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Natural Occurrence of Tomato Leaf Curl New Delhi Virus in Iranian Cucurbit Crops

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Natural Occurrence of Tomato Leaf Curl New Delhi Virus in Iranian Cucurbit Crops Plant Pathol. J. 32(3) : 201-208 (2016) http://dx.doi.org/10.5423/PPJ.OA.10.2015.0210 The Plant Pathology Journal pISSN 1598-2254 eISSN 2093-9280 ©The Korean Society of Plant Pathology Research Article Open Access Natural Occurrence of Tomato leaf curl New Delhi virus in Iranian Cucurbit Crops Sara Yazdani-Khameneh1, Samaneh Aboutorabi2, Majid Shoori2, Azin Aghazadeh2, Parastoo Jahanshahi2, Alireza Golnaraghi3*, and Mojdeh Maleki2 1Department of Plant Pathology, College of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University (IAU), Tehran P.O. Box 14515-775, Iran 2Department of Plant Protection, College of Agriculture, Varamin-Pishva Branch, Islamic Azad University (IAU), Varamin 33817-7489, Iran 3Department of Plant Protection, College of Agriculture and Natural Resources, Science and Research Branch, Islamic Azad University (IAU), Tehran P.O. Box 14515-775, Iran (Received on October 6, 2015; Revised on January 1, 2016; Accepted on January 27, 2016) The main areas for field-grown vegetable production fragments of the expected size of ca. 2.8 kb. The in Iran were surveyed during the years of 2012–2014 nucleotide sequences of the DNA amplicons derived to determine the occurrence of begomoviruses infec- from the isolate, Kz-Me198, were determined and ting these crops. A total of 787 leaf samples were compared with other sequences available in GenBank. collected from vegetables and some other host plants BLASTN analysis confirmed the begomovirus infection showing virus-like symptoms and tested by an enzyme- of the sample and showed 99% identities with Tomato linked immunosorbent assay (ELISA) using polyclonal leaf curl New Delhi virus (ToLCNDV); phylogenetic antibodies produced against Tomato yellow leaf curl analysis supported the results of the database searches. virus (TYLCV). According to the ELISA results, 81 This study reports the natural occurrence of TYLCV samples (10.3%) positively reacted with the virus in different hosts in Iran. Our results also reveal the antibodies. Begomovirus infections were confirmed emergence of ToLCNDV in Iranian cucurbit crops. by polymerase chain reaction (PCR) using previously described TYLCV-specific primer pair TYLCV-Sar/ Keywords : Begomovirus, phylogeny, Tomato leaf curl New TYLCV-Isr or universal primer pair Begomo-F/ Delhi virus, Tomato yellow leaf curl virus, vegetables Begomo-R. The PCR tests using the primer pair TYLCV-Sar/TYLCV-Isr resulted in the amplification of the expected fragments of ca. 0.67-kb in size for With regarding to proper role of vegetables in ensuring ELISA-positive samples tested from alfalfa, pepper, human health, these crops are considered as one of the spinach and tomato plants, confirming the presence of major food groups and grown all through the world (Dias, TYLCV. For one melon sample, having a week reaction 2011). Based on the per capita consumption of vegetables in ELISA and no reaction in PCR using TYLCV- in Iran, these products are widely grown in different specific primers, the PCR reaction using the primer regions of the country. The area harvested of vegetables pair Begomo-F/Begomo-R resulted in the amplification in Iran was estimated over 876 thousand hectares in 2012 as compared with about 57 million hectares in the *Corresponding author. world (Food and Agriculture Organization of the United Phone) +98-(0)21-44868536, FAX) +98-(0)21-44868550 Nations, 2012). Diseases and pests are often cited as one E-mail) [email protected] of the major limiting factors for vegetable production, cc This is an Open Access article distributed under the terms of the of which viral diseases account for more losses in such Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non- crops. Many viruses have been reported worldwide commercial use, distribution, and reproduction in any medium, provided from vegetable plants (Brunt et al., 1995). Also, several the original work is properly cited. viruses belonging to different virus genera and families Articles can be freely viewed online at www.ppjonline.org. such as Cauliflower mosaic virus, Cucumber mosaic 202 Yazdani-Khameneh et al. virus, Tomato mosaic virus, Tomato yellow fruit ring province, Iran. virus and Watermelon mosaic virus have been reported to infect Iranian vegetable crops (Farzadfar et al., 2005; Materials and Methods Golnaraghi et al., 2013; Massumi et al., 2007, 2009; Yazdani-Khameneh et al., 2013). Surveys and sample collection. During the growing The circular single stranded DNA viruses in the genus seasons of 2012–2014, the main areas for field-grown Begomovirus, family Geminiviridae, are amongst the vegetable crops in Iran, including Khuzestan and Tehran most important vegetable viruses (Brown and Bird, provinces were surveyed to determine the occurrence of 1992; Moriones and Navas-Castillo, 2000). This group TYLCV in these crops. Overall, 86 fields were surveyed of viruses known as one of the limiting factors of a and 777 leaf samples collected from different crops, number of economically important crops in many parts especially vegetables, showing virus-like symptoms of of the world, especially in tropical and sub-tropical mosaic, mottling, chlorosis, dwarfing, yellowing, leaf regions (Zerbini et al., 2005). Begomoviruses infect deformation, leaf curling, leaf rolling and necrosis (Table only dicotyledonous plant species and are transmitted 1). We also collected 10 symptomatic weed samples in exclusively by the whitefly Bemisia tabaci (Homoptera: or around fields surveyed. The collected leaves were Aleyrodidae) in a non-circulative manner (Briddon, immediately placed in plastic bags and transported to the 2015; King et al., 2012). These viruses are considered laboratory and kept at 4oC. For long-term storage, leaf as emerging plant viruses, due to their increasing samples were dried by using silica gel or a freeze-dryer incidence and the severity of the diseases they cause and stored at –20oC. (Polston and Anderson, 1997), which are frequently being reported to occur on new hosts and spread to more Serological assays. Samples were tested by a double- geographical locations (Mansoor et al., 2003; Varma and antibody sandwich enzyme-linked immunosorbent assay Malathi, 2003). Recombination and mutation may play (DAS-ELISA) (Clark and Adams, 1977) using TYLCV- important roles in the establishment and emergence of polyclonal antibodies (Bioreba, Reinach, Switzerland) new Begomovirus strains/species (Mansoor et al., 2006). according to the manufacturer’s instructions. All buffers Begomoviruses typically have genomes consisting of two as well as negative and positive controls used in these components, referred to as DNA-A and DNA-B, which assays were provided by the company. Briefly, microtiter encodes all virus functions required for DNA replication, plates were coated with 100 μl of 1:1,000 dilution of control of gene expression, insect transmission and TYLCV-immunoglobulin G (IgG) in carbonate coating movement in the plant (Hanley-Bowdoin et al., 1999). buffer and incubated overnight at 4oC. Plates were However, a small number of monopartite begomoviruses washed with washing buffer and then, leaf extracts have been discovered. For these viruses, all essential were added to the plate wells (100 μl in each well); the virus functions are encoded by a single component plates were incubated overnight at 4oC. After washing that resembles DNA-A of the bipartite begomoviruses the plates, 100 μl of alkaline phosphatase-conjugated (Navot et al., 1991; Rojas et al., 2001). To date, more TYLCV-IgG diluted in conjugate buffer (1:1,000) than two hundred confirmed and tentative species in the was added and incubated 3 hours at 37oC. Wells were genus Begomovirus have been noted by the International washed and incubated with 100 μl of substrate (1 mg/ Committee on Taxonomy of Viruses (ICTV; King et al., ml of p-nitrophenyl phosphate in substrate buffer) at 2012). room temperature. Color reactions were measured at In Iran, several begomoviruses have been reported to 405 nm using a microtiter plate reader (EL800; Bio- occur on vegetable crops in recent years; some of them Tek Instruments, Winooski, VT, USA). A sample was infect economically important crops such as tomato and considered virus-positive if its optical density (OD) was cucurbits (Fazeli et al., 2009; Heydarnejad et al., 2009; equal to or exceeded three times of the mean OD of the Kheyr-Pour et al., 2000; Massumi et al., 2009), and have negative controls (healthy). capacities to cause epidemics in and significant yield losses to these crops, e.g., Tomato yellow leaf curl virus Polymerase chain reaction (PCR). Total DNA was (TYLCV) (Moriones and Navas-Castillo, 2000). In this extracted from 0.04 g of dried leaf tissue using the paper, we detect TYLCV occurring in vegetables and CTAB protocol described earlier (Dellaporta et al., some other host plants in the country. We also detect and 1983) or DNeasy Plant Mini Kit (Qiagen, Hilden, describe the partial molecular characterization of Tomato Germany) according to those that recommended by the leaf curl New Delhi virus (ToLCNDV) infecting melon supplier; finally, DNA pellets were suspended in 25 μl of (Cucumis melo; family Cucurbitaceae) in Khuzestan diethylpyrocarbonate (DEPC)-treated water and stored Occurrence of ToLCNDV in Iranian Cucurbits 203 Table 1. Occurrence of Tomato yellow leaf curl virus (TYLCV) in different crops in the provinces surveyed* No. of fields, No. of samples, Province Host
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