First report of tomato ringspot virus infection of Krymsk 86 (Prunus cerasifera × P. persica), a hybrid rootstock, in California.

California produces 99% of prunes (Prunus domestica) in the U.S.A., valued at $148 million and accounting for 40% of world prune production. In the last decade, the rootstock Krymsk 86 (K86; P. cerasifera x P. persica) is increasingly used in prune production because of its high vigor, excellent anchorage and graft compatibility with a wide variety of Prunus crops. An estimated >50% of new prune plantings in the Sacramento Valley are on K86. In late spring of 2023, 'Improved French' prune trees in two Northern California counties, grafted on K86 and Lovell (P. persica) rootstocks, were declining with a pale colored canopy. One orchard was ~ 9-years-old and in the other, trees varied from ~ 20 years to newly planted. The percentage of affected trees in one orchard was 3.6% (n=1,824) and 4.6% (n=1,295) in trees on K86 and Lovell, respectively. Symptomatic trees were scattered throughout this orchard, with a higher density of affected trees in the northeast quadrant. Examination of the trunk revealed a necrotic brown line at the graft union, typical of prune brown line (PBL) disease (Mircetich and Hoy, 1981), caused by infection of rootstock by tomato ringspot virus (ToRSV), a member species of Nepovirus lycopersici (family Secoviridae. The virus is vectored by dagger nematodes (Teliz et al. 1967) and its presence was confirmed in one of the orchards. To confirm infection by ToRSV in declining trees, total RNA from leaf samples, feeder roots, and cambial tissue from bark samples from scion and rootstock of one tree each on K86 and Lovell, was obtained using RNeasy Plant Mini kit (www.qiagen.com) and tested by one step reverse transcription-polymerase chain reaction (RT-PCR) assay using primers previously described (Tang et al., 2014). The reaction conditions included RT at 54oC using random hexamers and Superscript III (Thermo Fisher Scientific, USA), followed by a denaturation step at 95°C for 5 min, and 35 amplification cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 sec; and a final extension of 72°C for 5 min. The expected size amplicons (176 bp) were visualized by agarose gel electrophoresis only from RNA obtained from the cambial tissue below the graft union confirming the ToRSV infection in K86 rootstock. Extracts from bark scrapings of the rootstocks of three symptomatic trees also tested positive for ToRSV with immunostrips (www.agdia.com). In subsequent RT-PCR tests, RNA extracts from cambial tissue from 9 of 10 symptomatic trees on K86 tested positive for ToRSV. The amplicons were purified using a gel extraction kit (Qiagen Inc., Valencia, CA), and subjected to Sanger sequencing (Azenta Life Sciences (South Plainfield, NJ, USA). The sequence of the ToRSV isolates 316 (GenBank accession PQ282959) and 318 (GenBank accession PQ2829560 exhibited 99.4% (175/176 base pairs) and 98.9 % (175/177) sequence identity, respectively, with ToRSV isolate Rasp1-2014 segment RNA2 (GenBank accession KM083895). Our results indicate that ToRSV can infect K86 and cause PBL. ToRSV and its nematode vectors have a wide host range that includes several crop plants and weeds, but this virus is not currently part of registration and phytosanitory certification of Prunus species in the state of California. This is the first report of ToRSV infection of the K86 rootstock. This information is important for the selection of a rootstock in prune orchards where the virus is endemic.