عنوان مقاله [English]
Introduction: Squash mosaic virus (SqMV) is a member of the genus Comovirus in the family Comoviridae. It is a seedborne and beetle-transmitted virus infecting most plants in the genera Cucurbita and Cucumis. Like other comoviruses, SqMV has a bipartite positive-strand RNA genome consisting of RNA1 and RNA2, which are separately encapsidated in isometric particles of 28 nm in diameter. The genomes contain a poly (A) tail at the 3-terminus and the genome-linked viral protein (VPg) attached to the 5end. ELISA has been used widely in plant virus diagnosis but it has relatively low sensitivity which is not suitable for detection of trace amounts of the virus in single viruliferous aphid vectors and mix infection. By contrast, PCR is an effective and efficient tool for in vitro amplification of DNA templates and has been extensively used for the diagnosis of viral and subviral pathogens with DNA and/or RNA genomes. The polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR) are powerful tools for highly sensitive detection of plant viruses with DNA and RNA. The first step in a successful PCR test is to have an extraction method and the major problem in RNA extraction is contamination by polyphenols and polysaccharides. So, in this study, we investigated the effectiveness of various extraction methods in identifying the Squash mosaic virus.
Materials and Methods:
Plant material and virus isolates RT-PCR and sequencing
Twenty-five samples of Melon from Khorasan Razavi and Jonubi provinces under the cultivation of Melon have been collected in spring and summer of 2017. The samples had typical virus types, severe mosaic spasms, complexity and deformity, and entered the process of extraction of the genome of the dandruff as a positive example. The leaves samples were used for different RNA extraction methods using Chang et al. method, Dena Zist and Qiagen Kit, Triasol and dsRNA cellulose method and were used directly or stored at minus 70 0C.
Two specific RT-PCR was set up for amplifying an amplicon of 1900 and 1300 bp in order to detect infected samples, as this region is conserved among all SqMV isolates, and determine the best method for extraction virus RNA. The SqMV partial coat protein gene and partial genome of RNA1 has been sequenced to confirm the results. Here, we employed the Chang et al. procedure which is based on CTAB buffer. We also followed the manufacturer's protocol to extract the genome by Dena Zist and Qiagen kit. Triasol is a mono-phasic solution of phenol and guanidine isothiocyanate. It is a ready-to-use reagent for the isolation of total RNA from cells and tissues. After addition of Triasol and chloroform, phase separation is created by centrifugation. RNA is present in the aqueous phase and can be recovered by precipitation with isopropanol or ethanol. The extraction protocol used for DsRNA is a modification of the non-phenol batch protocol reported by Morris et al. (1983) and was compared with two other dsRNA extraction protocols.
Results and Discussion: The destruction of the nucleic acid during the process of extracting from the plant tissue and the presence of inhibitory substances are the major problems in purifying the genome of the viral viruses. In order to effectively detect Squash mosaic virus from melon tissue at the contaminated fields, four RNA extraction methods were compared and then the nucleic acid extraction method was optimized. The quality of the nucleic acid was extracted by polymerase chain assay with reverse transcription and using two pairs of different primers. Among the methods of extraction, there were significant differences in the way that some of the methods were not able to detect the virus from the plant tissue. Among the investigated methods, the method of extracting dsRNA with cellulose resulted in the highest and most excellent nucleic acid due to the exclusive purification of the viral genomic from the plant tissue, making it possible to detect the virus in most of the samples. We concluded that the modified dsRNA extraction protocol is efficient, fast, economic, versatile, and requires small amounts of tissue. The protocol was successfully used to extract dsRNAs from the plants infected with acute and persistent viruses such as SqMV in Melon samples.