Differentiation of Cherry leaf roll virus isolates from walnut based on molecular analyses and reaction of herbaceous hosts

Walnut orchards in main production areas of Poland were surveyed in spring-early summer 2009-2018 to determine the incidence of Cherry leaf roll virus (CLRV). The virus was detected by DAS-ELISA in 46 out of 348 samples. One-step RT-PCR confirmed the positive results of DAS-ELISA. Primer pair CLRV62-F/CLRV1482-R designed during this study based on RNA2 complete sequence of the CLRV strain 739 from Actinidia chinensis (accession number: KC937026), was used to amplify similar to 1.4 kb fragment of 3' untranslated region (3' UTR) of ten isolates (O1, 8.1, 9.10, GP, O2, 06, O14, O15, 4.8, and 10.1). The RFLP and sequence analyses of this region showed the significant variability of the walnut isolates which were clustered into D1 and D2 phylogenetic groups. O2, O15 and GP isolates distinguished among the other classified to D2 group by possessing of the 8-nucleotide (nt) insert which was also present in nucleotide positions 6102-6109 of RNA2 sequence of the 739 strain. However, this mutation and another 5-nt insertion not present in nucleotide position 6067-6072 of RNA2 sequence of the 739 strain, did not significantly affect the phylogenetic position of walnut isolates. The reaction of Chenopodium quinoa, C. amaranticolor, Cucumis sativus, Nicotiana tabacum 'Samsun', and N. benthamiana on sap inoculation with CLRV varied dependently on host species and virus isolate.