The first report of PVY incidence in Iran pepper fields
S.Mostafae1*, G.H.Mosahebi2 & M. Kuhi Habibi3
Collage of Horticultural Science & Plant Protection University of Tehran, Karaj. Iran.
Accepted 03 Mach, 2012
Potato Virus Y(PVY) , Cucumber Mosaic Virus (CMV) and Alfalfa Mosaic Virus (AMV) are known as the main causes of sever systemic mosaic ,vein clearing ,vein necrosis and yellowing of 359 pepper samples were collected from pepper fields in Tehran province , using Double Antibody Sandwich Elisa (DAS-ELISA) and virus incidence for presence of CMV,PVY and AMV estimated . Biological properties including host range of the isolates was determined after biological purification. As this is the first report of PVY incidence in Iran pepper fields so we decided to investigate characterization of PVY isolated from pepper. The result of ELISA, Immuno electrophoresis and Immuno Capture ReverseTranscription Polymerase Chain Reaction (RT-PCR) indicated that pepper isolate of PVY shares the reported properties of the PVY in host range studies some differences were observed. for this reason the sequence analys and study of biological and molecular characterization of pepper-PVY isolated from Tehran pepper fields was performed. Keywords: Potato Virus Y, Cucumber Mosaic Virus, Alfalfa Mosaic Virus, Immuno Capture reverse transcription polymerase chain reaction (IC-RT-PCR), Immuno electrophoresis
INTRODUCTION Pepper is the most common solanaceous crop cultivated in Tehran province. Several regions were surveyed in 2 years and symptoms such as leaves yellowing and mosaic have been observed in pepper crops. Serological diagnosis revealed the presence of CMV, PVY and AMV in pepper growing areas throughout. CMV is one of the most important virus diseases of pepper worldwide. The virus exists as a number of strains, but all are apparently capable of infecting pepper and differ only in symptom.
PVY is the type member of the genus potyvirus in the family potyviridae. The symptoms induced by PVY and its host range are highly variable. These traits are used for the classification of PVY strains. PVY isolates have been historically classified according to symptomatology and aphid transmissibility in to PVY
O,
PVYN and PVY
C groups.
AMV or "calico mosaic" as the disease is called when this virus infects potato, can occasionally be recovered from pepper. AMV is aphid transmitted in a *Corresponding Authors E-mail: [email protected]
non persistent manner.
In this paper, we present identification of viruses causing mosaic and yellowing on pepper and biological and molecular characterization of typical pepper isolate of PVY especially in comparison with other PVY isolates. MATERIALS AND METHODS -Virus sources: Leaf samples were collected from different fields in Tehran Province. Samples were collected from plants showing virus-like symptoms as well as symptomless plants and carried to laboratory under standard conditions. -Serological virus identification: 359 Leaf samples with veinal necrosis, veinal clearing, mosaic and dwarfing symptoms were collected from 12 pepper farms in Tehran province. Pepper leaf samples were tested for the presence of PVY, CMV and AMV by
014 Glo. Adv. Res. J. Microbiol.
DAS-ELISA using polyclonal antibody (Clark and Adams, 1977). -Symptomatology Leaf extract from each pepper sample was mechanically inoculated to carborundum-dusted leaves of at least on 5 plants each Nicotiana tabacum cv.White Barley, N. rustica, N. glutinosa, Chenopodium amaranticolor, C. quinoa, Datura metel, D. stramonium , Physalis floridana, Capsicum annum, C. frutescense and Solanum tuberosum with phosphate buffer (0.1M, PH:7). Plants were maintained in an insect proof glasshouse for three weeks to observe symptom development. -Purification of pepper isolate of PVY: The virus was physically purified from propagative hosts: Datura metel, Nicotiana tabacum cv.White Barley and Capsicum annum separately using the method described by Leiser & Richter (1978) with some modifications.
100 gr leaf tissue was homogenized in 200 ml 0.5 M sodium citrate buffer, PH 7.4, containing 5mM EDTA and 15mM sodium DIECA. The homogenate juice was squeezed through cheesecloth and centrifuged for 15 min at 6000rpm.After removing the pellet triton x-100 was added to a final concentration of 3%(v/v) and stired
for 30 min at 4°C before centrifugation for 2h at 30,000g.
The pellets was resuspended in 10mM sodium citrate buffer,PH7.4,containing 1M urea and 0.1%(v/v) 2-mercaptoethanol.centrifuged for 15 min at 15,ooo rpm. layer the supernatant fluided over a cushion of 20%(w/v) sucrose and centrifuged for 2h at 50,000g.resuspend the pellets in 5mM borate buffer PH8,in 30mM Nacl,3mM sodium citrate. Protein analysis: Sample preparation, acrylamide gel preparation and running buffer for electrophoresis were as described by Sambrook et al.
Samples of 10 to 15 µl were run for 1.5 h at 20 milliamps through a 5% stacking and 12% resolving gel. The procedure for the detection of PVY protein bands involved the following steps: an over night blocking step(2%instant non fat dry milk in PBS at 4°C),overnight incubation with the primary antibody under the same condition, washing of the membrane three times in PBS, incubation with goat anti rabbit alkaline phosphatase conjugate at a dilution of 1:1000 in PBS for 90 min, and washing three times more. The membrane was then exposed to NBT/BCIP substrate until bands were clearly visible and the reaction stopped by rinsing with deionized water.
Fig (1):sever mosaic on C. annum
Fig (2)mosaic on C. quinoa
Mostafae et al. 015
-Immunocapture RT- PCR: Two primers (Nla/F,Nla/R) previously described(Glais etal.,2005) were used as a control to amplify PVY strain groups. Another primers pairs were selected to specifically amplify PVY
N, PVY
O, PVY
C and PVY
NTN
strains. The nucleotide sequences of the coding region of PCR product of PVY was determined and was analyzed by multiple alignment with Blast Software to other isolates that were available in the Gen Bank. RESULTS The percent age of the collected samples from different fields which reacted positively in DAS ELISA with
CMV, PVY, AMV polyclonal antiserum were 50.13%, 26.38% and 15.83% respectively. Host range studies showed that CMV isolated from pepper caused mosaic symptom on C. annum and C. quinoa (Fig. 12)
PVY isolated from pepper induced vein clearing and yellowing on C. annum(Fig.3), vein banding and mosaic on D. metel (Fig.4), mosaic on N. tabacum cv. White Burley (Fig. 5) and N.rustica (Fig. 6) but didn’t show any symptoms on P. floridana, C. amaranticolor, C. quinoa and Solanum tuberosum. AMV isolated from pepper caused yellowing on c. annum (Fig. 7).
After physical purification PVY isolated from pepper the A260/280 absorbance ratio was estimated. The A260/280 absorbance ratio of the isolate was 1.50 for purified virus preparation from N.tabacum cv.White Burley. SDS-PAGE of the coat protein extracted from
Fig(10): IC-RT-PCR with PVY primer1-6: infective samples(1000bp) 7: healthy sample M: Marke
Fig(11): IC-RT-PCR with PVYO primer 1: Healthy samples 2, 3, 4: infective samples(750bp), M: Marke
purified virus preparation gave bands at position of about 34 KDal (Fig. 8) and Western Blotting confirmed it as the PVY coat protein(Fig9).
The fragment of about 750 bp obtained after IC-RT-PCR by using specific primer pair of coat protein region of PVY(Fig. 10). Also for PVY strain detection primer pair o-8687F and o-9995R(Boonham etal.,2002) were used and the length of the amplified fragment was about 680 bp(Fig11) which determined this isolate as PVY
O but phylogenetic analysis of PVY isolates shows
that pepper-PVY isolates from Tehran pepper fields has
highest percentage of similarity with the non potato isolates :LYE84.2 (95%) and SON41 (90%). DISCUSSION This is the first report of PVY incidence in Iran pepper fields. We present a biological, serological and molecular characterization of pepper isolate of PVY. The symptomology results described above showed that typical pepper-PVY isolates were distinguished
018 Glo. Adv. Res. J. Microbiol. from potato isolates by host range. After phylogenetic analysis we noticed that pepper isolate has high similarity with LYE84.2 strain that was isolated from tomato and with SON41 strain witch was isolated from C. annuum. For this reason pepper infecting isolates could be classified within the PVY
NP group that
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