RESUMEN
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.
RESUMEN
Diverse Phytophthora species, including many important plant pathogens, have been widely detected among surface water irrigation sources. In the past decade, metabarcoding has been used to characterize waterborne Phytophthora populations. Metabarcoding typically involves amplification of portions of the nuclear ribosomal internal transcribed spacer (ITS)1 or ITS2 from Phytophthora species, followed by indexed high throughput sequencing. However, full-length sequences of the entire ITS region are required for resolution of many Phytophthora species. We used metabarcoding with PacBio sequencing of full-length ITS amplicons to analyze populations of Phytophthora in waterways of the Stockton East Water District (SEWD) in the northern San Joaquin Valley of California. This approach yielded species-level resolution of many members of the Phytophthora community. Results were compared to those obtained by using ITS1 or ITS2 regions alone and were found to provide superior species resolution for P. pini, P. capsici, and P. gregata. Samples were collected throughout the 2021 irrigation season from five waterways across the SEWD. Thirty-eight Phytophthora species were detected in the waterways, including tree-crop pathogens P. acerina, P. cactorum, P. pini, P. ×cambivora, P. niederhauserii, P. mediterranea, and P. taxon walnut. These pathogenic species were detected throughout the SEWD during most of the irrigation season. The results demonstrated the utility of full-length ITS amplicon sequencing for identifying Phytophthora species in environmental samples and suggested that some disease risk may be incurred by orchardists irrigating with SEWD water. Additional epidemiological studies will be required to critically evaluate this risk.
