First Report of the NA2 Clonal Lineage of Phytophthora ramorum in Indiana

The oomycete pathogen Phytophthora ramorum is the causal agent of ramorum leaf blight and sudden oak death (Rizzo et al. 2002). P. ramorum is known to cause leaf blight and stem dieback on nursery hosts including Rhododendron. The disease typically results in dark, brownish lesions on leaves or stems, which can cause wilting and death of the plant (Grunwald et al. 2008; Werres et al. 2001). Since the emergence of the disease, a federal quarantine was established to prevent the spread of the pathogen, and nurseries that export P. ramorum hosts are subject to a federally mandated certification program for interstate export (Grunwald et al. 2012). This disease has had a significant impact on the U.S. nursery industry via quarantine regulations imposed on nurseries infested with the pathogen. The pathogen has been introduced at least three times into the Western United States as clonal lineages NA1, NA2, and EU1 (Grunwald et al. 2009, 2019; Ivors et al. 2006). In the spring and summer of 2019, USDA-APHIS reported that a shipment of potentially P. ramorum-infested plants was delivered to several Eastern and Midwestern states. Rhododendron leaves from numerous counties in Indiana, showing characteristic necrotic leaf blight symptoms, were sampled by Indiana Department of Natural Resources (IDNR) nursery inspectors between April 18 and June 3, 2019. These samples were initially screened for the presence of Phytophthora at the Purdue Plant and Pest Diagnostic Laboratory using an ELISA test (Agdia). Subsamples from tissue producing a positive ELISA result were forwarded to the Michigan State University Plant & Pest Diagnostics laboratory for P. ramorum-specific PCR testing, which was later confirmed by USDA CPHST. Leaf surfaces of P. ramorum-positive samples were surface disinfested to remove contaminating organisms by washing vigorously in 50 ml of 70% ethanol for 10 s followed by three rinses in sterile water. Washed leaves were blotted dry on sterile paper towels. From each leaf, 5-mm leaf discs were punched out of the leading edge of leaf lesions, and the resulting leaf discs were submerged in selective V8 medium amended with pimaricin, ampicillin, and rifampicin in Petri dishes. Petri dishes were incubated at 20°C until growth was present. A plug of growth from the leading edge of the resulting colony was transferred to fresh V8 medium containing a 47-mm 0.4-micron polycarbonate filter (Nucleopore) and incubated for approximately 8 days at 20°C. DNA was extracted by the Center for Genome Research and Biocomputing at Oregon State University using the Omega BioTek Plant DNA DS kit (M1130), and part of the cellulose binding elicitor lectin gene (CBEL) was amplified and sequenced with primers CBEL5U and CBEL6L (Gagnon et al. 2014). Sequences were aligned with the CBEL reference sequences of EU1 (KF679685), EU2 (KF679716), NA1 (EU688908), and NA2 (KF679712) (Gagnon et al. 2014). All 26 of the Indiana isolates were unambiguously classified as belonging to the NA2 clonal lineage (GenBank accessions MN601787 to MN601812). This is the first report of the NA2 clonal lineage outside of British Columbia, Washington, and California, indicating that this pathogen was most likely moved West to East. Prior documented lineages detected obtained from P. ramorum-infected plants in Eastern receiving states have only harbored the NA1 clonal lineage (Goss et al. 2009, 2011). As a result of these detections, the IDNR ordered destruction of more than 6,100 Rhododendron plants at retail outlets in Indiana.