First report of Phytophthora ramorum on Cotoneaster sp. in the USA.

Cotoneaster (Rosaceae) is a genus of woody plants native to the Palearctic region which includes popular ornamental plants; some are invasive in parts of the USA. In May 2022 symptomatic leaves were detected on Cotoneaster pannosus (Silverleaf Cotoneaster) in Marin County, California (37.89165, -122.56755 ), an area infested heavily by Phytophthora ramorum, causal agent of Sudden Oak Death. Symptoms consisted of dark brown necrotic spots mostly near the tips and sometimes on the margin of the leaves, covering less than half of the leaf surface; no die-back or symptoms on twigs were detected. Diseased leaves were surface-sterilized with 70% ethanol, washed twice with de-ionized water, and placed on PARPH(V8) media. Two Phytophthora ramorum like isolates (NORS058 and NORS059) were obtained from different leaf samples from the same tree and the internal transcribed spacer (ITS) region was sequenced. Both sequences were deposited in GenBank (OR224345 and OR224346). NORS058 and NORS059 showed 99.88% and 99.75% sequence identity to P. ramorum strain Ex-type CPHST BL 55G (MG865581.1). Detached leaves of C. pannosus and C. lacteus (Milkflower Cotoneaster) were inoculated with mycelial plugs of P. ramorum NORS058, and incubated at 20°C. Both species developed necrotic leaf spots seven days post inoculation (dpi). Sporulation of the pathogen was observed on symptomatic leaves of C. lacteus. P. ramorum was reisolated from the symptomatic leaf tissue from both Cotoneaster species. Pathogenicity tests were also performed on whole plants of C. dammeri (Bearberry Cotoneaster) using the strain NORS058. Five plants each were inoculated using three different methods: 1) a zoospore solution (concentration 2.5 x10E5 spores/mL) were sprayed on the plant surface until run off. Ten leaves per plant were wounded with a needle, the remaining leaves were not wounded; 2) 200 µL of the zoospore solution in a PCR tube were attached to 5 leaves of each plant; and 3) 10 mL of the zoospore solution was drenched into the potting mix of the five plants. Control plants were treated as above but with water instead of the zoospore solution. Leaf spots developed 7 dpi on plants sprayed with zoospores on wounded leaves; and 10 dpi on plants treated with zoospores in the tube. P. ramorum was reisolated from symptomatic leaves treated with the first two methods mentioned above. Plants treated with a soil drench did not develop symptoms on the aerial parts or on roots that were sampled 50 dpi. Tests using AGDIA- immunostrips of the roots were negative. Control plants showed no aerial or root symptoms. To our knowledge, this is the first report of P. ramorum occurring on Cotoneaster in the USA. Previously, inoculation of detached leaves of C. dammeri and C. horizontalis with P. ramorum in Serbia resulted in symptom expression (Bulajic et al. 2010). P. ramorum was reported from a Cotoneaster sp. in the UK in 2010, but no further information were presented (FERA 2015). The tree sampled in 2022 showed symptoms again in spring 2023 and official regulatory samples were taken by the CDFA (California Department of Food and Agriculture) and confirmed by the USDA. During a survey in 2023, more symptomatic Cotoneaster plants were detected in Marin County, California, indicating Cotoneaster might play a role in the epidemiology of the disease. References: FERA 2015. https://planthealthportal.defra.gov.uk/pests-and-diseases/high-profile-pests-and-diseases/phytophthora/ Bulajic et al. 2010. Plant Dis. 94(6): 703.

Citizen Science Uncovers Phytophthora ramorum as a Threat to Several Rare or Endangered California Manzanita Species.

The Sudden Oak Death (SOD) Blitzes consist of yearly surveys led by citizen scientists designed to map the distribution of Phytophthora ramorum, cause of the forest disease called SOD, across northern California. During the 2017 Santa Cruz County SOD Blitz, six rare or endangered Arctostaphylos (manzanita) species were found to be possibly symptomatic for the first time. Symptoms included branch cankers and associated canopy mortality, and affected multiple individuals per species. Isolates of P. ramorum were obtained from each of the six species and, through a 30-day-long inoculation experiment on live plants, Koch's postulates were completed for each one of them, conclusively determining that they all are hosts of this pathogen. Two additional manzanita species were later found to be apparently symptomatic in Marin County. Inoculations on detached branches using an isolate of P. ramorum obtained from one of the six rare species from Santa Cruz County were successful, suggesting that these two species may also be hosts of P. ramorum. Detached leaves of all eight species were also successfully inoculated at the University of California-Berkeley in fall 2018 and then again in spring 2019. In these cases, the same isolate was used for all inoculations, in order to obtain information on the comparative susceptibility of the eight species in question. Both branch and leaf inoculations identified significant interspecific differences in susceptibility. The production of sporangia was low on all species but it was not zero, suggesting that sporulation may cause within-plant and limited across-plant contagion, especially in rainy years.

Detection and quantification of airborne conidia of Fusarium circinatum, the causal agent of pine pitch canker, from two California sites by using a real-time PCR approach combined with a simple spore trapping method.

Pinus radiata (Monterey pine), a tree native to coastal California and Mexico, is widely planted worldwide for timber production. A major threat to Monterey pine plantations is the fungal disease pine pitch canker, caused by Fusarium circinatum (Hypocreales). We present a novel trapping approach using filter paper in combination with a rapid molecular method to detect the presence of inoculum in the air. The assay is also useful for diagnosing the presence of the pathogen on plants. The test is based on the F. circinatum specific primer pair CIRC1A-CIRC4A, which amplifies a 360-bp DNA fragment in the intergenic spacer region of the nuclear ribosomal operon. Real-time PCR was used to calculate the number of fungal spores present in each reaction mixture by comparing the threshold cycle (Ct) of unknown spore samples to the Ct values of standards with known amounts of F. circinatum spores. The filter paper method allows prolonged and more sensitive spore sampling in the field compared to traditional traps using petri dishes filled with selective medium. A field test at two sites in coastal California infested with pine pitch canker was carried out during the summer and fall of 2002. Spore counts were in the range of ca. 1 x 10(3) to ca. 7 x 10(5)/m(2), with the highest spore counts in the fall, suggesting a seasonal fluctuation.