The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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A New Distinct Clade for Iranian Tomato spotted wilt virus Isolates Based on the Polymerase, Nucleocapsid, and Non-structural Genes

  • Abadkhah, Mahsa (Department of Plant Protection, Faculty of Agriculture, University of Zanjan) ;
  • Koolivand, Davoud (Department of Plant Protection, Faculty of Agriculture, University of Zanjan) ;
  • Eini, Omid (Department of Plant Protection, Faculty of Agriculture, University of Zanjan)
  • Received : 2018.04.16
  • Accepted : 2018.07.24
  • Published : 2018.12.01
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Abstract

Tomato spotted wilt virus (TSWV; Genus Orthotospovirus: Family Tospoviridae) is one of the most destructive viruses affecting a wide range of horticultural crops on a worldwide basis. In 2015 and 2016, 171 leaf and fruit samples from tomato (Solanum lycopersicum) plants with viral symptoms were collected from the fields in various regions of Iran. ELISA test revealed that the samples were infected by TSWV. The results of RT-PCR showed that the expected DNA fragments of about 819 bp in length were amplified using a pair of universal primer corresponding to the RNA polymerase gene and DNA fragments of ca 777 bp and 724 bp in length were amplified using specific primers that have been designed based on the nucleocapsid (N) and non-structural (NSs) genes, respectively. The amplified fragments were cloned into pTG19-T and sequenced. Sequence comparisons with those available in the GenBank showed that the sequences belong to TSWV. The high nucleotide identity and similarities of new sequences based on the L, N, and NSs genes showed that minor evolutionary differences exist amongst the isolates. The phylogenetic tree grouped all isolates six clades based on N and NSs genes. Phylogenetic analysis showed that the Iranian isolates were composed a new distinct clade based on a part of polymerase, N and NSs genes. To our knowledge, this is the first detailed study on molecular characterization and genetic diversity of TSWV isolates from tomato in Iran that could be known as new clade of TSWV isolates.

Keywords

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Fig. 1. Virus symptoms observed on tomato plants. (A) and (E) chlorotic and necrosis spots on leaf and fruit; (B) ring spots on fruit; (C) chlorotic blotches on fruits; (D) deformity and chlorotic spots on fruit; (F) chlorotic spots on fruits.

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Fig. 2. Symptoms production in plants inoculated with TSWV. (A) and (B) Solanum lycopersicum, (C) Nicotiana benthamiana, (D) N. tabacum cv samsun, (E) Vigna unguiculcta, (F) Cucurbita pepo.

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Fig. 3. Pairwise nucleotide sequence identity matrix of TSWV isolates from Iran and representative isolates from the GenBank, generated using SDT software for nucleocapsid gene (A) and non-structural gene (B).

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Fig. 4. Phylogenetic analysis based on the nucleotide sequences of a part of polymerase gene generated using Neighbor-Joining method by Mega 7. Bootstrap values on the branches represent the percentages out of 1000 bootstrap replicates program and Iranian isolates detected in this study have been marked by circle.

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Fig. 5. Phylogenetic analysis based on the nucleotide sequences of the nucleocapsid gene generated using Neighbor-Joining method by Mega 7. Bootstrap values on the branches represent the percentages out of 1000 bootstrap replicates program and Iranian isolates detected in this study have been marked by circle.

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Fig. 6. Phylogenetic analysis based on the nucleotide sequences of the non-structural gene generated using Neighbor-Joining method by Mega 7. Bootstrap values on the branches represent the percentages out of 1000 bootstrap replicates program and Iranian isolates detected in this study have been marked by circle.

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Fig. 7. Phylogenetic analysis based on the nucleotide sequences of nucleocapsid and non-structural genes generated using Neighbor-Joining method by Mega 7. Bootstrap values on the branches represent the percentages out of 1000 bootstrap replicates program and Iranian isolates detected in this study have been marked by circle.

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Fig. 8. Trend of polymorphism along the N and NSs genes in the TSWV populations, (A) N gene and (B) NSs gene comprising of 35 and 35 strains/isolates, respectively. Pi stands for nucleotide diversity. The curves were generated by sliding windows with 50 and 25 as the window and step sizes, respectively.

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Fig. 9. SLAC site graph to identify positively and negatively codons/sites.

Table 1. Reaction of the indicator plants to TSWV infection after mechanical inoculation

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Table 2. Primers used in this research

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Table 3. Origins, hosts, and accession numbers of Tomato spotted wilt virus isolates/strains analyzed in this research

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Table 4. Summary of genetic diversity and polymorphism analyses of TSWV N gene from different populations

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Table 5. Summary of genetic diversity and polymorphism analyses of TSWV NSs gene from different populations

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Table 6. Summary of demography test statistics between TSWV N gene populations

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Table 7. Summary of demography test statistics between TSWV NSs gene populations

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Table 8. Summary of gene flow and genetic differentiation estimates between TSWV Nucleocapsid gene populations

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Table 9. Summary of gene flow and genetic differentiation estimates between TSWV NSs gene populations

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