Molecular Identification of Beet Curly top Iran Virus Associated with Bean in Zanjan Province

Plant diseases caused by geminiviruses are one of the main constraints of legume production in the world. They are responsible for a constraint on production of various crops. Geminiviruses are characterized by their twinned icosahedral particles and their single-stranded DNA genome. The family Geminiviridae is grouped into four genera: Begomovirus, Mastrevirus, Curtovirus and Topocuvirus (Brown and Moriones, 2012). Recent reports for geminiviruses show that viruses such as Spinach curly top Arizona virus (SCTAV) and Beet curly top Iran virus (BCTIV) (Yazdi et al., 2008) can be grouped in a new genus, Becurtovirus (ICTV 2012;http://www.ictvonline.org/virusTaxonomy.asp). Zanjan Province is one of the main regions for growing bean in Iran. Various geminiviruses have been reported from bean included: Bean golden mosaic virus (BGMV), BCTIV, Bean Calico mosaic virus (BCaMV) and Bean dwarf mosaic virus (BDMV). In this study in order to determine the type and distribution of geminivirus/es causing bean diseases, samples were collected from Zanjan province and then tested for the presence of geminiviruses using polymerase chain reaction and rolling circle amplification systems.
Some of the geminiviruses cause disease in the bean. To identify geminiviruses in the bean, sixty samples were collected from Zanjan, Khdabadeh, KhoramDareh and Abhar in summer 2014. These samples were collected according to the symptoms such as golden mosaic, curling, malformation, blistering, yellowing of leaves and plant stunting. After DNA extraction, viral infection was tested by polymerase chain reaction (PCR) using degenerate primers, PAL1V 1978/ PAR1C 496 and Primer B/ Primer V181. Based on the disease symptoms and results of PCR, four samples were selected to confirm the presence of geminiviruses and also to amplify the full-length genome using a rolling cycle amplification (RCA) kit. The amplified DNA products were digested with EcoRI, PstI, and EcoRV enzymes. A 2800 bp DNA fragment was isolated from gel and cloned into EcoRI site of a pBlunt vector and then sequenced (Microgen, Korea). The resulted sequence was compared to other reported geminiviruses from databases such as Genbank. To find the phylogenetic relation of the identified virus with other reported geminiviruses, we made a phylogenetic tree using the aligned sequences in MEGA6 software by applying Neighbour-joining method with 1000 replicates.
Using degenerate primers more than 15 percent of the collected samples showed amplification of DNA fragments with expected sizes. Gel electrophoresis for PCR products using BC and PCR V181 primers for bean samples showed production of a 550bp fragment. The PCR products size was 900 and 1100 bp using PaR1C 496 and PAL1V 1978 primers. The phenotype for theses samples was included geminivirus-like symptoms such as: abnormal and yellowing (S37), cup shape (S60), yellowing and mosaic (S34), blistering and abnormality (S62). The same abnormal shape of leaves was reported from bean plants infected with geminiviruses such as BCTIV and BGMV (Gharouni K. S., 2012). According to the results of the PCR and type of the symptoms, four samples (S26, S37, S61 and S65) selected to confirm the geminiviral infection using Rolling Circle Amplification (RCA) reactions. The RCA reaction was performed using 200 ng of the DNA and followed the instruction of kit. The RCA product was digested with various restriction enzymes. Based on the digestion patterns of the amplified DNA in the presence of three restriction enzymes EcoRI, EcoRV, Pst1, a 2800 bp fragment from sample S37 was selected for cloning into a vector and sequencing. Analyzing of this sequence showed that the amplified DNA has the highest (98%) similarity to a Beet curly top Iran virus (BCTIV) isolated from sugar beet and a lower (89 %) similarity to an isolate of BCTIV from the bean in Khorasan-Razavi Province.
Geminiviruses are limiting factors for crop production in the bean. The most common geminiviruses are Bean golden mosaic virus and Bean calico mosaic virus. In Iran, BCTIV has been recently identified as a dominant and widespread curly top producing agent in important crops (Heydarnejad et al., 2007; Kardani et al., 2013; Soleimani et al., 2013). This diversity and wide occurrence of BCTIV made a big challenge for breeders to produce resistance or tolerant traits (Strausbaugh et al., 2008). Our results also confirmed the widespread occurrence of BCTIV in Iran and also the genetic variation of virus isolates from the same host plant in various geographical regions.