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
10.22067/jpp.2025.90010.1205
Abstract
Sugar beet 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 mild yellowing virus, Beet yellow stunt virus, Beet curly top virus, Cucumber mosaic virus, Beet mosaic virus, Beet leaf curl virus, Beet savoy virus, Lettuce infectious yellows virus, Chickpea chlorotic dwarf virus, Lettuce chlorosis virus, Beet pseudo-yellows virus, Beet curly top Iran virus, Tomato black ring virus (Beet ringspot virus), Beet cryptic virus 1, Beet cryptic virus 2, Beet cryptic virus 3, Beet black scorch virus and BvANV-1. The members of Betanucleorhabdoviruses have a monopartite genome and negative-strand RNA. In the genome of most of them, there are six genes (from '3 to '5) N (nucleocapsid protein), P (phosphoprotein), P3 (hypothetical motor protein), M (matrix protein), G (glycoprotein), L (large polymerase protein) respectively, but apple rootstock virus A and alfalfa-associated nucleorhabdovirus have seven genes, and this additional gene is located between M and G genes. Betanucleorhabdovirus genus has 14 species. Phylogenetic analyzes based on 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 Betanucleorhabdovirus 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 holding symptoms like mosaics and yellowing of the leaves in samples, were collected frommajor sugar beet cultivation areas of the country during September and October of 2021. Total RNA extraction followed by high-throughput sequencing based on the Illumina platform was done. 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 this 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 degrees Celsius for 15 minutes. The PCRproducts, which had an approximate size of 1208 bp, were sent to Fazapjoh Iran for sequencing. Phylogenetic analysis was performed based on complete genome sequence with MEGA 11, using Maximum likelihood method and General Time Reversible model (GTR+G+I) and bootstrap of 500 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 betanucleovirus was identified in sugar beet. This virus had the highest similarity among the viruses in the gene bank with Sambucus betanucleorhabdovirus 4 isolate B78-29 (72.86%) and had a nucleotide identity level below the demarcation criteria for delimiting ICTV species in the Betanucleorhabdovirus genus (nucleotide similarity of 75% for the whole genome) Therefore, a new species called beet betanucleorhbdovirus 1 was obtained in the genus Betanucleorhabdovirus. The genome length of beet betanucleorhabdovirus 1 was 13527 bp and 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, which after sequencing confirmed the beet betanucleorhabdovirus 1 infection. The phylogenetic tree showed that beet betanucleorhabdovirus 1 is a new member of betanucleorhabdoviruses. Sugar beet plants infected with beet betanucleorhabdovirus 1 showed leaf yellowing, 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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