First Report of Phytophthora ramorum Causing Leaf Blight on Nerium oleander in the United States

Nerium oleander L. (Apocynaceae; common oleander) is a woody plant native to the Palearctic and planted as an ornamental worldwide. In May 2024, small brown necrotic spots were detected on leaves of a residential oleanders, an area with many California Bay Laurel (Umbellularia californica) infested by P. ramorum, in Mill Valley, Marin County, CA (37. 90481ºN, 122. 55282ºW). Symptomatic leaves tested positive using a genus-specific lateral flow assay (LFA) for Phytophthora (Agdia Inc, Elkhart, IN, USA). Leaves were surface sterilized using 70% ethanol, plated on selective PARPH media (Jeffers et al. 1986) and incubated at 20ºC. After 6 days, a single culture was isolated (NORS074) from the necrotic spots and produced morphological structures typical of P. ramorum, including coralloid mycelium, chlamydospores and semi-papillate sporangia (Fig. 1) (Werres, S. , et al. 2001). The internal transcribed spacer (ITS) and beta tubulin regions were sequenced using the primers ITS1/ITS4 (White et al. 1990; accession no. PX270955) and TUBUF2/TUBUR1 (Kroon et al. 2004; accession no. PX551880), respectively. A BLAST search revealed 99. 50% identity for ITS and 99. 68% for the beta tubulin with P. ramorum ex-type strain CBS 101553 (accession no. NR₁47877. 1 and LC595884. 1, respectively). Ten unwounded and ten wounded leaves of N. oleander were inoculated with strain NORS074 using two methods: i) mycelial plugs and ii) zoospore solution (1. 72 x 105 zoospores/ml). All four treatments induced necrotic leaf spots; and P. ramorum was re-isolated from the leaves. Sporulation of the isolate on inoculated leaves was observed. Pathogenicity tests were also performed on whole plants of N. oleander variety Austin City Limits (N = 10; 40 cm tall and 35 cm wide) using two methods: i) a zoospore solution (1. 9 × 105 zoospores/ml) was sprayed on the plants until runoff; ii) leaf tips were immersed in 1. 5 ml of the zoospore solution in a 15 ml conical tube attached to the plant during entire experiment. Ten leaves per plant were wounded for each assay. The inoculations were carried out in an open-field environment to simulate natural condition. Plants were misted with water and kept individually in plastic bags for 72 hours to maintain humidity level. Leaves inoculated with all four different treatments developed symptoms 12 dpi consisting of small black necrotic spots and lesions along the midrib vein (Fig. 2). Symptomatic leaves tested positive using the same Phytophthora LFA, and P. ramorum was re-isolated on PARPH media. The identity of the re-isolates was confirmed morphologically and by ITS sequencing. No symptoms were detected on untreated control plants. To our knowledge, this is the first report of P. ramorum occurring on N. oleander in the United States, and the first time ever, that Koch’s postulates have been confirmed for this host-pathogen combination. Previously, P. ramorum was detected on N. oleander in Ireland, but no inoculation studies were reported (O'Hanlon et al. 2016). This study provides the evidence to reclassify N. oleander from an APHIS 'associated host' (APHIS 2022) to a proven host. An ‘associate host’ is a plant from which a pathogen was isolated; a ‘proven host’ is a plant, for which Koch’s Postulates were confirmed. The original infection, occurring during the exceptionally wet 2023/24 winter in a shaded area, was condition-dependent; failure to re-isolate P. ramorum in Spring 2025 suggests the occurrence was transient and driven by extreme environmental pressures.