یلدا سخن سنج

Chickpea chlorotic dwarf virus</em> (CpCDV, genus Mastrevirus</em>, family Geminiviridae</em>) is one of the most important viral agents which associated with yellowing symptom in the chickpea fields. During the surveys, 2015-2016, 248 chickpea and lentil plant samples showing yellowing symptom were collected from five provinces: Lorestan, Kermanshah, Zanjan, East and West Azarbaijan, in Iran. The samples were tested to CpCDV infection, using TBIA and positive samples in TBIA were tested by PCR. Total DNA of positive samples were extracted and enriched using rolling-circle amplification (RCA). RCA products were restricted using Xho</em> I enzyme and yielding products of ~2.5-6 kb. These fragments were used for polymerase chain reaction (PCR), using specific primers for RepA protein of CpCDV. PCR amplicons with an expected size of ~960 bp were amplified, cloned and sequenced.TBIA and PCR results showed the presence of CpCDV infection in all surveyed provinces with the highest infection in chickpea plants. Nucleotide sequence comparisons showed that Iranian isolates shared the highest identity to strain A, D and F from India, Pakistan, and Yemen. Phylogenetic anlyses showed that Iranian CpCDV strains were grouped with Sudan, Morrocco, India, and Pakistan (strain D, group III), Sudan, Oman, Yemen, and Pakistan (starin F, GroupIV), and Tunesia, Egypt, Syria, and Turkey (strain A, group V).

The legume crops such as chickpea and lentils are mainly cultivated in semi-arid tropical lands. Chickpea chlorotic dwarf virus (CpCDV) causes major losses to legumes throughout the world. Producing of specific antibody against this virus is crucial for surveys of disease in the fields and assessment of vial resistance in plant cultivars. Present article describes developing of specific antibody against the CpCDV virus by applying recombinant protein. In this study, coat protein of CpCDV was selected as a target for detection and preparation of polyclonal antibody. To achieve this aim CP gene encoding coat protein of CpCDV was initially PCR-amplified and inserted into bacterial expression vector. Expression of recombinant protein was performed in Bl21 strain of Escherichia coli. Purification was carried out under native conditions and the accuracy of recombinant protein production was confirmed by electrophoresis. The purified recombinant coat protein of CpCDV was used for immunization of rabbit. Purification of immunoglobulin molecules was performed by affinity chromatography using protein A column followed by conjugating of IgG to alkaline phosphatase enzyme. The capability of purified antibodies and conjugates for efficient detection of infected plants was assessed by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), western blotting and dot immunosorbent assay (DIBA). These results proved that prepared IgG and conjugate are able to distinguish with high efficiency CpCDV infected plants. To the best of our knowledge, this is the first report for production of anti-CpCDV antibodies raised through recombinant protein technology.

Yellowing is one of the most prevalent symptoms in chickpea fields, worldwide and Iran. Faba bean necrotic yellows virus (FBNYV, genus Nanovirus, family Nanoviridae) is associated with the yellowing symptoms in the chickpea fields. During the survey of the Lorestan and Kermanshah provinces in May 2015-2016, chickpea plants showing yellowing symptoms were collected and total DNAs were extracted. The samples were checked for nanovirus infection by PCR, using nanovirus-specific degenerate primers. Total DNA preparations from the nanovirus-positive samples were used for RCA, using φ29 polymerase and restriction endonuclease Aat II digests yielding products of ~1kb corresponding to linearized nanovirus DNA components. FBNYV genomic components were amplified using RCA products and specific primers of eight different U1, M, N, C, S, R, U2, and U4 components. The amplified fragments were purified and sequenced. Nucleotide sequences of the eight different components of three Iranian isolates of FBNYV (Lor-28, Lor-1, and Ker-21) were compared with FBNYV sequences in GenBank. Sequence comparison indicated that Iranian isolates of FBNYV are most similar to Azerbaijan isolates of FBNYV: FBNYV-[AZ; 12], and FBNYV-[AZ; 13.5]. Phylogenetic analyses of nucleotide and deduced amino acid sequences of DNA-S, C, and U1 revealed that Iranian FBNYV isolates clustered with Azerbaijan isolate FBNYV-[AZ;12 ](group II), while DNA-R, DNA-M, DNA-N, DNA-U2, and DNA-U4 clustered with FBNYV-[AZ; 13.5], (group I). Considering the genomic differences between two Azerbaijan isolates, which can be expected among Iranian isolates. These results may provide evidence of the reassortment occurrence among Iranian and Azerbaijan FBNYV isolates.

Tomato ring spot virus (ToRSV) is one of the most important viruses threatening vegetable production worldwide. During the years 2010 to 2012, a total of 110 asymptomatic and symptomatic pepper leaf samples with viral affecting like symptoms were randomly collected from fields and greenhouses of Tehran province. Plant samples were tested for the presence of ToRSV using Double Antibody Sandwich Elisa (DAS-ELISA) and Dot immunobinding assay (DBIA). Results indicated that 22% of the samples were infected with ToRSV. Bioassay was performed with the mechanical inoculation of herbaceous indicator plants to evaluate the biological properties of the detected isolates. The morphological features of the virus isolates were studied using Immunosorbent electron microscopy (ISEM). Specific pair of primers designed on the basis of a portion of the RdRP gene sequence and the presence of the ToRSV was confirmed with the reverse transcription-polymerase chain reaction (RT-PCR) for serologically detected samples. An PCR amplicon of a sequence related to the RdRP gene from a representative isolate, was selected and cloned into the pTZ57R/T plasmid vector. Recombinant plasmid contained ToRSV-RdRP gene was transformed into Escherichia coli DH5α. Recombinanat plasmid was isolated and the inserted DNA fragment was sequenced and submitted to NCBI with accession number (JQ972695). At the deduced amino acid and nucleotide sequence levels of the RdRP gene, Iranian ToRSV isolate showed 94-98% and 87- 94% identity to corresponded American isolates respectively. Results of this study could be useful for ToRSV detection, host range determination, distribution condition and designing of control management strategies against ToRSV in pepper fields of Iran.