- Email: [email protected]
Detection of potato mop-top virus in soils and potato tubers using bait-plant bioassay, ELISA and RT-PCR
Accepted Manuscript Title: Detection of potato mop-top virus in soils and potato tubers using bait-plant bioassay, ELISA and RT-PCR Author: Muhammad Arif Murad Ali Anayatur Rehman Muhammad Fahim PII: DOI: Reference:
S0166-0934(13)00426-6 http://dx.doi.org/doi:10.1016/j.jviromet.2013.10.022 VIRMET 12344
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
18-6-2013 4-10-2013 8-10-2013
Please cite this article as: Arif, M., Ali, M., Rehman, A., Fahim, M.,Detection of potato mop-top virus in soils and potato tubers using baitplant bioassay, ELISA and RT-PCR, Journal of Virological Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.10.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
PESHAWAR, PAKISTAN: 18 June 2013
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Arie J. Zuckerman The Editor-in-Chief, Journal of Virological Methods Email: [email protected] Tel: +44 (0) 20 7830 2579 Fax: +44 (0) 20 7830 2070
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Dear Sir/Madam,
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Attached please find a research paper to be published in Journal of Virological Methods. The title and authorship details are given below:
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Detection of Potato mop-top virus in soils and potato tubers using bait-plant bioassay, ELISA and RT-PCR by Muhammad Arif, Murad Ali, AnayaturRehman and Muhammad Fahim
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Potato mop-top virus (PMTV) is one of the most difficult to detect and control among all plant viruses. It is an emerging problem in this part of world (South Asia) and a limiting factor for seed and ware potato production. The virus is reported and cause serious losses in Europe, North and South America, Japan and China. Recently, PMTV and its vector, is one of the most important problem in seed potato production in Canada and USA. The virus is vectored by a soil-borne fungus-like organism, Sponsgospora subterranea, (the casual agent of powdery scab disease) and remained in resting spore (cystorosus) of the vector for more than 20 years in soils with out a plant host. Due to such properties of the virus, PMTV is one of most difficult problem to control in fields. The virus is evenly distributed in plant host and difficult to detect. The research paper submitted for publication in JVM reports methods and techniques for the rapid detection and identification of the virus in soils as well as in plant hosts. Due to the important nature of the problem, it is therefore requested that the manuscript may kindly be published in forthcoming issue of JVM. With best regards
Prof. Dr. Muhammad Arif Department of Plant Pathology The University of Agriculture Peshawar, Peshawar-25130, PAKISTAN Phone: +92 91 9216552 Fax: +92 91 9216520 Cell: +92 300 595 9748
e-mail: [email protected], [email protected]
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Manuscript
Detection of potato mop-top virus in soils and potato tubers using baitplant bioassay, ELISA and RT-PCR
Muhammad Arifa,*, Murad Alia, Anayatur Rehmana and Muhammad Fahimb a
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Department of Plant Pathology, The University of Agriculture Peshawar, Peshawar-25130, Pakistan b Institute of Biotechnology and Genetic Engineering, The University of Agriculture Peshawar, Peshawar-25130, Pakistan
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Keywords: Detection, PMTV, Plant Bioassay, ELISA, RT-PCR, Pakistan
__________________________________________________________________ Corresponding author at: Department of Plant Pathology, The University of
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Agriculture Peshawar, Peshawar-25130, Pakistan
Tel.: +92 91 9216552; fax: +92 91 9216520; E-mail address: [email protected] (M. Arif)
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ABSTRACT The hilly region of Northwest of Pakistan is leading seed potato producing areas of the country. Soil and plant samples were collected from the region and tested for PMTV using both conventional and molecular techniques. The bait plants exhibited PMTV-characteristic v-shaped yellow leaf markings in
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Nicotiana debneyi plants grown in putative viruliferious soils from 20/26
locations. The results were confirmed by back inoculation of sap from both
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roots and leaves of bait plant on indicator hosts (N. debneyi, N. benthamiana). The root samples of bait plants grown in soils of 25 locations and leaves of 24
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locations reproduced systemic infection on indicator hosts upon back inoculation. The virus was identified in bait plants grown in soils from 25/26
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locations using double antibody sandwich-enzyme linked immunosorbent assay (DAS)-ELISA and reverse transcription and polymerase chain reaction (RT-PCR) methods. The products of the 566 bp were amplified from coat
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protein region of PMTV RNA 3 in both root and leaf samples of baited plants. The virus was detected in 10 potato cultivars commercially grown in the region
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using DAS-ELISA and RT-PCR. The virus was also detected in zoospores of
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Spongospora subterranea derived from the peels of selected scabby tubers using triple antibody sandwich (TAS)-ELISA. The results indicate that a bait
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plant bioassay, infectivity assay, ELISA and RT- PCR can detect PMTV in roots and leaves of baited plants, field samples, zoospores of S. subterranea
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and tubers of 10 potato cultivars commercially grown in the region.
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1. Introduction Potato mop-top virus (PMTV) has fragile rod-shaped particles, transmitted by the soil-borne plasmodiophorid vector, Spongospora subterranea f. sp. subterranea (Wallr.) Lagerth. (Jones and Harrison, 1969; Harrison and Jones, 1970; Arif et al., 1995) and is a type species of genus Pomovirus, family
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Virgaviridae (King et al., 2012). PMTV has particles of two predominant
lengths of about 100-150 nm and 250-300 nm and diameters of 18-20 nm
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(Harrison and Jones, 1970; Harrison and Reavy, 2002). It is characterized by a tripartite, single-stranded RNA genome; the genomic components are
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separately encapsidated in capsid protein (Harrison and Jones, 1970; Savenkov et al., 2003). The virion contains three molecules of linear positive sense
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single-stranded RNA species (Kallender et al., 1990) of 6043nt (RNA 1; Savenkov et al., 1999), 2962 nt (RNA 2; Scott et al., 1994), and 2315 nt (RNA 3 of T isolate; Kashiwazaki et al., 1995). RNA 1 encodes polypeptides of 148
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kDa and 206 kDa, which are considered to be components of viral RNAdependent RNA polymerase, with the second protein probably being expressed
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translational read-through of the first cistron (Savenkov et al., 1999). RNA 2
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encodes the triple-gene-block (TGB) of movement protein and a putative small cysteine-rich 8 kDa protein (Scott et al., 1994; Kashiwazaki et al., 1995). RNA
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3 encodes the capsid protein and a read-through protein (Kashiwazaki et al., 1995; Sandgren et al., 2001). The read-through gene is expected to be involved in acquisition and transmission of the virus by the vector, S. subterranea (Reavy et al., 1998; Arif et al., 1999a). RNA 3 of newly isolated Scottish
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isolate S (PMTV-S) is at least 543 nt larger than that of T isolate (Reavy et al., 1998) and RNA 3 of a Swedish isolate (PMTV-Sw) is 3134 nt (Sandgren et al., 2001). The virus particles also contain a small amount of a read-through protein produced by suppression of coat protein termination codon (Cowan et al., 1997). The read-through protein has a Mr of 66,900 in the T isolate (Kashiwazaki et al., 1995), 87,000 in the S isolate (Reavy et al., 1998) and 91,000 in Sw isolate (Sandgren et al., 2001). The vector of PMTV, S. subterranea also causes powdery scab disease on tubers of susceptible cultivars (Hims and Preece, 1975; Harrison et al., 1997; Merz, 2008). Infections with virus can cause serious potato tuber ‘spraing’ 3
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disease symptoms (Sandgren, 1995; Sandgren et al., 2002) which can occur as brown arcs and circles in the flesh of tubers of susceptible cultivars upon potato planting in infested fields (primary infection) (Harrison and Jones, 1971; Kurppa, 1989). The secondary symptoms of PMTV are produced when potato plants are grown from virus infected seed tubers harvested after primary infection. The symptoms are production of chevron or yellow blotches on
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leaves and bunching effect due to shortening of internodes (mop-top) (Kurppa,
1989). The secondary infection, tubers can show cracks, blotchy surface
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markings or distortion (Calvert and Harrison, 1966; Harrison and Jones, 1970). The vector S. subterranea acquires and transmits PMTV in vivo through mono
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fungal culture (Arif et al., 1995). The virus can maintain its infectivity for more than 18 years inside cystosori without the host plant (Calvert, 1968), therefore,
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the control of the virus is a challenge.
PMTV occurs in potato growing regions of Northern and Central Europe, the Andean regions of South America, China and Japan (Jones, 1988).
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Recently, PMTV is reported as an important pathogen of seed potato in Canada (Xu et al., 2004), Poland (Budziszewska et al., 2010) and the United States of
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America (Lambert et al., 2003; Xu et al., 2004; David et al., 2010; Crosslin,
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2011).
Several surveys have been carried out in the past for the assessment of
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powdery scab in Northwest (Khyber Pakhtunkhwa) and Northern areas (Gilgit Baltistan) (Ahmad et al., 1996). However, no work has been done on detection, identification and characterization of PMTV in Pakistan. Here in this paper, we report detection, identification and characterization
of PMTV using
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conventional and molecular techniques.
2. Materials and Methods 2.1. Field surveys: collection of plant and soil samples Soil and plant samples were collected from major seed/ware potato producing areas of the Northwest of Pakistan (Table 1). The Northwest was divided into six different zones. The zones A-G were further divided into locations viz A-E were divided into four locations each while each location in zone F-G were further divided into three locations. Each location in all six
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zones was further divided into three sites/fields. Plant sampling was made in an estimated area of four meter square of each selected site/field. After harvest, tubers were washed, examined for superficial spraing symptoms and a total of 10-15 scabby tubers (one or more scab pustules) were selected from each site of the selected field. Ten scabby tubers of each potato cultivar commercially grown in the region (Table 2) were also selected separately. Tubers were stored
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for 3-5 months at 7-8°C. Approximately 500 g soil samples were collected
from cropping layer (5-10 cm) below the surface of each of three sites/
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locations, about 20-25m intervals and 3-4m inside the boundaries of each field.
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The soil was mixed thoroughly and kept moist at 4°C until used for air drying.
2.2. Soil-bait test
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Field soil samples were spread out on paper sheet and air-dried for seven to 10 days at 22-24°C. The dried soil samples were ground into fine powder using pestle and mortar and passed through 300 and 200 micro meters sieves,
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respectively. Twenty grams of fine soil powder was placed in each of four depressions in stream-sterilized potting mixture (sand: clay: compost, 1:1:1
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ratio v/v) in one liter plastic pots. The pots were soaked 10-15 h with tap water
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in a steel tray (4 pots/tray/treatment). Four Nicotiana debneyi seedlings were transplanted into each pot. The pots were placed in glasshouse at 18-20°C. The
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pots were flooded to half their height in water for three days, then the water was allowed to drain and the pots were left with out water for four days. The plants were uprooted after six to eight weeks for virus testing through back inoculation of sap from leaves and roots to indicator plants (N. debneyi, N.
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benthamiana), by triple antibody sandwich/double antibody sandwich-enzymelinked immunosorbent assay (TAS/DAS-ELISA), and by reverse transcription and polymerase chain reaction (RT-PCR).
2.3. Serological detection of PMTV in Spongospora subterranea through TASELISA TAS-ELISA was done as described previously (Torrance et al., 1992; Arif et al., 1994). Leaf and root samples (1 g/5 ml) and tuber tissues (1 g/ml) were extracted in extraction buffer [0.01 M phosphate buffered saline (PBS) containing 0.05% Tween-20 and 0.1 -0.2% skimmed milk powder (EveryDay, 5
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Nestlé Pakistan, Lahore, Pakistan)]. Samples were added to microtitre plates
(NUNC
Immunoplate
II)
pre-coated
with
polyclonal
anti-PMTV
immunoglobulin (IgG) diluted 1:500 and monoclonal anti-PMTV IgG (1:1000) with coating buffer pH 9.6. For the virus detection in zoospores, preparation were obtained by air drying scabby potato peels and incubating 0.1-0.2 g of cystosori in 10 ml of sterile distilled water (SDW) at 12-15°C for 7-10 days.
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The germinated zoospore suspensions were filtered through fine plastic mesh
to remove debris. Between c 1x107 and 6x107 zoospores per ml were present in
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the suspensions. The zoospores were centrifuged at 800 g for five min, resuspended in 2 ml extraction buffer, frozen at -70°C for 20 min, then thawed
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on ice and used in TAS-ELISA.
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2.4. Serological detection of PMTV in potato and bait plant through DASELISA
DAS-ELISA was used for the detection of PMTV using commercial
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ELISA kit (Bioreba, Switzerland). The tests were performed in polystyrene micro-plates (NUNC, Immunoplate II, Thermal Scientific, Waltham, MA,
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USA) as essentially described by Clark and Adams (1977). The plates were
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coated with 200 µl aliquots of PMTV–specific antibody (Bioreba, Chr. Merian, Switzerland) with coating buffer, pH 9.6. The plates were kept at RT in a
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humid box for 3-4 h. Leaf samples were extracted by crushing through pestle and mortar in extraction buffer, pH 7.4. Leaf and root samples were extracted in extraction buffer at 1:5 (w/v) while tuber 1:1 (w/v) ratio and 200µl of prepared sample was dispensed in each well after three times washing. After
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first incubation was completed the plates were washed with 1 x PBST, pH 7.4. Controls were used as provided and recommended by the supplier (Bioreba, Chr. Merian, Switzerland). ELISA plates were incubated inside a humid box for 4 h at RT or 4-6°C overnight in refrigerator. After washing, 200ul of enzyme conjugate (Bioreba, Chr. Merian, Switzerland) (1:1000) was dispensed in each well of plate and incubated in a humid box for 4 h. at RT. The plates were washed three times with washing buffer. 200µl of substrate solution (dissolve para-nitrophenyl-phosphate (pNPP) at 1 mg/ml) and plates were incubated at RT in the dark. Absorbance at 405 nm (A405) was measured with Titertek Multiskan, Model MC (Flow Laboratories, Covina, CA, USA) after 1 6
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h or 2 h of substrate incubation at RT 24-25°C, and overnight (c. 16 h) at 4°C. Samples were considered to be positive when the A405 values exceed the virusfree samples by at least a factor of three.
2.5. Molecular detection of PMTV in bait plants and potato tubers 2.5.1. RNA Extraction and first cDNA strand synthesis
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Total RNA extracts were made from roots and leaves of N. debneyi (bait plants) showing typical yellow v-shaped markings in their leaves and internal
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necrosis (brown arc) in potato tuber flesh as described by Verwoerd et al.
(1989) and Barker et al. (1993), respectively. In most cases, RNA extraction kit
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(Invitrogen; Life Technologies, Carlsbad, CA, USA) was used with the extraction procedure based on manufacture’s instructions. RNA pellet was
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dissolved in RNase-free water (50µl). The final RNA concentration was measured with a GeneQuartTM photometer (Pharmacia, Stockholm, Sweden). First stand cDNA was synthesized by mixing two micro grams of total
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RNA with one micro gram of downstream primer, 1 micro liter 10 x PCR buffer (10mM Tris-HCl, pH 8.4 containing 50 mM KCl and 2 mM MgCl2), and
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2 mM of each of four deoxynucleoside triphosphates (dNTPs) in a total volume
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of 10 µl. The mixture was heated to 65°C for 2 minutes followed by slow cooling to 42°C on heating block, then 20 Units of RNase inhibitor and 20
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Units reverse transcriptase (Boehringer-Mannheim, Germany) were added and the mixture incubated at 42°C for 2 h. The total cDNA product was then amplified by PCR.
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2.5.2. Polymerase chain reaction (RT-PCR) The genome of PMTV consists of three ssRNA species and the coat protein
gene is located on RNA 3 (Kashiwazaki et al., 1995). Olignucleotides primers were designed to amplify the coat protein gene, with additional (underlined) nucleotides to create a BamHI site. Primer 1 (downstream) had the sequence: 5`-CGGGATCCTATGCACCAGCCCAGCGT-3` complementary to residues 801 to 819 of PMTV (isolate T) RNA 3 , and primer 2 (upstream) had the sequence:
5`-TCGGATCCTCTCGGATACCACCCTT-3` identical to residues
268 to 284 of PMTV (isolate T) RNA 3.
The oligonucleotides were
synthesized on an Applied BioSystems, Model 391 PCRmate DNA synthesizer 7
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(Applied Biosystems, Foster City, CA, USA). The primers were expected to amplify a band of 566 bp corresponding to the PMTV coat protein gene with additional BamHI sites (Arif et al., 1994). The PCR mixture (50µl) contained one micro liter each of down-steam and upstream primers, 2µl of a 2 mm solution of each of the four dNTPs, 1.5 µl of 50mM MgCl2 , 10 µl 10 x PCR buffer, 1 µl (5 units) Taq DNA polymerase
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(Promega, Madison, WI, USA) and 28µl water. The mixture was subjected to 40 cycles of heating and cooling in PTC-200 Thermal Cycler (M J Research,
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Watertown, MA, USA). Each cycle consisted of 1.5 min at 94°C for
denaturation, 1.5 min at 54°C for primer annealing, and 2.5 min. at 72°C for
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primer extension, followed by a 10 minutes final extension at 72°C. The PCR products (10 µl) were electrophoreses in 1% agrose gels in Tris-borate-EDTA
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buffer containing 0.5 µl/ml ethidium bromide. Molecular size markers (Promega, Madison, WI, USA) were used as control. The specific bands were
Laboratories, Hercules, CA, USA)
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3. Results
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visualized under UV light using Quantity One for Windows (Bio-Rad
3.1. Detection of PMTV through soil-bait tests
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Root and leaves samples from each of the four N. debneyi plants (in one
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pot/location) were tested after 6-8 wk by symptomatology, ELISA, RT-PCR and back inoculation of sap to indicator plants. The bait plants (N. debneyi) exhibited v-shaped yellow leaf markings (Fig. 1a). A total 20/26 locations soil samples from seven zones showed PMTV infected bait plants on the basis of
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symptoms (Table 1). In TAS/DAS-ELISA, PMTV was detected in bait plants grown in putative viruliferious soils from 25/26 locations. The A405 values in samples considered positive was quite higher than that of the values of healthy sap (Table 1).
RT-PCR analysis of pooled samples of all four N. debneyi bait plants indicated that out of 26 locations, PMTV was detected in 25 and 24 roots and leaves of bait plants, respectively. The root sample of bait plants of one location (Mankyal) was positive while virus was not detected in leaves samples using RT-PCR (Table 1; Fig 2). A 566 bp band was visible in agarose gels from both roots and leaves of baited plants (Fig 2). The results were further 8
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confirmed by back inoculation of sap from both roots and leaves of bait plants on indicator plants [N. debneyi or N. benthamiana (Fig. 1b)]. The root samples of bait plants grown in soils of 25 locations and leaves of 24 locations reproduced systemic infection on indicator host upon back indexing (Table 1). Both roots and leaves samples of bait plants from two locations (Ashrait –zone C and Kalam-zone E) produced mosaic symptoms similar to PVX and infected
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indicator plants were killed two weeks after back inoculation. However, no specific PVX symptoms were observed in bait plants (N. debneyi) grown in
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infested soils of both locations.
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3.2. Detection of PMTV in tubers of commercial potato cultivars grown in Northwest of Pakistan
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PMTV was detected through symptomatology, ELISA and RT-PCR in scabby tubers of 10 cultivars commercially grown in Northwest of Pakistan
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(Table 2). Composite samples were taken from brown arc or necrotic tissues, rose and stolon ends of each tuber of 10 cultivars for both ELISA and RT-PCR. Potato peels of scabby tubers were separately made for ELISA detection of
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PMTV in zoospores of S. subterranea. PMTV was also detected in almost all
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scabby tubers of commercial cultivars grown in Northwest of Pakistan, when tested through ELISA (Table 2). The results further confirmed by the detection
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of PMTV in zoospores of S. subterranea derived from the peels of selected scabby tubers of 10 cultivars showing powdery scab symptoms viz blisters and pustules on surface and internal browning in flesh of tubers. PMTV was not
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detected through ELISA in scabby tubers of any cultivar where there was no internal browning in the flesh of tubers or zoospores derived from scabby tubers with out internal browning symptoms (Table 2). PMTV internal browning was positively correlated with the presence of the virus but external symptoms of the virus or the vector were not positively correlated with PMTV infection in the tubers (Table 2). The results were further confirmed through RT-PCR (Fig. 3). RT-PCR amplified 566 bp band of CP region in scabby tubers of all 10 potato cultivars showing internal browning or brown arc symptoms. However, the virus was not detected in scabby tubers of any cultivar without internal browning or brown arc symptoms (result not shown).
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4. Discussion In Pakistan, potato is being produced though out the year with wide range of different agro-ecological zones but seed potato produced mainly in autumn and summer. Both formal and informal seed potato production systems exist in Pakistan. The bulk of the seed produced in the country is derived form informal
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seed sector and over 97% of total seed requirements are met by the home
grown seed potatoes and less than 3% are met by the certified seed including
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local and imported sources (Hussain and Farooq, 1995). The Northwest of Pakistan is leading seed potato producing areas for both informal and formal
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seed systems in the country. The long presence of powdery scab disease in potato in the region (Ahmad et al., 1996) led to the investigation of the
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occurrence and distribution of PMTV in potato germplasm commercially grown in the region. Occurrence and distribution of any of plant virus is important to determine the economic importance and significance of viral
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disease in target areas. Significance of the virus will be very high when crop propagated through seed tubers and the pathogen is tuber-perpetuated. The
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seriousness of the problem can be assessed from the fact that the virus is
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persistent in resting spore (cystosorus) of the vector (S. subterranea) and remains viable in viruliferious cytosorus for more than 18 years without the
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host plant (Calvert, 1968). The resistance has not been identified yet in potato germplasm against the virus (Tenorio et al., 2006). As mentioned above, the farmers in Northwest of Pakistan use to multiply their own seed. The flow of virus-free certified seed is limited in the system due to several factors and the
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practice of growing multiple crops (3-4 crops/year) without proper implementation of quarantine regulations in the region. With such limitations, occurrence of PMTV and its vector in major seed/ware potato producing areas (Northwestern region) is extremely important and need immediate attention. The results of this investigation indicates that PMTV is prevalent in Northwest including major seed producing areas of Malakand and Hazara divisions, presumably extended to seed producing areas of Northern Areas and the Azad Jammu and Kashmir. In this study, bait test supplemented with ELISA or RT-PCR or both, detected successfully PMTV in infested soil. Arif et al. (1994) used bait test for 10
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the detection of PMTV in infested soils. The use of TAS/DAS ELISA and RTPCR of the bait test plants further confirm the identity of the virus. Back indexing of sap from both roots and leaves of bait plants (especially roots) on test plants (N. debneyi or N. benthamiana) allow multiplication of a few virus particles and serves as a successful bio-assay for the detection of PMTV and other viruses transmitted through plasmodiophorid vector (Chen and Wilson,
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1995; Dessens and Meyer, 1996).
The results revealed that powdery scab symptoms on potato tubers such as
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blisters and pustules does not guaranteed the presence of PMTV (Table 2). However, PMTV was readily detected by ELISA or PCR or both in potato
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tubers with brown arc or internal browning symptoms in tuber flesh. It was concluded that ELISA or PCR could not detect PMTV in scabby tubers or
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tubers with spraing symptoms without internal browning or brown arc in tuber flesh. In a previous attempt, it was reported that TAS-ELISA failed to detect PMTV in tubers that displayed spraing symptoms and RT-PCR results from
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such tubers were extremely weak (Arif et al., 1994). Torrance et al. (1992) reported that TAS-ELISA on recently harvested symptoms less tubers from
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infected mother plants detected PMTV in some extract but not others parts of
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the same tuber. It may be due to physiological age or temperature at which tubers were stored might affect virus replication and spread or mother plant
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was infected late and has low virus titer or even high specificity of TASELISA. The possibility of infection of aviruiliferous S. subterranea producing blister on tubers could not be avoided. Although we did not carry out any assay for the detection of other viruses,
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the possibility of PVX contamination on limited scale in soil samples from Ashrait and Kalam, Malakand division, cannot be ignored. Tenorio et al. (2006) reported PVX infection in bait plants growing in infested soils and shown the possibility of presence of Synchytrium endobioticum spores that transmits PVX under greenhouse and field conditions. Some of the aspects that need further investigation the how much density of S. subterranea carrying PMTV and the phenomenon whether a PMTV infected potato plant through secondary infection play a role to make S. subterranea viruliferous or a viruliferous vector delivers virus in the plant through primary infection or both. Answers to theses questions may help to develop control 11
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strategies against viruses transmitted through plasmodiophoroid vectors. Although natural resistance in potato is not available, the availability of transgenic resistance can be one of the best options to minimize losses (Reavy et al., 1995; Arif et al., 1999b; Reavy et al., 1997). The CP gene of the virus incorporated in to potato and transgenic potato plants were again highly resistant (immune) to vector-inoculated virus in screen-house tests (Barker et
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al., 1998). Exploitation of such approaches may lead toward development of effective and durable resistance against PMTV.
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Improvement in diagnostic techniques of PMTV (Torrance et al., 1992;
Arif et al., 1994; Arif, 1995; Nielsen and Mølgaard, 1997; Nakayama et al.,
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2010) lead to proper detection and identification of the virus in soils, plants, tubers and zoospores of S. subterranea but development of effective control
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measure for such pathogen is still a challenge (Arif, 2000). The facts mentioned above in connection with introduction and spread of PMTV in Northwest of Pakistan is an open challenge to the stakeholders and the
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scientific community of the country. Recently, farmers have started abandoning growing potatoes and have moved to other valuable cash crops in
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the areas such as peas and cabbage, etc. (M. Arif and M. Fahim, personal
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communication). Though it will greatly assist in reducing the active inoculums pressure; potatoes might still remain in practice as alternative crop for the
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purpose of crop rotation. It is therefore high time to adopt every measure to check both the virus and the vector in seed producing areas of Northwest and elsewhere to save potato production in Pakistan. Emphasize should be given to produce virus-free seed potato in small pockets that are virus free, while at the
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same time, strict quarantine measures should be adopted to avoid introduction of potato viruses in the area. This investigation will serve as a baseline to stimulate research on PMTV, its vector and other soil-borne viruses, in Pakistan.
Acknowledgements This work was funded by Higher Education Commission, Islamabad, Pakistan under National Research Program for Universities (NRPU) through Grant No 20-1182/ R & D /08.
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Arif, M., Torrance, L., Reavy, B., 1995. Acquisition and transmission of potato mop-top furovirus by a culture of Spongospora subterranea f. sp. subterranea derived from single cystosorus. Ann. Appl. Biol. 126, 493-503.
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Arif, M., Reavy, B. Torrance, L., 1999a. Read-through protein gene of potato mop-top furovirus is associated with acquisition and transmission of the virus by Spongospora substerranea f.sp subterranea. Pak. J. Bot. 31, 225-236.
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Arif, M., Reavy, B. Barker, H., 1999b. Engineering high level of coat protein mediated resistance to fungal transmission of potato mop-top furovirus in Nicotiana benthamiana. Pak. J. Biol. Sci. 2, 478-483
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Arif, M., 2000. Fungally-Transmitted Rod-shaped Viruses, Biology, Transmission and Molecular Pathology. Pak. J. Biol. Sci. 3, 1194-1212.3. Barker, H., Webster, K. D., Reavy, B., 1993. Detection of Potato virus Y in potato tubers: a comparison of polymerase chain reaction and enzyme-linked immunosorbant assay. Potato Res. 36, 13-20.
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Barker, H., Reavy, B., McGeachy, K. D., Dawson, S., 1998. Transformation of Nicotiana benthamiana with potato mop-top virus coat protein gene produces a novel resistance phenotype mediated by the coat protein. Mol. Plant-Micro. Inter. 11, 626-633.
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Budziszewska, M., Wieczorek, P., Nowaczyk, K., Borodynko, N., Pospieszny, H., Obrepalska-Steplowska, A., 2010. First report of Potato mop-top virus on potato in Poland. Pl. Dis. 94, 920.
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Calvert, E. L., 1968. The reaction of potato varieties to mop-top virus. Record of Agricultural Research of the Ministry of Agriculture for the Northern Ireland 17, 31-40.
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Calvert, E. L., Harrison, B. D., 1966. Potato mop-top, a soil-borne virus. Plant Path. 15, 134-139. Chen, J. P., Wilson, T. M. A., 1995. Taxonomy of rigid rod-shaped viruses transmitted by fungi. Agronomie 15, 421-426. Clark, M. F., Adams, A. N., 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for detection of plant viruses. J. Gen. Virol. 34, 475-483.
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Cowan, G. H., Torrance, L., Reavy, B., 1997. Detection of potato mop-top virus capsid readthrough protein in virus particles. J. Gen. Virol. 78, 1779-1783. Crosslin, J. M., 2011. First report of Potato mop-top virus on potatoes in Washington State. Pl. Dis. 95, 1483. David, N., Mallik, I., Crosslin, J. M., Gudmestad, N. C., 2010. First report of Potato moptop virus in North Dakota. Pl. Dis. 94, 1506. Dessens, J. T., Meyer, M., 1996. Identification of structural similarities between putative transmission proteins of Polymyxa and Spongospora transmitted bymoviruses and furoviruses. Virus Gen. 12, 95-99. Harrison, B. D., Reavy, B., 2002. Potato mop-top virus. CMI/AAB Descriptions of Plant Viruses, No. 389. Commonwealth Mycological Institute/ Association of Applied Biologists, Kew, Surrey, England. Harrison, B. D., Jones, R. A. C., 1970. Host range and properties of potato mop-top virus. Ann. Appl. Biol. 65, 393-402.
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Harrison, B. D., Jones, R. A. C., 1971. Factor affecting the development of spraing in potato tubers infected with potato mop-top virus. Ann. Appl. Biol. 68, 281-289. Harrison, J. G., Searle, R. J., Williams, N. A., 1997. Powdery scab disease of potato-a review. Plant Path. 46, 1-25. Hims, M. J., Preece, T. F., 1975. Spongospora subterranea f. sp. subterranea. No. 477. In: Descriptions of Pathogenic Fungi and Bacteria. Commonwealth Mycological Insititute/ Association of Applied Biolobists, Kew, Surrey, England.
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Hussain, A., Farooq, K., 1995. Seed potato system in Pakistan. In: Research and Development of Potato Production in Pakistan. pp-41-50. Ed. A. Hussain. Proceeding of the National Seminar held at NARC, Islamabad during 23-25 April 1995. Pakistan Swiss Potato Development Project: PARC, Islamabad, Pakistan.
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Jones, R. A. C., 1988. Epidemiology and control of potato mop-top virus. In: Developments in Applied Biology II. Viruses with Fungal Vectors. pp.255-270. Eds. J. I. Cooper and M. J. C. Asher. Wellesbourne, UK: Association of Applied Biologists.
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Jones, R. A. C., Harrison, B. D., 1969. The behaviour of potato mop-top virus in soil, and evidence for its transmission by Spongospora subterranea (Wallr.) Lagerh. Ann. Appl. Biol. 63, 1-17. Kallender, H., Buck, K. W., Brunt, A. A., 1990. Association of three RNA molecules with potato mop-top virus. Netherl. J. Plant Path. 96, 47-50.
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Kashiwazaki, S., Scott, K. P., Reavy, B., Harrison, B. D., 1995. Sequence analysis and gene content of potato mop-top virus RNA 3: further evidence of heterogeneity in the genome organization of furoviruses. Virology 206, 701-706.
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King, A., Lefkowitz, M. Q., E., Adams, M. J., Carstens, E. B., (Eds). 2012. Virus Taxonomy-Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press.
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Kurppa, A. H. J., 1989. The distribution and incidence of mop-top virus in Finland as determined in 1987 and on the variation of disease symptoms in infected potatoes. Ann. Agric. Fenn. 28, 285-295.
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Lambert, D. H., Levy, L., Mavrodieva, V. A., Johnson, S. B., Babcock, M. J., Vayda, M. E., 2003. First report of Potato mop-top virus on potato from the United States. Pl. Dis. 87, 872.
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Merz, U., 2008. Powdery scab of potato-occurrence, life cycle and epidemiology. Amer. J. Pot. Res. 85, 241-246. Nakayama, T., Maoka, T., Hataya, T., Shimizu, M., Fuwa, H. Tsuda, S. Mori, M., 2010. Diagnosis of Potato mop-top virus in soil using bait plant bioassay and RT-PCRmicroplate hybridization. Amer. J. Pot. Res. 87, 218-225
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Nielsen, S. L., Mølgaard, J. P., 1997. Incidence, appearance and development of potato moptop furovirus-induced spraing in potato cultivars and the influence on yield, distribution in Denmark and detection of the virus in tubers by ELISA. Potato Res. 40, 101-110. Reavy, B., Arif, M., Kashiwazaki, S., Webster, K. D., Barker, H., 1995. Immunity to potato mop-top virus in Nicotiana benthamiana plants expressing the coat protein gene is effective against fungal inoculation of the virus. Mol. Plant-Micro. Inter. 8, 286-291. Reavy, B., Sandgren, M., Barker, H., Heino, P., Oxelfelt, P., 1997. A coat protein transgene from a Scottish isolate of potato mop-top virus mediates strong resistance against Scandinavian isolates which have similar coat protein genes. Euro. J. Plant Path. 103, 829834. Reavy, B., Arif, M., Cowan, G. H., Torrance, L., 1998. Association of sequences in the coat protein/readthrough domain of potato mop-top virus with transmission by Spongospora subterrane. J. Gen. Virol. 79, 2343-2347. Sandgren, M., Savenkov, E. I., Valkonen, J. P. T., 2001. The readthrough region of Potato mop-top virus (PMTV) coat protein encoding RNA, the second largest RNA of PMTV
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genome, undergoes structural changes in naturally infected and experimentally inoculated plants. Arch. Virol.. 146, 467-477. Sandgren, M., Plaisted, R. L., Watanabe, K. N., Olsson, S., Valkonen, J. P. T., 2002. Evaluation of some North and South American potato breeding lines for resistance to Potato mop-top virus in Sweden. Amer. Pot. J. 79, 205-210. Sandgren, M., 1995. Potato mop-top virus (PMTV) distribution in Sweden, development of symptoms during storage and cultivar trials in field and glasshouse. Potato Res. 38, 379389.
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Savenkov, E. I., Sandgren, M., Valkonen, J. P. T., 1999. Complete sequence of RNA 1 and the presence of tRNA-like structure in all RNAs of Potato mop-top virus, genus Pomovirus. J. Gen.Virol. 80, 2779-2784.
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Savenkov, E. I., Germundsson, A., Zamyatnin Jr., A.A., Sandgren, M.,Valkonen, J. P. T., 2003. Potato mop-top virus: the coat protein-encoding RNA and the gene for cysteine-rich protein are dispensable for systemic virus movement in Nicotiana benthamiana. J. Gen. Virol. 84, 1001-1005.
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Scott, K. P., Kashiwazaki, S. Reavy, B., Harrison, B. D., 1994. The nucleotide sequence of potato mop-top virus RNA 2: a novel type of genome organization for a furovirus. J. Gen.Virol. 75, 3561-3568.
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Tenorio, J., Franco, Y., Chuquillanqui, C., Owens, R. A., Salazar, L. F., 2006. Reaction of potato varieties to Potato mop-top virus infection in the Andes. Amer. J. Pot. Res. 83, 423-431.
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Torrance, L., Cowan, G. H., Scott, K. P., Pereira, L. G., Roberts, I. M., Reavy, B., Harrison, B. D., 1992. Detection and diagnosis of potato mop-top virus. Annual Report of Scottish Crop Research Institute for 1991, pp. 80-82. Verwoerd, T. C., Dekker, B. M. M., Hoekema, A., 1989. A small scale procedure for rapid isolation of plant RNAs. Nucl. Acid Res. 17, 2362.
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Xu, H., DeHaan, T. –L., Boar, S. H. D., 2004. Detection and confirmation of Potato mop-top virus in potatoes produced in the United States and Canada. Pl. Dis. 88, 363-367.
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Table 1 Detection of PMTV in N. debneyi-bait plants grown 6-8 wk in putative viruliferious soils collected from various zones/locations of Northwest of Pakistan. Detection of PMTV through: ELISA RT-PCR
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E
F
Root + + + + + + + + + + + + + + + + + + + + + +
Leaf + + + + + + + + + + + + + + + + + + + + +
+ +
0.870 (0.670) 0.954 (0.660)
1.764 (0.890) 1.141 (0.960)
+ +
+ +
+ +
+ +
+ -
1.310 (0.860) 0.110 (0.040) 1.876 (1.130)
1.623 (1.180) 0.124 (0.045) 1.980 (1.136)
+ -
+ -
+ -
+ -
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Leaf 1.540 (0.860) 1.130 (0.636) 1.141 (0.710) 1.632 (0.880) 1.427 (0.860) 0.120 (0.065) 1.123 (0.690) 1.130 (0.770) 1.134 (0.923) 1.154 (0.860) 0.983 (0.730) 0.132 (0.036) 1.132 (0.880) 0.865 (0.560) 1.321 (0.990) 1.675 (1.160) 1.768 (1.211) 1.868 (1.119) 0.923 (0.660) 1.543 (0.992) 1.432 (0.880) 1.872 (0.860) 1.321 (0.860)
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C
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B
Root 1.2031,2 (0.736) 1.120 (0.560 ) 1.132 (0.670) 1.128 (0.712) 0.987 (0.550) 0.110 (0.060) 1.350 (0.756 ) 1.123 (0.856) 0.876 (0.640) 1.142 (0.860) 1.247 (0.843) 0.698 (0.498) 1.112 (0.864) 0.981 (0.660) 1.211 (0.960) 1.342 (0.945) 1.564 (0.980) 1.432 (0.965) 1.345 (0.885) 0.876 (0.560) 1.220 (0.920) 0.987 (0.860) 1.121 (0.860)
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Shin Fatehpur Madyan Miandam Kherabad Jukhtai Senay Shahgram Cham Ghari Ashrait Mankyal Kaidam Balakot Ghruna Laikot Pishmal Kalam Gabral Ashuran Tarhana Bilal Town Academy Town Doraha Ghitti Ghatti Baffa Healthy Infected
+ + + + + + + + + + + + + + + + +
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Khwazakhela
A
Infectivity assay Root Leaf + + + + + + + + + + + + + + + + + + +3 +3 + + + + + + + + + + + +3 +3 + + + + + + + + + +
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Systemic symptoms
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Locations
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Zones
1
Mean values (A405) of four replicates Values (A405) after 1 h at room temperature and overnight (c. 16 h) in parentheses 3 Test plant back inoculated with sap from root and leaves from baited plant exhibited mosaic symptoms similar to PVX instead PMTV 2
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Table 2 Detection PMTV in potato cultivars commercially grown in various zones of Northwest of Pakistan Potato Symptoms Detection of PMTV 2 Cultivars Powdery PMTV ELISA scab1 External Internal Tubers Zoospores Desiree Blisters No symptom Necrotic lesions 0.9523,4 (0.493) 0.781 (0.421) Blisters No symptom No symptom 0.150 (0.049) 0.134 (0.043) No symptom No symptom No symptom 0.142 (0.047) 0.130 (0.042) Diamant Blisters, Necrotic rings Brown arc 1.683 (0.590) 0.965 (0.580) Pustules Blisters No symptom No symptom 0.145 (0.046) 0.133 (0.047) No symptom No symptom No symptom 0.150 (0.049) 0.134 (0.043) Cardinal Blisters, Necrotic rings Brown arc 1.780 (0.701) 1.264 (0.778) Pustules Blisters No symptom No symptom 0.152 (0.059) 0.136 (0.044) No symptom No symptom No symptom 0.140 (0.056) 0.131 (0.048) Raja Blisters, Necrotic rings Necrotic lesions, 1.670 (0.569) 1.141 (0.680) Pustules Brown arc Blisters No symptom No symptom 0.156 (0.049) 0.134 (0.043) No symptom No symptom No symptom 0.160 (0.049) 0.144 (0.041) Kuroda Blisters No symptom Necrotic lesions 0.891 (0.452) 1.234 (0.770) Blisters No symptom No symptom 0.150 (0.050) 0.134 (0.040) No symptom No symptom No symptom 0.150 (0.040) 0.134 (0.042) Rocco Pustules Necrotic rings Necrotic lesions 1.121 (0.590) 1.432 (0.876) Blisters No symptom No symptom 0.141 (0.044) 0.130 (0.040) No symptom No symptom No symptom 0.150 (0.047) 0.134 (0.042) Sarpomira Blisters, Necrotic rings Necrotic lesion, 1.682 (0.660) 1.342 (0.860) Pustules Brown arc Blisters No symptom No symptom 0.148 (0.048) 0.134 (0.041) No symptom No symptom No symptom 0.145 (0.049) 0.134 (0.043) Peramount Pustules Necrotic rings Necrotic lesions 1.313 (0.821) 1.786 (0.990) Blisters No symptom No symptom 0.151 (0.047) 0.139 (0.042) No symptom No symptom No symptom 0.140 (0.044) 0.134 (0.045) Ultimas Blisters, Necrotic rings Brown arc 1.840 (0.801) 1.344 (0.870) Pustules Blisters No symptom No symptom 0.152 (0.059) 0.136 (0.044) No symptom No symptom No symptom 0.140 (0.056) 0.131 (0.048) Asterix Blisters, Necrotic rings Brown arc 1.708 (0.690) 1.165 (0.660) Pustules Blisters No symptom No symptom 0.145 (0.046) 0.133 (0.047) No symptom No symptom No symptom 0.150 (0.049) 0.134 (0.043) Healthy cv 0.124 (0.040) Desiree Infected 1.980 (1.121) 1
Assessment of powdery scab (Spongospora subterranea) and PMTV on the basis of symptoms: Blister-like swelling (blisters) on surface of tubers, or open pustules (pustules) usually 2-10 mm in diameter containing powdery mass of brown to black in colour. 2 PMTV symptoms: necrotic rings on surface of infected tuber while brown arc or internal browning in tuber flesh 3 Mean values (A405) of four tubers of each variety with or with spraing (brown arc) symptoms 4 Values (A405 nm) after overnight (c. 16 h) and 1 h at room temperature (in parentheses)
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(a)
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(b)
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Fig. 1. Detection of potato mop-top virus through soil-bait bio-assay. (a) Nicotiana debneyi plants showing chlorotic v-shaped leaf markings, 4-6 wk after growing seedlings in infested soils using bait test bio-assay. The N. debneyi seedlings were transplanted in infested soils collected from various zones/locations of potato growing areas of Northwest of Pakistan, (b) N. benthamiana plant showing mosaic/mottling symptoms 2-3 wk after back inoculation of sap from roots of N. debneyi baited plants.
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M 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 1819 20 2122 23 24 25 26 C
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603 bp-
2 3
4 5 6 7 8 9 10 1112131415 16 17 1819 20 2122 232425 26 C
603 bp-
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B
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M 1
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A
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Fig. 2. Detection of potato mop-top virus by RT-PCR in Nicotiana debneyi bait plants (A) roots (B) leaves after 6 to 8 weeks of growing in infested soils collected from various zones/locations of major potato growing areas on Northwest of Pakistan. Lanes contain: M, DRIgest III molecular size markers, the 603 bp band is indicated; each lane represents sampling areas as: 1, Khwazakhela, 2, Shin, 3, Fatehpur, 4, Madyan, 5, Miandam, 6, Kher Abad, 7, Jukhtai, 8, Senay, 9, Shahgram, 10, Cham Ghari, 11, Ashrait, 12, Mankyal, 13, Kaidam, 14, Balakot, 15, Ghruna, 16, Laikot, 17, Peshmal, 18, Kalam, 19, Gabral, 20, Ashuran, 21, Tarhana, 22, Bilal Town, 23, Academy Town, 24, Doraha, 25, Ghitti Ghatti, 26, Baffa, C, virus free control. Arrow indicates the position of the PMTV specific 566 bp band.
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2
3
4
5
6
7
8
9 10 C
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603 bp-
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Fig. 3. Detection of potato mop-top virus RNA sequence by RT-PCR in potato tubers of 10 cultivars mainly grown in Northwest of Pakistan, Lanes contain: M, DRIgest III molecular size markers, the 603 bp band is indicated; 1, cv Desiree; 2, cv Diamant; 3, cv Cardinal; 4, cv Raja; 5, cv Kuroda; 6, cv Rocco; 7, cv Sarpomira; 8, cv Peramount; 9, cv Ultimas; 10, cv Asterix; C, virus-free control. Arrow indicates the position of the PMTV specific 566 bp band.
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S0166-0934(13)00426-6 http://dx.doi.org/doi:10.1016/j.jviromet.2013.10.022 VIRMET 12344
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
18-6-2013 4-10-2013 8-10-2013
Please cite this article as: Arif, M., Ali, M., Rehman, A., Fahim, M.,Detection of potato mop-top virus in soils and potato tubers using baitplant bioassay, ELISA and RT-PCR, Journal of Virological Methods (2013), http://dx.doi.org/10.1016/j.jviromet.2013.10.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
*Highlights (for review)
PESHAWAR, PAKISTAN: 18 June 2013
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Arie J. Zuckerman The Editor-in-Chief, Journal of Virological Methods Email: [email protected] Tel: +44 (0) 20 7830 2579 Fax: +44 (0) 20 7830 2070
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Dear Sir/Madam,
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Attached please find a research paper to be published in Journal of Virological Methods. The title and authorship details are given below:
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Detection of Potato mop-top virus in soils and potato tubers using bait-plant bioassay, ELISA and RT-PCR by Muhammad Arif, Murad Ali, AnayaturRehman and Muhammad Fahim
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Potato mop-top virus (PMTV) is one of the most difficult to detect and control among all plant viruses. It is an emerging problem in this part of world (South Asia) and a limiting factor for seed and ware potato production. The virus is reported and cause serious losses in Europe, North and South America, Japan and China. Recently, PMTV and its vector, is one of the most important problem in seed potato production in Canada and USA. The virus is vectored by a soil-borne fungus-like organism, Sponsgospora subterranea, (the casual agent of powdery scab disease) and remained in resting spore (cystorosus) of the vector for more than 20 years in soils with out a plant host. Due to such properties of the virus, PMTV is one of most difficult problem to control in fields. The virus is evenly distributed in plant host and difficult to detect. The research paper submitted for publication in JVM reports methods and techniques for the rapid detection and identification of the virus in soils as well as in plant hosts. Due to the important nature of the problem, it is therefore requested that the manuscript may kindly be published in forthcoming issue of JVM. With best regards
Prof. Dr. Muhammad Arif Department of Plant Pathology The University of Agriculture Peshawar, Peshawar-25130, PAKISTAN Phone: +92 91 9216552 Fax: +92 91 9216520 Cell: +92 300 595 9748
e-mail: [email protected], [email protected]
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Manuscript
Detection of potato mop-top virus in soils and potato tubers using baitplant bioassay, ELISA and RT-PCR
Muhammad Arifa,*, Murad Alia, Anayatur Rehmana and Muhammad Fahimb a
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Department of Plant Pathology, The University of Agriculture Peshawar, Peshawar-25130, Pakistan b Institute of Biotechnology and Genetic Engineering, The University of Agriculture Peshawar, Peshawar-25130, Pakistan
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Keywords: Detection, PMTV, Plant Bioassay, ELISA, RT-PCR, Pakistan
__________________________________________________________________ Corresponding author at: Department of Plant Pathology, The University of
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Agriculture Peshawar, Peshawar-25130, Pakistan
Tel.: +92 91 9216552; fax: +92 91 9216520; E-mail address: [email protected] (M. Arif)
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ABSTRACT The hilly region of Northwest of Pakistan is leading seed potato producing areas of the country. Soil and plant samples were collected from the region and tested for PMTV using both conventional and molecular techniques. The bait plants exhibited PMTV-characteristic v-shaped yellow leaf markings in
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Nicotiana debneyi plants grown in putative viruliferious soils from 20/26
locations. The results were confirmed by back inoculation of sap from both
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roots and leaves of bait plant on indicator hosts (N. debneyi, N. benthamiana). The root samples of bait plants grown in soils of 25 locations and leaves of 24
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locations reproduced systemic infection on indicator hosts upon back inoculation. The virus was identified in bait plants grown in soils from 25/26
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locations using double antibody sandwich-enzyme linked immunosorbent assay (DAS)-ELISA and reverse transcription and polymerase chain reaction (RT-PCR) methods. The products of the 566 bp were amplified from coat
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protein region of PMTV RNA 3 in both root and leaf samples of baited plants. The virus was detected in 10 potato cultivars commercially grown in the region
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using DAS-ELISA and RT-PCR. The virus was also detected in zoospores of
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Spongospora subterranea derived from the peels of selected scabby tubers using triple antibody sandwich (TAS)-ELISA. The results indicate that a bait
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plant bioassay, infectivity assay, ELISA and RT- PCR can detect PMTV in roots and leaves of baited plants, field samples, zoospores of S. subterranea
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and tubers of 10 potato cultivars commercially grown in the region.
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1. Introduction Potato mop-top virus (PMTV) has fragile rod-shaped particles, transmitted by the soil-borne plasmodiophorid vector, Spongospora subterranea f. sp. subterranea (Wallr.) Lagerth. (Jones and Harrison, 1969; Harrison and Jones, 1970; Arif et al., 1995) and is a type species of genus Pomovirus, family
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Virgaviridae (King et al., 2012). PMTV has particles of two predominant
lengths of about 100-150 nm and 250-300 nm and diameters of 18-20 nm
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(Harrison and Jones, 1970; Harrison and Reavy, 2002). It is characterized by a tripartite, single-stranded RNA genome; the genomic components are
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separately encapsidated in capsid protein (Harrison and Jones, 1970; Savenkov et al., 2003). The virion contains three molecules of linear positive sense
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single-stranded RNA species (Kallender et al., 1990) of 6043nt (RNA 1; Savenkov et al., 1999), 2962 nt (RNA 2; Scott et al., 1994), and 2315 nt (RNA 3 of T isolate; Kashiwazaki et al., 1995). RNA 1 encodes polypeptides of 148
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kDa and 206 kDa, which are considered to be components of viral RNAdependent RNA polymerase, with the second protein probably being expressed
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translational read-through of the first cistron (Savenkov et al., 1999). RNA 2
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encodes the triple-gene-block (TGB) of movement protein and a putative small cysteine-rich 8 kDa protein (Scott et al., 1994; Kashiwazaki et al., 1995). RNA
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3 encodes the capsid protein and a read-through protein (Kashiwazaki et al., 1995; Sandgren et al., 2001). The read-through gene is expected to be involved in acquisition and transmission of the virus by the vector, S. subterranea (Reavy et al., 1998; Arif et al., 1999a). RNA 3 of newly isolated Scottish
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isolate S (PMTV-S) is at least 543 nt larger than that of T isolate (Reavy et al., 1998) and RNA 3 of a Swedish isolate (PMTV-Sw) is 3134 nt (Sandgren et al., 2001). The virus particles also contain a small amount of a read-through protein produced by suppression of coat protein termination codon (Cowan et al., 1997). The read-through protein has a Mr of 66,900 in the T isolate (Kashiwazaki et al., 1995), 87,000 in the S isolate (Reavy et al., 1998) and 91,000 in Sw isolate (Sandgren et al., 2001). The vector of PMTV, S. subterranea also causes powdery scab disease on tubers of susceptible cultivars (Hims and Preece, 1975; Harrison et al., 1997; Merz, 2008). Infections with virus can cause serious potato tuber ‘spraing’ 3
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disease symptoms (Sandgren, 1995; Sandgren et al., 2002) which can occur as brown arcs and circles in the flesh of tubers of susceptible cultivars upon potato planting in infested fields (primary infection) (Harrison and Jones, 1971; Kurppa, 1989). The secondary symptoms of PMTV are produced when potato plants are grown from virus infected seed tubers harvested after primary infection. The symptoms are production of chevron or yellow blotches on
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leaves and bunching effect due to shortening of internodes (mop-top) (Kurppa,
1989). The secondary infection, tubers can show cracks, blotchy surface
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markings or distortion (Calvert and Harrison, 1966; Harrison and Jones, 1970). The vector S. subterranea acquires and transmits PMTV in vivo through mono
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fungal culture (Arif et al., 1995). The virus can maintain its infectivity for more than 18 years inside cystosori without the host plant (Calvert, 1968), therefore,
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the control of the virus is a challenge.
PMTV occurs in potato growing regions of Northern and Central Europe, the Andean regions of South America, China and Japan (Jones, 1988).
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Recently, PMTV is reported as an important pathogen of seed potato in Canada (Xu et al., 2004), Poland (Budziszewska et al., 2010) and the United States of
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America (Lambert et al., 2003; Xu et al., 2004; David et al., 2010; Crosslin,
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2011).
Several surveys have been carried out in the past for the assessment of
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powdery scab in Northwest (Khyber Pakhtunkhwa) and Northern areas (Gilgit Baltistan) (Ahmad et al., 1996). However, no work has been done on detection, identification and characterization of PMTV in Pakistan. Here in this paper, we report detection, identification and characterization
of PMTV using
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conventional and molecular techniques.
2. Materials and Methods 2.1. Field surveys: collection of plant and soil samples Soil and plant samples were collected from major seed/ware potato producing areas of the Northwest of Pakistan (Table 1). The Northwest was divided into six different zones. The zones A-G were further divided into locations viz A-E were divided into four locations each while each location in zone F-G were further divided into three locations. Each location in all six
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zones was further divided into three sites/fields. Plant sampling was made in an estimated area of four meter square of each selected site/field. After harvest, tubers were washed, examined for superficial spraing symptoms and a total of 10-15 scabby tubers (one or more scab pustules) were selected from each site of the selected field. Ten scabby tubers of each potato cultivar commercially grown in the region (Table 2) were also selected separately. Tubers were stored
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for 3-5 months at 7-8°C. Approximately 500 g soil samples were collected
from cropping layer (5-10 cm) below the surface of each of three sites/
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locations, about 20-25m intervals and 3-4m inside the boundaries of each field.
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The soil was mixed thoroughly and kept moist at 4°C until used for air drying.
2.2. Soil-bait test
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Field soil samples were spread out on paper sheet and air-dried for seven to 10 days at 22-24°C. The dried soil samples were ground into fine powder using pestle and mortar and passed through 300 and 200 micro meters sieves,
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respectively. Twenty grams of fine soil powder was placed in each of four depressions in stream-sterilized potting mixture (sand: clay: compost, 1:1:1
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ratio v/v) in one liter plastic pots. The pots were soaked 10-15 h with tap water
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in a steel tray (4 pots/tray/treatment). Four Nicotiana debneyi seedlings were transplanted into each pot. The pots were placed in glasshouse at 18-20°C. The
ce p
pots were flooded to half their height in water for three days, then the water was allowed to drain and the pots were left with out water for four days. The plants were uprooted after six to eight weeks for virus testing through back inoculation of sap from leaves and roots to indicator plants (N. debneyi, N.
Ac
benthamiana), by triple antibody sandwich/double antibody sandwich-enzymelinked immunosorbent assay (TAS/DAS-ELISA), and by reverse transcription and polymerase chain reaction (RT-PCR).
2.3. Serological detection of PMTV in Spongospora subterranea through TASELISA TAS-ELISA was done as described previously (Torrance et al., 1992; Arif et al., 1994). Leaf and root samples (1 g/5 ml) and tuber tissues (1 g/ml) were extracted in extraction buffer [0.01 M phosphate buffered saline (PBS) containing 0.05% Tween-20 and 0.1 -0.2% skimmed milk powder (EveryDay, 5
Page 6 of 21
Nestlé Pakistan, Lahore, Pakistan)]. Samples were added to microtitre plates
(NUNC
Immunoplate
II)
pre-coated
with
polyclonal
anti-PMTV
immunoglobulin (IgG) diluted 1:500 and monoclonal anti-PMTV IgG (1:1000) with coating buffer pH 9.6. For the virus detection in zoospores, preparation were obtained by air drying scabby potato peels and incubating 0.1-0.2 g of cystosori in 10 ml of sterile distilled water (SDW) at 12-15°C for 7-10 days.
ip t
The germinated zoospore suspensions were filtered through fine plastic mesh
to remove debris. Between c 1x107 and 6x107 zoospores per ml were present in
cr
the suspensions. The zoospores were centrifuged at 800 g for five min, resuspended in 2 ml extraction buffer, frozen at -70°C for 20 min, then thawed
us
on ice and used in TAS-ELISA.
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2.4. Serological detection of PMTV in potato and bait plant through DASELISA
DAS-ELISA was used for the detection of PMTV using commercial
M
ELISA kit (Bioreba, Switzerland). The tests were performed in polystyrene micro-plates (NUNC, Immunoplate II, Thermal Scientific, Waltham, MA,
d
USA) as essentially described by Clark and Adams (1977). The plates were
te
coated with 200 µl aliquots of PMTV–specific antibody (Bioreba, Chr. Merian, Switzerland) with coating buffer, pH 9.6. The plates were kept at RT in a
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humid box for 3-4 h. Leaf samples were extracted by crushing through pestle and mortar in extraction buffer, pH 7.4. Leaf and root samples were extracted in extraction buffer at 1:5 (w/v) while tuber 1:1 (w/v) ratio and 200µl of prepared sample was dispensed in each well after three times washing. After
Ac
first incubation was completed the plates were washed with 1 x PBST, pH 7.4. Controls were used as provided and recommended by the supplier (Bioreba, Chr. Merian, Switzerland). ELISA plates were incubated inside a humid box for 4 h at RT or 4-6°C overnight in refrigerator. After washing, 200ul of enzyme conjugate (Bioreba, Chr. Merian, Switzerland) (1:1000) was dispensed in each well of plate and incubated in a humid box for 4 h. at RT. The plates were washed three times with washing buffer. 200µl of substrate solution (dissolve para-nitrophenyl-phosphate (pNPP) at 1 mg/ml) and plates were incubated at RT in the dark. Absorbance at 405 nm (A405) was measured with Titertek Multiskan, Model MC (Flow Laboratories, Covina, CA, USA) after 1 6
Page 7 of 21
h or 2 h of substrate incubation at RT 24-25°C, and overnight (c. 16 h) at 4°C. Samples were considered to be positive when the A405 values exceed the virusfree samples by at least a factor of three.
2.5. Molecular detection of PMTV in bait plants and potato tubers 2.5.1. RNA Extraction and first cDNA strand synthesis
ip t
Total RNA extracts were made from roots and leaves of N. debneyi (bait plants) showing typical yellow v-shaped markings in their leaves and internal
cr
necrosis (brown arc) in potato tuber flesh as described by Verwoerd et al.
(1989) and Barker et al. (1993), respectively. In most cases, RNA extraction kit
us
(Invitrogen; Life Technologies, Carlsbad, CA, USA) was used with the extraction procedure based on manufacture’s instructions. RNA pellet was
an
dissolved in RNase-free water (50µl). The final RNA concentration was measured with a GeneQuartTM photometer (Pharmacia, Stockholm, Sweden). First stand cDNA was synthesized by mixing two micro grams of total
M
RNA with one micro gram of downstream primer, 1 micro liter 10 x PCR buffer (10mM Tris-HCl, pH 8.4 containing 50 mM KCl and 2 mM MgCl2), and
d
2 mM of each of four deoxynucleoside triphosphates (dNTPs) in a total volume
te
of 10 µl. The mixture was heated to 65°C for 2 minutes followed by slow cooling to 42°C on heating block, then 20 Units of RNase inhibitor and 20
ce p
Units reverse transcriptase (Boehringer-Mannheim, Germany) were added and the mixture incubated at 42°C for 2 h. The total cDNA product was then amplified by PCR.
Ac
2.5.2. Polymerase chain reaction (RT-PCR) The genome of PMTV consists of three ssRNA species and the coat protein
gene is located on RNA 3 (Kashiwazaki et al., 1995). Olignucleotides primers were designed to amplify the coat protein gene, with additional (underlined) nucleotides to create a BamHI site. Primer 1 (downstream) had the sequence: 5`-CGGGATCCTATGCACCAGCCCAGCGT-3` complementary to residues 801 to 819 of PMTV (isolate T) RNA 3 , and primer 2 (upstream) had the sequence:
5`-TCGGATCCTCTCGGATACCACCCTT-3` identical to residues
268 to 284 of PMTV (isolate T) RNA 3.
The oligonucleotides were
synthesized on an Applied BioSystems, Model 391 PCRmate DNA synthesizer 7
Page 8 of 21
(Applied Biosystems, Foster City, CA, USA). The primers were expected to amplify a band of 566 bp corresponding to the PMTV coat protein gene with additional BamHI sites (Arif et al., 1994). The PCR mixture (50µl) contained one micro liter each of down-steam and upstream primers, 2µl of a 2 mm solution of each of the four dNTPs, 1.5 µl of 50mM MgCl2 , 10 µl 10 x PCR buffer, 1 µl (5 units) Taq DNA polymerase
ip t
(Promega, Madison, WI, USA) and 28µl water. The mixture was subjected to 40 cycles of heating and cooling in PTC-200 Thermal Cycler (M J Research,
cr
Watertown, MA, USA). Each cycle consisted of 1.5 min at 94°C for
denaturation, 1.5 min at 54°C for primer annealing, and 2.5 min. at 72°C for
us
primer extension, followed by a 10 minutes final extension at 72°C. The PCR products (10 µl) were electrophoreses in 1% agrose gels in Tris-borate-EDTA
an
buffer containing 0.5 µl/ml ethidium bromide. Molecular size markers (Promega, Madison, WI, USA) were used as control. The specific bands were
Laboratories, Hercules, CA, USA)
d
3. Results
M
visualized under UV light using Quantity One for Windows (Bio-Rad
3.1. Detection of PMTV through soil-bait tests
te
Root and leaves samples from each of the four N. debneyi plants (in one
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pot/location) were tested after 6-8 wk by symptomatology, ELISA, RT-PCR and back inoculation of sap to indicator plants. The bait plants (N. debneyi) exhibited v-shaped yellow leaf markings (Fig. 1a). A total 20/26 locations soil samples from seven zones showed PMTV infected bait plants on the basis of
Ac
symptoms (Table 1). In TAS/DAS-ELISA, PMTV was detected in bait plants grown in putative viruliferious soils from 25/26 locations. The A405 values in samples considered positive was quite higher than that of the values of healthy sap (Table 1).
RT-PCR analysis of pooled samples of all four N. debneyi bait plants indicated that out of 26 locations, PMTV was detected in 25 and 24 roots and leaves of bait plants, respectively. The root sample of bait plants of one location (Mankyal) was positive while virus was not detected in leaves samples using RT-PCR (Table 1; Fig 2). A 566 bp band was visible in agarose gels from both roots and leaves of baited plants (Fig 2). The results were further 8
Page 9 of 21
confirmed by back inoculation of sap from both roots and leaves of bait plants on indicator plants [N. debneyi or N. benthamiana (Fig. 1b)]. The root samples of bait plants grown in soils of 25 locations and leaves of 24 locations reproduced systemic infection on indicator host upon back indexing (Table 1). Both roots and leaves samples of bait plants from two locations (Ashrait –zone C and Kalam-zone E) produced mosaic symptoms similar to PVX and infected
ip t
indicator plants were killed two weeks after back inoculation. However, no specific PVX symptoms were observed in bait plants (N. debneyi) grown in
cr
infested soils of both locations.
us
3.2. Detection of PMTV in tubers of commercial potato cultivars grown in Northwest of Pakistan
an
PMTV was detected through symptomatology, ELISA and RT-PCR in scabby tubers of 10 cultivars commercially grown in Northwest of Pakistan
M
(Table 2). Composite samples were taken from brown arc or necrotic tissues, rose and stolon ends of each tuber of 10 cultivars for both ELISA and RT-PCR. Potato peels of scabby tubers were separately made for ELISA detection of
d
PMTV in zoospores of S. subterranea. PMTV was also detected in almost all
te
scabby tubers of commercial cultivars grown in Northwest of Pakistan, when tested through ELISA (Table 2). The results further confirmed by the detection
ce p
of PMTV in zoospores of S. subterranea derived from the peels of selected scabby tubers of 10 cultivars showing powdery scab symptoms viz blisters and pustules on surface and internal browning in flesh of tubers. PMTV was not
Ac
detected through ELISA in scabby tubers of any cultivar where there was no internal browning in the flesh of tubers or zoospores derived from scabby tubers with out internal browning symptoms (Table 2). PMTV internal browning was positively correlated with the presence of the virus but external symptoms of the virus or the vector were not positively correlated with PMTV infection in the tubers (Table 2). The results were further confirmed through RT-PCR (Fig. 3). RT-PCR amplified 566 bp band of CP region in scabby tubers of all 10 potato cultivars showing internal browning or brown arc symptoms. However, the virus was not detected in scabby tubers of any cultivar without internal browning or brown arc symptoms (result not shown).
9
Page 10 of 21
4. Discussion In Pakistan, potato is being produced though out the year with wide range of different agro-ecological zones but seed potato produced mainly in autumn and summer. Both formal and informal seed potato production systems exist in Pakistan. The bulk of the seed produced in the country is derived form informal
ip t
seed sector and over 97% of total seed requirements are met by the home
grown seed potatoes and less than 3% are met by the certified seed including
cr
local and imported sources (Hussain and Farooq, 1995). The Northwest of Pakistan is leading seed potato producing areas for both informal and formal
us
seed systems in the country. The long presence of powdery scab disease in potato in the region (Ahmad et al., 1996) led to the investigation of the
an
occurrence and distribution of PMTV in potato germplasm commercially grown in the region. Occurrence and distribution of any of plant virus is important to determine the economic importance and significance of viral
M
disease in target areas. Significance of the virus will be very high when crop propagated through seed tubers and the pathogen is tuber-perpetuated. The
d
seriousness of the problem can be assessed from the fact that the virus is
te
persistent in resting spore (cystosorus) of the vector (S. subterranea) and remains viable in viruliferious cytosorus for more than 18 years without the
ce p
host plant (Calvert, 1968). The resistance has not been identified yet in potato germplasm against the virus (Tenorio et al., 2006). As mentioned above, the farmers in Northwest of Pakistan use to multiply their own seed. The flow of virus-free certified seed is limited in the system due to several factors and the
Ac
practice of growing multiple crops (3-4 crops/year) without proper implementation of quarantine regulations in the region. With such limitations, occurrence of PMTV and its vector in major seed/ware potato producing areas (Northwestern region) is extremely important and need immediate attention. The results of this investigation indicates that PMTV is prevalent in Northwest including major seed producing areas of Malakand and Hazara divisions, presumably extended to seed producing areas of Northern Areas and the Azad Jammu and Kashmir. In this study, bait test supplemented with ELISA or RT-PCR or both, detected successfully PMTV in infested soil. Arif et al. (1994) used bait test for 10
Page 11 of 21
the detection of PMTV in infested soils. The use of TAS/DAS ELISA and RTPCR of the bait test plants further confirm the identity of the virus. Back indexing of sap from both roots and leaves of bait plants (especially roots) on test plants (N. debneyi or N. benthamiana) allow multiplication of a few virus particles and serves as a successful bio-assay for the detection of PMTV and other viruses transmitted through plasmodiophorid vector (Chen and Wilson,
ip t
1995; Dessens and Meyer, 1996).
The results revealed that powdery scab symptoms on potato tubers such as
cr
blisters and pustules does not guaranteed the presence of PMTV (Table 2). However, PMTV was readily detected by ELISA or PCR or both in potato
us
tubers with brown arc or internal browning symptoms in tuber flesh. It was concluded that ELISA or PCR could not detect PMTV in scabby tubers or
an
tubers with spraing symptoms without internal browning or brown arc in tuber flesh. In a previous attempt, it was reported that TAS-ELISA failed to detect PMTV in tubers that displayed spraing symptoms and RT-PCR results from
M
such tubers were extremely weak (Arif et al., 1994). Torrance et al. (1992) reported that TAS-ELISA on recently harvested symptoms less tubers from
d
infected mother plants detected PMTV in some extract but not others parts of
te
the same tuber. It may be due to physiological age or temperature at which tubers were stored might affect virus replication and spread or mother plant
ce p
was infected late and has low virus titer or even high specificity of TASELISA. The possibility of infection of aviruiliferous S. subterranea producing blister on tubers could not be avoided. Although we did not carry out any assay for the detection of other viruses,
Ac
the possibility of PVX contamination on limited scale in soil samples from Ashrait and Kalam, Malakand division, cannot be ignored. Tenorio et al. (2006) reported PVX infection in bait plants growing in infested soils and shown the possibility of presence of Synchytrium endobioticum spores that transmits PVX under greenhouse and field conditions. Some of the aspects that need further investigation the how much density of S. subterranea carrying PMTV and the phenomenon whether a PMTV infected potato plant through secondary infection play a role to make S. subterranea viruliferous or a viruliferous vector delivers virus in the plant through primary infection or both. Answers to theses questions may help to develop control 11
Page 12 of 21
strategies against viruses transmitted through plasmodiophoroid vectors. Although natural resistance in potato is not available, the availability of transgenic resistance can be one of the best options to minimize losses (Reavy et al., 1995; Arif et al., 1999b; Reavy et al., 1997). The CP gene of the virus incorporated in to potato and transgenic potato plants were again highly resistant (immune) to vector-inoculated virus in screen-house tests (Barker et
ip t
al., 1998). Exploitation of such approaches may lead toward development of effective and durable resistance against PMTV.
cr
Improvement in diagnostic techniques of PMTV (Torrance et al., 1992;
Arif et al., 1994; Arif, 1995; Nielsen and Mølgaard, 1997; Nakayama et al.,
us
2010) lead to proper detection and identification of the virus in soils, plants, tubers and zoospores of S. subterranea but development of effective control
an
measure for such pathogen is still a challenge (Arif, 2000). The facts mentioned above in connection with introduction and spread of PMTV in Northwest of Pakistan is an open challenge to the stakeholders and the
M
scientific community of the country. Recently, farmers have started abandoning growing potatoes and have moved to other valuable cash crops in
d
the areas such as peas and cabbage, etc. (M. Arif and M. Fahim, personal
te
communication). Though it will greatly assist in reducing the active inoculums pressure; potatoes might still remain in practice as alternative crop for the
ce p
purpose of crop rotation. It is therefore high time to adopt every measure to check both the virus and the vector in seed producing areas of Northwest and elsewhere to save potato production in Pakistan. Emphasize should be given to produce virus-free seed potato in small pockets that are virus free, while at the
Ac
same time, strict quarantine measures should be adopted to avoid introduction of potato viruses in the area. This investigation will serve as a baseline to stimulate research on PMTV, its vector and other soil-borne viruses, in Pakistan.
Acknowledgements This work was funded by Higher Education Commission, Islamabad, Pakistan under National Research Program for Universities (NRPU) through Grant No 20-1182/ R & D /08.
12
Page 13 of 21
References Ahmad, I., Iftikhar, S., Soomro, M. H., Merz, U., 1996. First report of Spongospora subterranea f.sp. subterranea on potato in Pakistan. Pl. Dis. 80, 959. Arif, M., 1995. Studies on Fungus Transmission and Molecular Pathology of Potato MopTop Furovirus. Ph. D. Thesis. The University of Edinburgh, UK. Arif, M., Torrance, L., Reavy, B., 1994. Improved efficiency of detection of potato mop-top furovirus in potato tubers and in the roots and leaves of soil-bait plants. Potato Res. 37, 373-381.
ip t
Arif, M., Torrance, L., Reavy, B., 1995. Acquisition and transmission of potato mop-top furovirus by a culture of Spongospora subterranea f. sp. subterranea derived from single cystosorus. Ann. Appl. Biol. 126, 493-503.
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Arif, M., Reavy, B. Torrance, L., 1999a. Read-through protein gene of potato mop-top furovirus is associated with acquisition and transmission of the virus by Spongospora substerranea f.sp subterranea. Pak. J. Bot. 31, 225-236.
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Arif, M., Reavy, B. Barker, H., 1999b. Engineering high level of coat protein mediated resistance to fungal transmission of potato mop-top furovirus in Nicotiana benthamiana. Pak. J. Biol. Sci. 2, 478-483
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Arif, M., 2000. Fungally-Transmitted Rod-shaped Viruses, Biology, Transmission and Molecular Pathology. Pak. J. Biol. Sci. 3, 1194-1212.3. Barker, H., Webster, K. D., Reavy, B., 1993. Detection of Potato virus Y in potato tubers: a comparison of polymerase chain reaction and enzyme-linked immunosorbant assay. Potato Res. 36, 13-20.
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Barker, H., Reavy, B., McGeachy, K. D., Dawson, S., 1998. Transformation of Nicotiana benthamiana with potato mop-top virus coat protein gene produces a novel resistance phenotype mediated by the coat protein. Mol. Plant-Micro. Inter. 11, 626-633.
d
Budziszewska, M., Wieczorek, P., Nowaczyk, K., Borodynko, N., Pospieszny, H., Obrepalska-Steplowska, A., 2010. First report of Potato mop-top virus on potato in Poland. Pl. Dis. 94, 920.
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Calvert, E. L., 1968. The reaction of potato varieties to mop-top virus. Record of Agricultural Research of the Ministry of Agriculture for the Northern Ireland 17, 31-40.
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Calvert, E. L., Harrison, B. D., 1966. Potato mop-top, a soil-borne virus. Plant Path. 15, 134-139. Chen, J. P., Wilson, T. M. A., 1995. Taxonomy of rigid rod-shaped viruses transmitted by fungi. Agronomie 15, 421-426. Clark, M. F., Adams, A. N., 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for detection of plant viruses. J. Gen. Virol. 34, 475-483.
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Cowan, G. H., Torrance, L., Reavy, B., 1997. Detection of potato mop-top virus capsid readthrough protein in virus particles. J. Gen. Virol. 78, 1779-1783. Crosslin, J. M., 2011. First report of Potato mop-top virus on potatoes in Washington State. Pl. Dis. 95, 1483. David, N., Mallik, I., Crosslin, J. M., Gudmestad, N. C., 2010. First report of Potato moptop virus in North Dakota. Pl. Dis. 94, 1506. Dessens, J. T., Meyer, M., 1996. Identification of structural similarities between putative transmission proteins of Polymyxa and Spongospora transmitted bymoviruses and furoviruses. Virus Gen. 12, 95-99. Harrison, B. D., Reavy, B., 2002. Potato mop-top virus. CMI/AAB Descriptions of Plant Viruses, No. 389. Commonwealth Mycological Institute/ Association of Applied Biologists, Kew, Surrey, England. Harrison, B. D., Jones, R. A. C., 1970. Host range and properties of potato mop-top virus. Ann. Appl. Biol. 65, 393-402.
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Harrison, B. D., Jones, R. A. C., 1971. Factor affecting the development of spraing in potato tubers infected with potato mop-top virus. Ann. Appl. Biol. 68, 281-289. Harrison, J. G., Searle, R. J., Williams, N. A., 1997. Powdery scab disease of potato-a review. Plant Path. 46, 1-25. Hims, M. J., Preece, T. F., 1975. Spongospora subterranea f. sp. subterranea. No. 477. In: Descriptions of Pathogenic Fungi and Bacteria. Commonwealth Mycological Insititute/ Association of Applied Biolobists, Kew, Surrey, England.
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Hussain, A., Farooq, K., 1995. Seed potato system in Pakistan. In: Research and Development of Potato Production in Pakistan. pp-41-50. Ed. A. Hussain. Proceeding of the National Seminar held at NARC, Islamabad during 23-25 April 1995. Pakistan Swiss Potato Development Project: PARC, Islamabad, Pakistan.
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Jones, R. A. C., 1988. Epidemiology and control of potato mop-top virus. In: Developments in Applied Biology II. Viruses with Fungal Vectors. pp.255-270. Eds. J. I. Cooper and M. J. C. Asher. Wellesbourne, UK: Association of Applied Biologists.
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Jones, R. A. C., Harrison, B. D., 1969. The behaviour of potato mop-top virus in soil, and evidence for its transmission by Spongospora subterranea (Wallr.) Lagerh. Ann. Appl. Biol. 63, 1-17. Kallender, H., Buck, K. W., Brunt, A. A., 1990. Association of three RNA molecules with potato mop-top virus. Netherl. J. Plant Path. 96, 47-50.
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Kashiwazaki, S., Scott, K. P., Reavy, B., Harrison, B. D., 1995. Sequence analysis and gene content of potato mop-top virus RNA 3: further evidence of heterogeneity in the genome organization of furoviruses. Virology 206, 701-706.
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King, A., Lefkowitz, M. Q., E., Adams, M. J., Carstens, E. B., (Eds). 2012. Virus Taxonomy-Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier/Academic Press.
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Kurppa, A. H. J., 1989. The distribution and incidence of mop-top virus in Finland as determined in 1987 and on the variation of disease symptoms in infected potatoes. Ann. Agric. Fenn. 28, 285-295.
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Lambert, D. H., Levy, L., Mavrodieva, V. A., Johnson, S. B., Babcock, M. J., Vayda, M. E., 2003. First report of Potato mop-top virus on potato from the United States. Pl. Dis. 87, 872.
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Merz, U., 2008. Powdery scab of potato-occurrence, life cycle and epidemiology. Amer. J. Pot. Res. 85, 241-246. Nakayama, T., Maoka, T., Hataya, T., Shimizu, M., Fuwa, H. Tsuda, S. Mori, M., 2010. Diagnosis of Potato mop-top virus in soil using bait plant bioassay and RT-PCRmicroplate hybridization. Amer. J. Pot. Res. 87, 218-225
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Nielsen, S. L., Mølgaard, J. P., 1997. Incidence, appearance and development of potato moptop furovirus-induced spraing in potato cultivars and the influence on yield, distribution in Denmark and detection of the virus in tubers by ELISA. Potato Res. 40, 101-110. Reavy, B., Arif, M., Kashiwazaki, S., Webster, K. D., Barker, H., 1995. Immunity to potato mop-top virus in Nicotiana benthamiana plants expressing the coat protein gene is effective against fungal inoculation of the virus. Mol. Plant-Micro. Inter. 8, 286-291. Reavy, B., Sandgren, M., Barker, H., Heino, P., Oxelfelt, P., 1997. A coat protein transgene from a Scottish isolate of potato mop-top virus mediates strong resistance against Scandinavian isolates which have similar coat protein genes. Euro. J. Plant Path. 103, 829834. Reavy, B., Arif, M., Cowan, G. H., Torrance, L., 1998. Association of sequences in the coat protein/readthrough domain of potato mop-top virus with transmission by Spongospora subterrane. J. Gen. Virol. 79, 2343-2347. Sandgren, M., Savenkov, E. I., Valkonen, J. P. T., 2001. The readthrough region of Potato mop-top virus (PMTV) coat protein encoding RNA, the second largest RNA of PMTV
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genome, undergoes structural changes in naturally infected and experimentally inoculated plants. Arch. Virol.. 146, 467-477. Sandgren, M., Plaisted, R. L., Watanabe, K. N., Olsson, S., Valkonen, J. P. T., 2002. Evaluation of some North and South American potato breeding lines for resistance to Potato mop-top virus in Sweden. Amer. Pot. J. 79, 205-210. Sandgren, M., 1995. Potato mop-top virus (PMTV) distribution in Sweden, development of symptoms during storage and cultivar trials in field and glasshouse. Potato Res. 38, 379389.
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Savenkov, E. I., Sandgren, M., Valkonen, J. P. T., 1999. Complete sequence of RNA 1 and the presence of tRNA-like structure in all RNAs of Potato mop-top virus, genus Pomovirus. J. Gen.Virol. 80, 2779-2784.
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Savenkov, E. I., Germundsson, A., Zamyatnin Jr., A.A., Sandgren, M.,Valkonen, J. P. T., 2003. Potato mop-top virus: the coat protein-encoding RNA and the gene for cysteine-rich protein are dispensable for systemic virus movement in Nicotiana benthamiana. J. Gen. Virol. 84, 1001-1005.
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Scott, K. P., Kashiwazaki, S. Reavy, B., Harrison, B. D., 1994. The nucleotide sequence of potato mop-top virus RNA 2: a novel type of genome organization for a furovirus. J. Gen.Virol. 75, 3561-3568.
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Tenorio, J., Franco, Y., Chuquillanqui, C., Owens, R. A., Salazar, L. F., 2006. Reaction of potato varieties to Potato mop-top virus infection in the Andes. Amer. J. Pot. Res. 83, 423-431.
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Torrance, L., Cowan, G. H., Scott, K. P., Pereira, L. G., Roberts, I. M., Reavy, B., Harrison, B. D., 1992. Detection and diagnosis of potato mop-top virus. Annual Report of Scottish Crop Research Institute for 1991, pp. 80-82. Verwoerd, T. C., Dekker, B. M. M., Hoekema, A., 1989. A small scale procedure for rapid isolation of plant RNAs. Nucl. Acid Res. 17, 2362.
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ce p
te
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Xu, H., DeHaan, T. –L., Boar, S. H. D., 2004. Detection and confirmation of Potato mop-top virus in potatoes produced in the United States and Canada. Pl. Dis. 88, 363-367.
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Page 16 of 21
Table 1 Detection of PMTV in N. debneyi-bait plants grown 6-8 wk in putative viruliferious soils collected from various zones/locations of Northwest of Pakistan. Detection of PMTV through: ELISA RT-PCR
D
E
F
Root + + + + + + + + + + + + + + + + + + + + + +
Leaf + + + + + + + + + + + + + + + + + + + + +
+ +
0.870 (0.670) 0.954 (0.660)
1.764 (0.890) 1.141 (0.960)
+ +
+ +
+ +
+ +
+ -
1.310 (0.860) 0.110 (0.040) 1.876 (1.130)
1.623 (1.180) 0.124 (0.045) 1.980 (1.136)
+ -
+ -
+ -
+ -
Ac
ce p
G
Leaf 1.540 (0.860) 1.130 (0.636) 1.141 (0.710) 1.632 (0.880) 1.427 (0.860) 0.120 (0.065) 1.123 (0.690) 1.130 (0.770) 1.134 (0.923) 1.154 (0.860) 0.983 (0.730) 0.132 (0.036) 1.132 (0.880) 0.865 (0.560) 1.321 (0.990) 1.675 (1.160) 1.768 (1.211) 1.868 (1.119) 0.923 (0.660) 1.543 (0.992) 1.432 (0.880) 1.872 (0.860) 1.321 (0.860)
an
C
M
B
Root 1.2031,2 (0.736) 1.120 (0.560 ) 1.132 (0.670) 1.128 (0.712) 0.987 (0.550) 0.110 (0.060) 1.350 (0.756 ) 1.123 (0.856) 0.876 (0.640) 1.142 (0.860) 1.247 (0.843) 0.698 (0.498) 1.112 (0.864) 0.981 (0.660) 1.211 (0.960) 1.342 (0.945) 1.564 (0.980) 1.432 (0.965) 1.345 (0.885) 0.876 (0.560) 1.220 (0.920) 0.987 (0.860) 1.121 (0.860)
d
Shin Fatehpur Madyan Miandam Kherabad Jukhtai Senay Shahgram Cham Ghari Ashrait Mankyal Kaidam Balakot Ghruna Laikot Pishmal Kalam Gabral Ashuran Tarhana Bilal Town Academy Town Doraha Ghitti Ghatti Baffa Healthy Infected
+ + + + + + + + + + + + + + + + +
te
Khwazakhela
A
Infectivity assay Root Leaf + + + + + + + + + + + + + + + + + + +3 +3 + + + + + + + + + + + +3 +3 + + + + + + + + + +
ip t
Systemic symptoms
cr
Locations
us
Zones
1
Mean values (A405) of four replicates Values (A405) after 1 h at room temperature and overnight (c. 16 h) in parentheses 3 Test plant back inoculated with sap from root and leaves from baited plant exhibited mosaic symptoms similar to PVX instead PMTV 2
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M
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Table 2 Detection PMTV in potato cultivars commercially grown in various zones of Northwest of Pakistan Potato Symptoms Detection of PMTV 2 Cultivars Powdery PMTV ELISA scab1 External Internal Tubers Zoospores Desiree Blisters No symptom Necrotic lesions 0.9523,4 (0.493) 0.781 (0.421) Blisters No symptom No symptom 0.150 (0.049) 0.134 (0.043) No symptom No symptom No symptom 0.142 (0.047) 0.130 (0.042) Diamant Blisters, Necrotic rings Brown arc 1.683 (0.590) 0.965 (0.580) Pustules Blisters No symptom No symptom 0.145 (0.046) 0.133 (0.047) No symptom No symptom No symptom 0.150 (0.049) 0.134 (0.043) Cardinal Blisters, Necrotic rings Brown arc 1.780 (0.701) 1.264 (0.778) Pustules Blisters No symptom No symptom 0.152 (0.059) 0.136 (0.044) No symptom No symptom No symptom 0.140 (0.056) 0.131 (0.048) Raja Blisters, Necrotic rings Necrotic lesions, 1.670 (0.569) 1.141 (0.680) Pustules Brown arc Blisters No symptom No symptom 0.156 (0.049) 0.134 (0.043) No symptom No symptom No symptom 0.160 (0.049) 0.144 (0.041) Kuroda Blisters No symptom Necrotic lesions 0.891 (0.452) 1.234 (0.770) Blisters No symptom No symptom 0.150 (0.050) 0.134 (0.040) No symptom No symptom No symptom 0.150 (0.040) 0.134 (0.042) Rocco Pustules Necrotic rings Necrotic lesions 1.121 (0.590) 1.432 (0.876) Blisters No symptom No symptom 0.141 (0.044) 0.130 (0.040) No symptom No symptom No symptom 0.150 (0.047) 0.134 (0.042) Sarpomira Blisters, Necrotic rings Necrotic lesion, 1.682 (0.660) 1.342 (0.860) Pustules Brown arc Blisters No symptom No symptom 0.148 (0.048) 0.134 (0.041) No symptom No symptom No symptom 0.145 (0.049) 0.134 (0.043) Peramount Pustules Necrotic rings Necrotic lesions 1.313 (0.821) 1.786 (0.990) Blisters No symptom No symptom 0.151 (0.047) 0.139 (0.042) No symptom No symptom No symptom 0.140 (0.044) 0.134 (0.045) Ultimas Blisters, Necrotic rings Brown arc 1.840 (0.801) 1.344 (0.870) Pustules Blisters No symptom No symptom 0.152 (0.059) 0.136 (0.044) No symptom No symptom No symptom 0.140 (0.056) 0.131 (0.048) Asterix Blisters, Necrotic rings Brown arc 1.708 (0.690) 1.165 (0.660) Pustules Blisters No symptom No symptom 0.145 (0.046) 0.133 (0.047) No symptom No symptom No symptom 0.150 (0.049) 0.134 (0.043) Healthy cv 0.124 (0.040) Desiree Infected 1.980 (1.121) 1
Assessment of powdery scab (Spongospora subterranea) and PMTV on the basis of symptoms: Blister-like swelling (blisters) on surface of tubers, or open pustules (pustules) usually 2-10 mm in diameter containing powdery mass of brown to black in colour. 2 PMTV symptoms: necrotic rings on surface of infected tuber while brown arc or internal browning in tuber flesh 3 Mean values (A405) of four tubers of each variety with or with spraing (brown arc) symptoms 4 Values (A405 nm) after overnight (c. 16 h) and 1 h at room temperature (in parentheses)
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ip t cr
(a)
M
an
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(b)
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Fig. 1. Detection of potato mop-top virus through soil-bait bio-assay. (a) Nicotiana debneyi plants showing chlorotic v-shaped leaf markings, 4-6 wk after growing seedlings in infested soils using bait test bio-assay. The N. debneyi seedlings were transplanted in infested soils collected from various zones/locations of potato growing areas of Northwest of Pakistan, (b) N. benthamiana plant showing mosaic/mottling symptoms 2-3 wk after back inoculation of sap from roots of N. debneyi baited plants.
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M 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 1819 20 2122 23 24 25 26 C
ip t
603 bp-
2 3
4 5 6 7 8 9 10 1112131415 16 17 1819 20 2122 232425 26 C
603 bp-
M
an
B
us
M 1
cr
A
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Fig. 2. Detection of potato mop-top virus by RT-PCR in Nicotiana debneyi bait plants (A) roots (B) leaves after 6 to 8 weeks of growing in infested soils collected from various zones/locations of major potato growing areas on Northwest of Pakistan. Lanes contain: M, DRIgest III molecular size markers, the 603 bp band is indicated; each lane represents sampling areas as: 1, Khwazakhela, 2, Shin, 3, Fatehpur, 4, Madyan, 5, Miandam, 6, Kher Abad, 7, Jukhtai, 8, Senay, 9, Shahgram, 10, Cham Ghari, 11, Ashrait, 12, Mankyal, 13, Kaidam, 14, Balakot, 15, Ghruna, 16, Laikot, 17, Peshmal, 18, Kalam, 19, Gabral, 20, Ashuran, 21, Tarhana, 22, Bilal Town, 23, Academy Town, 24, Doraha, 25, Ghitti Ghatti, 26, Baffa, C, virus free control. Arrow indicates the position of the PMTV specific 566 bp band.
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2
3
4
5
6
7
8
9 10 C
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1
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M
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M
an
603 bp-
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Fig. 3. Detection of potato mop-top virus RNA sequence by RT-PCR in potato tubers of 10 cultivars mainly grown in Northwest of Pakistan, Lanes contain: M, DRIgest III molecular size markers, the 603 bp band is indicated; 1, cv Desiree; 2, cv Diamant; 3, cv Cardinal; 4, cv Raja; 5, cv Kuroda; 6, cv Rocco; 7, cv Sarpomira; 8, cv Peramount; 9, cv Ultimas; 10, cv Asterix; C, virus-free control. Arrow indicates the position of the PMTV specific 566 bp band.
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