First report of Raspberry leaf mottle virus infecting raspberry in Poland.

Abstract

Raspberry leaf mottle virus (RLMV) in Europe has been associated with raspberry mosaic disease (Martin et al. 2013) and crumbly fruit disease (Quito-Avila et al. 2014) in red raspberry (Rubus idaeus L.) beside Rubus yellow net virus (RYNV) and Black raspberry necrosis virus (BRNV). RLMV has also been reported infecting blackberry (Jones and McGavin 1998; Thekke-Veetil et al. 2016). A survey of virus diseases was conducted from 2016 to 2018 in 34 raspberry commercial plantations in Central and southeastern Poland. Some of the plants showed chlorotic spots, vein yellows, mosaic patterns and deformation of leaves and fruits. Leaf samples from 146 plants were tested using RT-PCR for RLMV, RYNV, BRNV, Raspberry vein chlorosis virus (RVCV), Raspberry leaf blotch virus (RLBV), and Raspberry bushy dwarf virus (RBDV). The pairs of primers CPhF and CPhR (Tzanetakis et al. 2007) as well as 1436F and 1437R (McGavin and MacFarlane 2010) were used to amplify a 452-bp fragment of the coat protein homologue (CPh) gene and a 681-bp of the 1 a helicase gene, respectively. RT-PCR products were obtained for five samples from cultivars: Malling Promise (MPrOK1 and MPrOk2; plantation A), Canby (CanKop; plantation B), Polka (PolKop; plantation B) and Malling Seedling (MSeWa; plantation C) grown in southeastern Poland. MPrOK1 and MSeWa were co-infected with RYNV while the other three samples were mixed infected with RBDV. Each of these plants showed chlorotic spots and mosaic symptoms of different intensity, leaf deformation and reduced growth. The RLMV-specific amplicons obtained for all five positive samples were directly sequenced at Genomed company, Warsaw, Poland. The obtained sequences were aligned using ClustalW of the Lasergene 7.1 software (DNASTAR Inc., USA) and were compared with sequences available in GenBank using the BLASTn algorithm. The identity of nucleotide (nt) sequences of Cph gene of RLMV isolates found in Poland was revealed. They shared 97.7% and 99% nt sequence similarity with 'HCRL Glen Clova' from USA (DQ357218) and 'SCRI stock' isolate from United Kingdom (FN391551) strains respectively The nucleotide sequence of isolates from Poland indicates a closer relationship with the strain found in UK. However, the amino acid (aa) sequence of both reference strains was identical and shared 99.2% identity with the corresponding fragment of the isolates from Poland. The representative sequence of PolKop isolate was deposited in GenBank under accession No. MT241265. The similarity of nucleotide sequence of the 1 a helicase gene ranged from 98.5% (between MseWa and four other isolates) to 100% (between MPrOk1 and MPrOk2). The amino acids sequences of MPrOk1, MPrOk2 and MSeWa isolates were identical whereas they shared 99.5% similarity with CanKop and PolKop isolates. The nt and aa sequences within this region of all five isolates were in 98% homologous with the sequences of 'HCRL Glen Clova' strain. Nucleotide sequences of the 1 a helicase gene of the CanKop, PolKop, MseWa, MPrOk1, MPrOk2 isolates were deposited in GenBank (acc. Nos. MT847633-MT8347637). Considering the low incidence of RLMV (five out of 146 positively tested), it can be assumed that there is no emergency of the RLMV large-scale occurrence in Poland. However, as RLMV is transmitted by Amphorophora idaei aphids found in our country, infected plants near raspberry plantations may play an important role in virus spread. This is the first report of RLMV infecting red raspberry in Poland. Acknowledgments This work was performed in the frame of multiannual programme 2015-2020 "Actions to improve the competitiveness and innovation in the horticultural sector with regard to quality and food safety and environmental protection" (task 2.1), financed by the Polish Ministry of Agriculture and Rural Development. I would like to thank Mrs. Dorota Starzec for technical assistance. References Jones, A. T, and McGavin, W. J. 1998. Ann. Appl. Biol. 132:239. https://doi.org/10.1111/j.1744-7348.1998.tb05200.x. Martin, R. R. et al. 2013. Plant Dis. 98:176. http://dx.doi.org/10.1094/PDIS-04-12-0362-FE. McGavin, W. J. and MacFarlane, S. A. 2010. Ann. Appl. Biol. 156:439. doi:10.1111/j.1744-7348.2010.00401.x. Quito-Avila, D. F., et al. 2014. Plant Dis. 98:176. http://dx.doi.org/10.1094/PDIS-05-13-0562-RE. Thekke-Veetil, T., et al. 2016. Plant Dis. 101:265. https://doi.org/10.1094/PDIS-07-16-1014-PDN Tzanetakis, I. E., et al. 2007. Virus Res. 127:26. doi:10.1016/j.virusres.2007.03.010.