In this study, the prevalence and genome diversity of apple stem pitting virus (ASPV) in commercial pear orchards in South Africa were investigated. Leaves were collected from trees in two geographically distinct regions, representing six different pear cultivars covering 12 orchards. Total RNA was extracted and assayed for ASPV using RT-PCR. A subset of 18 samples was subjected to high-throughput sequencing (HTS) for a more detailed analysis. Bioinformatic analyses of the HTS data, led to the identification and sequencing of a divergent ASPV variant with 85.10

HomePlant DiseaseVol. 106, No. 5First Report of Apple rubodvirus 2 Infecting Pear (Pyrus communis) in South Africa Previous DISEASE NOTE OPENOpen Access licenseFirst Report of Apple rubodvirus 2 Infecting Pear (Pyrus communis) in South AfricaK. Bougard, H. J. Maree, G. Pietersen, J. Meitz-Hopkins, and R. BesterK. BougardDepartment of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaSearch for more papers by this author, H. J. Mareehttps://orcid.org/0000-0001-9639-4558Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaCitrus Research International, P.O. Box 2201, Matieland, 7602, South AfricaSearch for more papers by this author, G. PietersenDepartment of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaSearch for more papers by this author, J. Meitz-Hopkinshttps://orcid.org/0000-0002-8127-6978Department of Plant Pathology, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaSearch for more papers by this author, and R. Bester†Corresponding author: R. Bester; E-mail Address: [email protected]https://orcid.org/0000-0002-2226-0968Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaCitrus Research International, P.O. Box 2201, Matieland, 7602, South AfricaSearch for more papers by this authorAffiliationsAuthors and Affiliations K. Bougard1 H. J. Maree1 2 G. Pietersen1 J. Meitz-Hopkins3 R. Bester1 2 † 1Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa 2Citrus Research International, P.O. Box 2201, Matieland, 7602, South Africa 3Department of Plant Pathology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa Published Online:14 Mar 2022https://doi.org/10.1094/PDIS-08-21-1631-PDNAboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Apple rubbery wood virus 2 (ARWV-2; Rott et al. 2018) belongs to the species Apple rubodvirus 2, a member of the genus Rubodvirus (family Phenuiviridae; Kuhn et al. 2020). ARWV-2 was first identified in apples and is associated with apple rubbery wood disease (ARWD), which is characterized by unusual flexibility of stems and branches, reduced growth, shortened internodes, and increased cold sensitivity (Jakovljevic et al. 2017; Rott et al. 2018). ARWD was first reported in 1935 in England on apple and has since been found on quince and pear (Jakovljevic et al. 2017; Rott et al. 2018). In January 2021, leaves were collected from a pear tree (Pyrus communis cv. ‘Forelle’, F514) in a commercial orchard near Villiersdorp, South Africa. The tree displayed no foliar or tree branch symptoms except for malformed fruits potentially due to insect feeding damage or pear stony pit disease previously associated with infection of apple stem pitting virus (ASPV) (Paunovic et al. 1999). Leaf petioles (1 g) were used for total RNA extraction, using a modified CTAB extraction protocol (Ruiz-García et al. 2019). A sequencing library was constructed (Illumina TruSeq Stranded Total RNA with plant Ribo-Zero) and sequenced on an Illumina HiseqX instrument (Macrogen, South Korea). A total of 30,709,182 paired-end reads (100 nt) were obtained and trimmed for quality with Trimmomatic (SLIDINGWINDOW:3:20, MINLEN:20) (Bolger et al. 2014). De novo assembly, using default parameters of CLC Genomics Workbench 11.0.1 (Qiagen), resulted in 97,294 contigs. BLASTn analysis identified 17 viral contigs, with 14 contigs having high nucleotide identity to ASPV and three to ARWV-2. The latter contigs included all three segments of ARWV-2. The L contig was 7,371 nt, M was 1,289 nt, and S was 1,463 nt in length, generated with 7,341, 626, and 9,161 reads for segments L, M, and S, respectively. Segment S had the highest read coverage (524.87×), followed by segments L (88.07×) and M (36.60×). The ARWV-2 GenBank accessions with the highest percentage identity to the contigs were MF062128.1 from the United States of America (98.2% to segment L), MN163134.1 from China (97.5% to segment M), and NC_055535.1 from Germany (93.5% to segment S). The contigs spanned 100, 80.92, and 100% of these accessions of segments L, M, and S, respectively, and were deposited in GenBank as accessions MZ593725 to MZ593727. Reverse transcription polymerase chain reaction was used to validate the presence of ARWV-2 in sample F514, using primers directed at segments L (con708_178F/con708_666R), M (ARWaV-2S1_38F/ARWaV-2S1_682R), and S (ARWaV-2M567F/ARWaV-2M1342R) (Rott et al. 2018). Amplicon sequences (510 bp [L], 645 bp [M], and 799 bp [S]) were confirmed with bidirectional Sanger sequencing. Fifty-nine additional pear samples were surveyed in 2021 for ARWV-2 using the M segment assay mentioned above. The survey included the Koue Bokkeveld and Elgin areas, and cultivars ‘Bosc’ (22 samples), ‘Abate’ (10 samples), ‘Rosemarie’ (3 samples), ‘Forelle’ (9 samples), ‘Packham’s Triumph’ (12 samples), and ‘Early Bon Chretien’ (3 samples). A total of 27 samples (11 samples from the Koue Bokkeveld region and 16 samples from the Elgin region) tested positive for ARWV-2, demonstrating the common presence of this virus in pears in South Africa. This is the first report of ARWV-2 infecting pear in South Africa. Although no association with disease symptoms were observed, this study expands the data on the incidence and distribution of this virus in South Africa.The author(s) declare no conflict of interest.References:Bolger, A. M., et al. 2014. Bioinformatics 30:2114. https://doi.org/10.1093/bioinformatics/btu170 Crossref, ISI, Google ScholarJakovljevic, V., et al. 2017. Eur. J. Plant Pathol. 148:637. https://doi.org/10.1007/s10658-016-1119-z Crossref, ISI, Google ScholarKuhn, J. H., et al. 2020. Arch. Virol. 165:3023. https://doi.org/10.1007/s00705-020-04731-2 Crossref, ISI, Google ScholarPaunovic, S., et al. 1999. J. Phytopathol. 147:695. https://doi.org/10.1046/j.1439-0434.1999.00449.x Crossref, ISI, Google ScholarRott, M. E., et al. 2018. Plant Dis. 102:1254. https://doi.org/10.1094/PDIS-06-17-0851-RE Link, ISI, Google ScholarRuiz-García, A. B., et al. 2019. Page 163 in: Citrus Tristeza Virus. Humana, New York, NY. Crossref, Google ScholarFunding: The project was funded by Hortgro (PO-20-USGEN-PM01).The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 106, No. 5 May 2022SubscribeISSN:0191-2917e-ISSN:1943-7692 Download Metrics Article History Issue Date: 28 Apr 2022Published: 14 Mar 2022First Look: 14 Oct 2021Accepted: 13 Oct 2021 Page: 1535 Information© 2022 The American Phytopathological SocietyFundingHortgroGrant/Award Number: PO-20-USGEN-PM01KeywordsApple rubbery wood virus 2 (ARWV-2)tree fruitsviruses and viroidsThe author(s) declare no conflict of interest.PDF downloadCited byOccurrence of apple rubbery wood virus 1 and apple rubbery wood virus 2 in pear and apple in Campania (southern Italy) and development of degenerate primers for the rapid detection of rubodviruses7 February 2023 | Journal of Plant PathologyApple rubbery wood virus 2CABI Compendium, Vol. CABI CompendiumIdentification and Characterization of Citrus Concave Gum-Associated Virus Infecting Citrus and Apple Trees by Serological, Molecular and High-Throughput Sequencing Approaches5 November 2021 | Plants, Vol. 10, No. 11

HomePlant DiseaseVol. 106, No. 2First Report of Coguvirus eburi Infecting Pear (Pyrus communis) in South Africa PreviousNext DISEASE NOTE OPENOpen Access licenseFirst Report of Coguvirus eburi Infecting Pear (Pyrus communis) in South AfricaK. Bougard, H. J. Maree, G. Pietersen, J. Meitz-Hopkins, and R. BesterK. BougardDepartment of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaSearch for more papers by this author, H. J. Mareehttps://orcid.org/0000-0001-9639-4558Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaCitrus Research International, PO Box 2201, Matieland, 7602, South AfricaSearch for more papers by this author, G. PietersenDepartment of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaSearch for more papers by this author, J. Meitz-Hopkinshttps://orcid.org/0000-0002-8127-6978Department of Plant Pathology, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaSearch for more papers by this author, and R. Bester†Corresponding author: R. Bester; E-mail Address: rachelle@sun.ac.zahttps://orcid.org/0000-0002-2226-0968Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South AfricaCitrus Research International, PO Box 2201, Matieland, 7602, South AfricaSearch for more papers by this author AffiliationsAuthors and Affiliations K. Bougard1 H. J. Maree1 2 G. Pietersen1 J. Meitz-Hopkins3 R. Bester1 2 † 1Department of Genetics, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa 2Citrus Research International, PO Box 2201, Matieland, 7602, South Africa 3Department of Plant Pathology, Stellenbosch University, Private Bag X1, Matieland, 7602, South Africa Published Online:17 Jan 2022https://doi.org/10.1094/PDIS-08-21-1630-PDNAboutSectionsView articlePDFPDF Plus ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleCoguvirus eburi is a member of the genus Coguvirus in the family Phenuviridae (Kuhn et al. 2020). The species Coguvirus eburi was established to include citrus virus A (CiVA), which is a negative-sense, single-stranded RNA virus that was first found infecting sweet orange in southern Italy via high-throughput sequencing (HTS) (Navarro et al. 2018). This virus was also found to infect pome fruits in France, such as pear (Svanella-Dumas et al. 2019). More recently, CiVA infections have been associated with impietratura disease in citrus (Beris et al. 2021). In the summer of 2021, leaf samples were collected from a pear tree (Pyrus communis cv. Bosc, B175) in the Koue Bokkeveld, South Africa, as part of a virus survey. Sample B175 displayed no visual disease symptoms. One gram of leaf petioles was used for total RNA extraction, using a modified CTAB extraction protocol (Ruiz-García et al. 2019). Ribo-depleted RNA was prepared (Ribo-Zero Plant kit) and a sequencing library constructed (Illumina TruSeq Stranded Total RNA). The RNA library was paired-end (2 × 100 bp) sequenced on an Illumina HiSeqX instrument (Macrogen, South Korea). A total of 47,750,152 reads were obtained. Raw data were trimmed for quality with Trimmomatic (SLIDINGWINDOW:3:20, MINLEN:20) (Bolger et al. 2014). De novo assembly performed with CLC Genomics Workbench 11.0.1 (Qiagen) (default parameters) using high quality reads yielded 75,250 contigs. BLASTn analysis identified two viral contigs with high nucleotide (nt) identity to apple stem pitting virus (ASPV) and CiVA. The CiVA contig was 9,400 nt and on closer examination, a concatemer of CiVA RNA1 and RNA2. The concatenation occurred due to the characteristic near-identical nucleotides shared at the 5′ and 3′ ends of RNA1 and RNA2 of these negative-stranded RNA viruses (Navarro et al. 2018). After splitting and curation, the RNA1 contig was 6,664 nt and the RNA2 contig 2,686 nt. A total of 51,397 and 34,820 reads were used to construct these contigs, resulting in an average depth of coverage of 761 and 1,281 for RNA1 and RNA2, respectively. The contigs had the highest nt identity to the complete CiVA GenBank accessions MT720885.1 (95.53%) and MW148460.1 (96.03%), spanning 99.6% and 98.1% of the genomes of RNA1 and RNA2, respectively. These contigs were submitted as partial genomes to GenBank as accessions MZ463039 and MZ463040. RT-PCR was used to validate the presence of CiVA in sample B175. Two RT-PCR assays, directed at RNA1 and RNA2, respectively (Bester et al. 2021), were used to generate amplicons. Amplicon sequences were confirmed with bidirectional Sanger sequencing. Twenty-one additional samples from the same orchard as B175 as well as other samples from the Koue Bokkeveld and Elgin areas, including cultivars Abate (10 samples), Forelle (10 samples), Early Bon Chretien (3 samples), Packham’s Triumph (12 samples), and Rosemarie (3 samples), were all surveyed for CiVA using the same RT-PCR assays as mentioned above. Thirty-six of the 59 samples tested were positive for CiVA, which further confirms the presence and widespread distribution of this virus in the limited survey conducted in pears in South Africa. However, no association with any disease symptoms or specific cultivar were identified. This is the first report of CiVA infecting pear in South Africa. This study therefore contributed to investigating the distribution of this virus and will assist the South African plant material certification scheme to assess the incidence of CiVA in South Africa.The author(s) declare no conflict of interest.References:Beris, D., et al. 2021. Phytopathology 111:1782. https://doi.org/10.1094/PHYTO-01-21-0027-R Link, ISI, Google ScholarBester, R., et al. 2021. J. Citrus Pathol. 8. https://doi.org/10.5070/C481049000 Crossref, Google ScholarBolger, A. M., et al. 2014. Bioinformatics 30:2114. Crossref, ISI, Google ScholarKuhn, J. H., et al. 2020. Arch. Virol. 165:3023. https://doi.org/10.1007/s00705-020-04731-2 Crossref, ISI, Google ScholarNavarro, B., et al. 2018. Front. Microbiol. 9:2340. https://doi.org/10.3389/fmicb.2018.02340 Crossref, ISI, Google ScholarRuiz-García, A. B., et al. 2019. Page 163 in: Citrus Tristeza Virus. Methods in Molecular Biology. Humana, New York. https://doi.org/10.1007/978-1-4939-9558-5_12 Crossref, Google ScholarSvanella-Dumas, L., et al. 2019. Plant Dis. 103:2703. https://doi.org/10.1094/PDIS-01-19-0028-PDN Link, ISI, Google ScholarFunding: The project was funded by Hortgro (PO-20-USGEN-PM01).The author(s) declare no conflict of interest.DetailsFiguresLiterature CitedRelated Vol. 106, No. 2 February 2022SubscribeISSN:0191-2917e-ISSN:1943-7692 DownloadCaptionWater-soaked lesions on bougainvillea caused by Robbsia andropogonis (O. Morales-Galván et al.). Photo credit: L. F. Flores-López. Soybean leaf showing crinkling and downward curling, characteristic of infection by soybean mosaic virus (SMV) (S. van Bentum et al.). Photo credit: S. van Bentum. Metrics Downloaded 230 times Article History Issue Date: 28 Feb 2022Published: 17 Jan 2022First Look: 29 Aug 2021Accepted: 26 Aug 2021 Page: 772 Information© 2022 The American Phytopathological SocietyFundingHortgroGrant/Award Number: PO-20-USGEN-PM01KeywordsCiVAcitrus virus Aviruses and viroidstree fruitsThe author(s) declare no conflict of interest.Cited ByIdentification and Characterization of Citrus Concave Gum-Associated Virus Infecting Citrus and Apple Trees by Serological, Molecular and High-Throughput Sequencing Approaches5 November 2021 | Plants, Vol. 10, No. 11