Document Type : Research Article
Authors
1
Department of plant protection,, school of agriculture, ferdowsi university of Mashhad
2
Ferdowsi University of Mashhad
3
Department of plant protection, school of agriculture, ferdowsi university of Mashhad
Abstract
Introduction[1]
Sugar beet (Beta vulgaris) is one of the most important agricultural crops in Iran. Viruses that cause disease in sugar beet include Beet necrotic yellow vein virus, Beet soil-borne mosaic virus, Beet soil-borne virus, Beet virus Q, Beet yellows virus, Beet western yellows virus, Beet chlorosis virus, Beet curly top virus, Beet curly top Iran virus and Beet black scorch virus. Members of the genus Betanucleorhabdovirus have a monopartite genome and negative-strand RNA. The genome of betanucleorhabdoviruses is 12.9 to 14.4 kb in length and contains six to seven genes. The genus Betanucleorhabdovirus has 18 species. Phylogenetic analyzes based on the sequence of the L gene have shown that betanucleorhabdoviruses form a monophyletic group and are placed in a sister group with dichorhaviruses. So far, comprehensive and complete research has not been done regarding the identification and determination of molecular characteristics of betanucleorhabdoviruses infecting sugar beet in Iran. Therefore, this research was carried out in order to identify and determine the molecular characteristics of these viruses to pave the way for other research fields, especially their pathogenesis and management.
Materials and Methods
In order to identify the casual agent of the viral diseases of sugar beet in Iran showing symptoms like mosaics and yellowing of the leaves in samples, were collected from major sugar beet cultivation areas of the country during September and October of 2023. Total RNA was extracted from 60 mg of sugar beet leaf and root tissue using SV Total RNA Extraction Kit (Promega, USA). After determining the quantity and quality of the samples using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), one sample from the purified total RNA samples that had an optical absorption ratio of 260 nm to 280 nm of approximately 2 was sent to Macrogen Company (Seoul, South Korea) for high-throughput sequencing based on the Illumina platform. High-throughput sequencing reads were imported to the CLC Genomics Workbench (v.12) software, and after editing, assembling the reads based on the software's default, contig fragments were made. Contig fragments were annotated in NCBI database (GenBank) using BLASTX and BLASTN algorithms. Reverse transcription polymerase chain reaction was used to confirm the presence of the virus in the collected samples. Total RNA was used with Random Hexamer primer to convert RNA to cDNA. Polymerase chain reaction was performed using specific primers designed for the N gene of the virus. The polymerase chain reaction included the initial annealing step at 95°C for 5 minutes and then 35 cycles including 95°C for 1 minute, 54°C for 1 minute, and 72°C for 2 minutes. Then the reaction mixture was kept at 72°C for 15 minutes. The PCR products, which had an approximate size of 1208 bp, were sequenced. Phylogenetic analysis was performed based on complete genome sequence with MEGA 11, using Maximum likelihood method and General Time Reversible model (GTR+G+I) with 1000 bootstrap replications.
Results and Discussion
After receiving the high-throughput sequencing results from South Korea's Macrogen Company and their preliminary analysis, the complete genome of a new Betanucleorhabdovirus was identified in sugar beet. The identified isolates showed 86.13% nucleotide identity, and isolate IR1 (accession number OR227650) shared the highest nucleotide identity among the viruses in the GenBank with tomato betanucleorhabdovirus 2 isolate SKO20ST1 (70.3%) and had a nucleotide identity level below the demarcation criteria for delimiting ICTV species in the genus Betanucleorhabdovirus (nucleotide identity of 75% for the whole genome) Therefore, a new species tentatively called beet betanucleorhbdovirus 1 (beet leaf curl betanucleorhabdovirus) was identified in the genus Betanucleorhabdovirus. The full-length genome of beet betanucleorhabdovirus 1 had six open reading frames (3'-N-P-P3-M-G-L-5'). The beet betanucleorhabdovirus 1 specific primers amplified a fragment with the expected size of 1208 bp from the sugar beet sample followed by sequencing of the PCR product. The result confirmed the infection of the original beet samples with beet betanucleorhabdovirus 1. The phylogenetic analysis showed that beet betanucleorhabdovirus 1 is most closely related to members of the genus Betanucleorhabdovirus. Sugar beet plants infected with beet betanucleorhabdovirus 1 showed curling and mild yellowing of leaf, although these symptoms could be caused by infection of the host plant with other infecting viruses.
Conclusion
This research identified a new virus with a negative single-stranded RNA genome, which belongs to the genus Betanucleorhabdovirus as a new species.
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