Evaluation the Efficiency of Different Methods of RAN Extraction for the Identification of the Squash Mosaic Virus

پژوهش های حفاظت گیاهان ایران

Article 1, Volume 34, Issue 2 - Serial Number 48, June 2020, Pages 145-152   

Document Type: Research Article

DOI: 10.22067/jpp.v34i2.72088

Authors

M. Gerami Nooghabi; M. Zakiaghl;

Ferdowsi University of Mashhad

Abstract

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.
 

Keywords

Keywords: Extraction RNA; Squash mosaic virus; Khorasan

References

1- Bahloul M., and Burkard G. 1993. An improved method for the isolation of total RNA from spruce tissues. Plant Molecular Biology Reporter 11: 212-215.

2- Bananej K., and Vahdat A. 2008. Identification, distribution and incidence of viruses in field grown cucurbit crops of Iran. Phytopathology Mediterranean 47: 247-257.

3- Campbell R.N. 1971. Squash mosaic virus. Description of plant viruses. Applied Biology 43-47.

4- Chang S., Puryear J., and Cairney J. 1993. A simple and efficient method for isolation RNA from Pine trees. Journal of Plant Molecular Biology 11: 113-116.

5- Fenby N.S., Scott N.W., Slater A., Elliott M.C. 1995. PCR and nonisotopic labeling techniques for plant virus detection. Cell Molecular Biology 41: 639–652.

6- Freitag J.H. 1956. Beetle transmission, host range, and properties of Squash mosaic virus. Phytopathology 46: 73-81.

7- Gambino G., Perrone I., and Gribaudo I. 2008. A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochemical Analysis 19: 520–525.

8- Gawande S.J., Shukla A., Chimote V.P., Kaushal N., Kaunda P.I., Garg D., and Chimote, K.P. 2011. Development of PCR-Based Techniques for the Detection of Immobilised Potato Virus Y Virions. Journal of Plant Pathology 93: 127-132.

9- Gholampour Z., and Zaki Aghl M. 2016. Comparison of RNA extraction methods for identification of Grapevine fan leaf virus. Journal of Plant Protection 30: 127-133.

10- Goldbach R.W., and Wellink J. 1996. Comoviruses: molecular biology and replication. In The Plant viruses. Polyhedral Virions and Bipartite RNA Genomes 4: 35-76.

11- Haudenshield J.S., and Palukaitis P. 1998. Diversity among isolates of squash mosaic virus. Journal of Gen Virology 79: 2331–2341.

12- Hebert T.T. 1963. Precipitation of plant viruses by polyethylene glycol. Phytopathology 53: 362-365.

13- Henson J.M., and French R. 1993. The polymerase chain reaction and plant disease diagnosis. Annual Review Phytopathology 31: 81–109.

14- Hull, R. 2002. Mathews Plant Virology. Academic press. New York.

15- Khankhum S., Escalante C., Rodrigues E., Souto R., and Valverde R.A. 2016. Extraction and electrophoretic analysis of large dsRNAs from desiccated plant tissues infected with plant viruses and biotrophic fungi. European Journal of Plant Pathology 53: 362–369.

16- Lequin R.M. 2005. "Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clinical Chemistry 51: 2415–2418.

17- Nelson M.R., and Knuhtsen H.K. 1973. Squash mosaic virus variability: review and serological comparisons of six biotypes. Phytopathology 63: 920–926.

18- Privvidenti R., Gonsalves D., and Humaydan H.S. 1984. Occurrence of Zucchini yellow mosaic virus in cucurbits from Connecticut, New York, Florida, and California. Plant Disease 68: 443-446.

19- Salzman R.A., Fujita T., Zhu-Salzman K., Hasegawa P.M., and Bressan R.A. 1999. An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Molecular Biology Reporter 17: 11-17.

20- Schmidt S.D., Mazzella M.J., Nixon R.A., and Mathews P.M. 2012. Measurement by enzyme-linked immunosorbent assay. Methods in Molecular Biology 849: 507–527.

21- Singh R.P. 1998. Reverse-transcription polymerase chain reaction for the detection of viruses from plants and aphids. Journal of Virological Methods 74: 125–138.

22- Svoboda J., and Leisova-Svobodova L. 2011. First report of Squash mosaic virus in ornamental pumpkin in the Czech Republic. Plant Disease 95(10): 1321-1321.

23- Van Kammen A. 1967. Purification and properties of the components of cowpea mosaic virus. Virology 31: 633–642.

24- Wellink J. 1998. Comovirus isolation and RNA extraction. Methods in Molecular Biology 81: 205-209.