###Canada###Ca508###S. tuberosum###95.8%###93.2%

DQ314755###Canada###Ca518###S. tuberosum###95.4%###93.0%

DQ314756###Canada###Ca616###S. tuberosum###95.8%###93.2%

FJ527748###Serbia###95-08###M. sativa###96.1%###92.9%

FJ858265###Brazil###-###M. sativa###98.6%###95.4%

HM807304###Australia###N20###M. sativa###98.3%###95.4%

HM807305###Australia###S30###M. sativa###96.8%###94.0%

HM807306###Australia###S40###M. sativa###98.3%###95.4%

HM807307###Australia###W1###M. sativa###98.3%###95.4%

JX112757###Australia###EW###M. sativa###98.0%###94.7%

JX112758###Australia###Aq###M. sativa###98.8%###95.6%

JX112759###Australia###HU###M. sativa###98.6%###95.4%

JQ685858###Iran###Ke.Sh.Al###M. sativa###96.3%###94.2%

JQ685859###Iran###Ke.Si.Al###M. sativa###96.4%###94.4%

JQ685860###Iran###Ke.Ba.Po###S. tuberosum###96.4%###94.1%

HQ288892###Egypt###-###S. tuberosum###96.1%###93.5%

JQ281522###China###-###M. sativa###98.1%###95.4%

KC434083###Saudi Arabia###Saj###M. sativa###99.7%###96.4%

KC434084###Saudi Arabia###Wd###M. sativa###94.0%###97.4%

KC569796###Saudi Arabia###PSA-Wd###S. tuberosum###96.5%###100%

KC569797###Saudi Arabia###PSA-Ha###S. tuberosum###100%###96.5%

Dot blot hybridization show that the DIG-labeled cDNA probe is sensitive enough to allow for detection of AMV in infected potato plants. Moreover the hybridization was more sensitive than ELISA as more samples tested positive for AMV infection. For routine testing of large numbers of samples the dot blot hybridization assays are more convenient. Dot blot hybridization assays have been used widely to detect AMV and other viruses and viroids in infected plants (Peiro et al. 2012).Sequencing analysis of both Saudi Arabian AMV isolates showed slight differences in the nucleotide sequences of their CP genes. Sequence comparison revealed that the two Saudi Arabian isolates of AMV isolated from potato plants shared 93.2% to 99.7% sequence similarity with the twenty-eight reported isolates of AMV obtainedfrom GenBank. The occurrence of AMV isolates that havenucleotide sequences similar to the Saudi isolates isolated from potato plants may suggest the common origin of distantly distributed isolates which can be attributed to the widespread tuber and mechanical transmission of the two virus isolate.This study confirmed that the AMV is still threatening the potato crop production in Saudi Arabia. DNA analysis showed that there was high genetic similarity of the CP gene of the collected isolates indicating that the virus may spread through potato tubers among the two geographic distantpotato-growing regions in the Kingdom. Also as long as thevirus was isolated from alfalfa further studies are needed to investigate the presence of this virus in weeds and other crops in order to study the epidemic of the virus in the Kingdom.

Acknowledgement

The authors are grateful to the Agricultural Research Center College of Food and Agricultural Sciences King Saud University for the supporting this research. Thanks to Dr. Ahmed Ali Al-Qahtani at Biological and Medical Research Department King Faisal Specialist Hospital and Research Center Riyadh Saudi Arabia for cooperation and provision of the necessary facilities valuable for sequencing.

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Author:Al-Saleh, Mohammed A.; Amer, Mahmoud A.; Al-Shahwan, Ibrahim M.; Abdalla, Omer A.; Shakeel, M.T.
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:7SAUD
Date:Oct 31, 2014
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