First report of Phytophthora ramorum causing leaf blight on Ceratonia siliqua in the United States

Abstract

Ceratonia siliqua (Fabaceae; carob) is a woody plant native to the Palearctic and widely planted as an ornamental worldwide. In February 2024, large, irregularly shaped black necrotic lesions were detected along the leaf margins of a mature carob tree in Larkspur, Marin Co., CA (37.94779 ºN, 122.54812 ºW). Symptomatic leaves tested positive using a genus-specific lateral flow assay (LFA) for Phytophthora (Agdia Inc, Elkhart, IN, USA). Tissue segments were surface sterilized using 70% Ethanol, plated on selective PARPH media (Jeffers et al. 1986) and incubated at 20ºC. A single culture (NORS075) was isolated from the leaves, which produced morphological structures typical of P. ramorum, including coralloid mycelium with distinct hyphal swellings, chlamydospores, and semi-papillate sporangia (Fig.1) (Werres, S., et al. 2001). The isolate was identified by sequencing the internal transcribed spacer (ITS) and beta tubulin regions using the primers ITS1/ITS4 (White et al. 1990; GenBank Accession No. PX564765.1) and TUBUF2/TUBUR1 (Kroon et al. 2004; GenBank Accession No. PX614639), respectively. A BLAST search revealed 99.50% identity for ITS (796/800 bp) and 99.77% for the beta tubulin (878/880 bp) with P. ramorum ex-type strain CBS 101553 (accession no. NR_147877.1 and LC595884.1, respectively). Detached leaves of C. siliqua were inoculated with strain NORS075 using two methods: i) mycelial plugs and ii) zoospore solution (1.13 x 105 zoospores/ml). Ten unwounded and ten wounded leaves were inoculated for each inoculation method. All four treatments resulted in necrotic leaf spots (Fig.2), and P. ramorum was successfully re-isolated. Sporulation was observed on the inoculated leaves. Pathogenicity tests were also performed on whole plants (N = 20; 12cm tall and 8cm wide) of C. siliqua using two methods: i) a zoospore solution (1.9 × 105 zoospores/mL) was sprayed on ten plants until runoff; ii) leaf tips of ten other plants were immersed in 0.75 mL of the zoospore solution in a 1.5 mL microcentrifuge tube attached to the leaves. For both experiments, five leaves per plant were wounded, while the remaining leaves were left unwounded. Control plants were treated as described above but with water. Leaves inoculated by any of the four different treatments developed symptoms 5 dpi consisting of small black necrotic spots and lesions around the inoculated area (Fig.2). Symptomatic leaves tested positive using the same Phytophthora LFA, and P. ramorum was re-isolated from them. The identity of the isolates was confirmed morphologically and by sequencing the ITS region. No symptoms were detected on the control plants. To our knowledge, this is the first report of P. ramorum occurring on C. siliqua globally and the first confirmation of Koch’s postulates for this host-pathogen combination. Previously P. ramorum has been isolated from other members of the Fabaceae, such as Pickeringia montana and Cercis chinensis (APHIS 2022). Later in 2024, symptoms associated with the presence of P. ramorum were detected on approximately 90% of all ornamental carob trees in Larkspur and the neighboring town of Mill Valley, but not in other areas within Marin Co. During 2025, symptoms were detected in the same areas, but at much lower rates. The exceptionally rainy winter of 2023–2024 in the area likely contributed to high inoculum pressure and favorable infection conditions not typically found in the open, drier areas characteristic of carob habitats. Nurseries growing carob trees should survey them regularly to avoid spreading the disease to new areas.