Chile is the main exporter of sweet cherries (Prunus avium), with a total of 228.6 thousand tons exported in the 2019-20 season, and a production from the Coquimbo to the Aysén region (http://www.iqonsulting.com/yb/). In January 2019, cherry trees from a commercial orchard located near Osorno city (40°37'S, 72°54'W), Region de Los Lagos, Chile, showed symptoms such as the presence of wood cankers, necrotic spots in leaves, and premature defoliation, with a mean disease incidence near 40%. Symptomatic leaves with necrotic spots were collected for analysis, from which all the necrotic spots were extracted by incision with a sterile scalpel, macerated in 30 mL of AFT buffer and subsequently, 100 µL of the suspension was plated on King's B (KB) agar and incubated for 48 to 72 h at 27°C, obtaining a total of two bacterial colonies identified as 7684.1 and 7684.2. Afterward, each colony was stroked in a new KB agar plate, incubated for 16 h at 27°C, and the obtained biomass was used in subsequent experiments. In KB agar, both colonies exhibited fluorescence under UV light and, according to the LOPAT method (Lelliott et al., 1966), they were gram negative, positive to levan and tobacco hypersensitivity tests and negative to oxidase, potato soft rot, arginine dihydrolase and gelatin tests, and were confirmed as Pseudomonas syringae. Then, the 16s and gyrB genes of each isolate were amplified by PCR, sequenced, and compared with the NCBI Genbank database (Weisburg et al., 1991; Sarkar and Guttman, 2004), finding a 99,93% genetic similarity (1064/1065) with a previously reported 16s sequence of a Pseudomonas syringae pv. morsprunorum (Psm) isolate (accession number CP026558.1), and a 99,69% (636/638) with a previously reported gyrB gene of Psm (accession number LC364094.1), respectively. Additionally, the closest pathovar different to morsprunorum aligned with our gyrB sequence was P. syringae pv. aesculin, with 97,8% of identity (624/638). Our sequences were deposited in Genbank with the accession numbers MN528473 (16s), MN535696 (gyrB) for 7684.1, and MN528474 (16s), MN535697 (gyrB) for 7684.2. To identify if the isolates correspond to Psm races 1 (Psm1) or 2 (Psm2), race-specific conventional PCRs and qPCRs assays were carried out using the specific primers described by Kaluzna et al., (2016), showing that the two isolates were positive to Psm1 in both PCR assays. Pathogenicity was tested by inoculating immature cherry fruitlets (cv. Sweetheart) with bacterial suspension at 108 CFU/mL. For each strain, ten fruitlets were inoculated by pricking with a sterile needle previously immersed in the bacterial suspension (Ruinelli et al., 2019). Sterile distilled water was used as negative control. Seven to fourteen days post-inoculation, necrotic and water-soaked brown lesions with yellow margins were observed on the fruits inoculated with bacterial strains. The pathogen was reisolated and confirmed as Pseudomonas syringae pv. morsprunorum by 16s and gyrB sequencing, and as race 1 by race-specific PCRs. Our results were confirmed by the National Plant Protection Organization, (Servicio Agrícola y Ganadero de Chile, SAG), generating the first report of Psm race 1 in Chile. Thus, SAG established new protocols for quarantine of absent pests in the national territory (Resol. N°3080, SAG, Chile), and an immediate phytosanitary program for Psm (Resol. Exenta N°8948/2019, SAG, Chile). In conclusion, our discovery contributes to the monitoring and control of the disease in Chile.
Keywords: Causal Agent; Crop Type; Etiology; Fruit; Prokaryotes; Subject Areas; tree fruits.