ABSTRACT
Cornus capitata Wall. ex Roxb. (evergreen dogwood) is a bushy evergreen tree or shrub native to East Asia grown for its showy creamy bracts in late spring followed by attractive red fruit. In Feb 2023, a sample of foliage with leaf spots and tip dieback from C. capitata 'Mountain Moon' was submitted from a Humboldt Co. nursery as part of a CDFA inspection program for Phytophthora ramorum. The leaf spots were medium to dark brown, irregularly shaped, and ranged from 5 to 8 mm in diameter. They were located primarily along the leaf midrib and covered up to 1/4 of the leaf surface. Six 6-mm-diameter leaf discs taken from the margins of brown lesions and tip dieback were plated on Phytophthora selective CMA-PARP (PARP) media (Jeffers and Martin 1986). After 6 to 10 days, colonies resembling P. ramorum, with coralloid coenocytic hyphae, chlamydospores, ellipsoidal semi-papillate and caducous sporangia, and a relatively slow growth rate were recovered. Abundant sporangia formed on agar singly or in clusters on sympodially branched sporangiophores (n = 50), varying in size from 35 to 60 µm × 20 to 30 µm (mean 45.6 × 24.8 µm) with a length/breadth ratio ranging from 1.3 to 2.3 (mean 1.8). Chlamydospores (n = 50) ranged from 35 to 62 µm in diameter (mean 51.9 µm) on 14-day-old PARP cultures. The internal transcribed spacer region (ITS) using primers ITS5/ITS4 (White et al. 1990; accession no. OR636225) and cytochrome oxidase subunit 1 region (cox1) using primers OomCox1Levup/Fm85mod (Robideau et al. 2011; OR635665) of one isolate (0254-32A) were amplified and sequenced. BLAST analysis showed 100% identity of both regions to P. ramorum ex-type strain (MG865581 and MH136973). Microsatellite loci placed the P. ramorum isolate in the NA2 clonal lineage (Goss et al, 2011). Pathogenicity of P. ramorum isolate 0254-32A was tested using five C. capitata plants (2.5-year-old, 28-cm-tall, 3.78-liter pot). Zoospore inoculum was produced as described in Blomquist et al. (2021). Above ground parts of each plant were sprayed with inoculum (15 ml, 1.3 × 105 zoospores/ml). Inoculated plants were incubated in a dew chamber in the dark at 23°C for 72 h and then placed in a 23±1°C growth chamber with a 12-h photoperiod. Five control plants were treated as above but with sterile water instead of the zoospore suspension. Two days after inoculation, brown spots were visible on leaves on all inoculated plants, initiating from where the drops of inoculum had persisted. After 3 days, brown lesions, from water drop- to majority of entire leaf-sized, were observed on approximately 75% of inoculated leaves. After 6 days, lesions expanded to the edges of leaves, causing leaf curling and defoliation. Lesions stopped expanding after 3 weeks, and by 4 weeks, most infected leaves had abscised, with no new infections observed. Phytophthora ramorum was consistently isolated from foliar lesions of inoculated plants on PARP. It was not isolated from leaf or stem tissues of control plants, which remained asymptomatic during the 4-week experiment period. Phytophthora ramorum was detected on C. capitata in the UK in 2015 (DEFRA 2015). To our knowledge, this is the first report of P. ramorum infecting C. capitata in the United States and the completion of Koch's postulates on any Cornus spp. Incidence on C. capitata in the California nursery was low. However, their proximity to other infected foliar hosts suggests Cornus spp. may present a potential risk for the spread of P. ramorum.
ABSTRACT
Brisbane box,Lophostemon confertus (Myrtaceae) is a frost tender evergreen tree planted for its upright form, large ovate leaves and attractive white flowers which bloom in the spring. In June of 2017, the Plant Pest Diagnostics Center lab received a call from an arborist who described Brisbane box street trees dying in central Sausalito, Marin Co., California. Trees ranged from containing 10% to nearly 80% dead hanging leaves. Six trees along the same street were affected. Wilted brown leaves remained attached to branchlets covered in black cankers. Some healthy branchlets had leaves with angular spots which crossed the veins and were surrounded by yellow halos. Isolations were made onto CMA-PARP (Jeffers and Martin, 1986) from the canker and leaf spot margins. A Phytophthora species resemblingPhytophthora ramorum grew on CMA-PARP media with coralloid coenocytic hyphae, chlamydospores, and ellipsoidal semi-papillate sporangia. The internal transcribed spacer region (ITS) of rDNA was amplified and sequenced using primers PHY.OO.18F and PHY.OO.28SR (Rooney-Latham, et al. 2019). BLAST analysis of 770 base pairs of the sequenced amplicon (GenBank MK993541) showed 100% identity with the ITS sequence of the P. ramorum ex-type (MG865581). A portion of the cox2 gene was amplified and sequenced (Hudspeth et al. 2000) (GenBank MK994528) and 530 base pairs matched with 100% identity GU222130. Pathogenicity was confirmed by inoculating 3, initially 1.8-meter-tall trees in 18.9-liter pots. Prior to inoculations, trees were cut so they would fit into 122 cm high dew and growth chambers. For each tree, 3 lower branchlets measuring from 4 to10 mm in diameter were inoculated by wounding with a 6 mm punch, placing a colonized agar plugs in the wound, then wrapping with Parafilm. Lower branches were covered in plastic to protect them from subsequent zoospore inoculation. Branchlet inoculum was prepared by growing P. ramorum on V8 juice agar (V8) for 4 days at 22°C. Zoospores were prepared for leaf inoculation by taking 6 mm agar plugs from the margin of 6-day old cultures and flooding plugs in soil water for four days. Zoospores were released by transferring plugs to sterile distilled water at 4°C for 1.5 h. Leaves on the same three trees that were inoculated with the plugs were sprayed with 350 mL of zoospores (2 × 105 zoospores/mL), and placed in a dew chamber at 23°C for 48 h. Afterwards, they were transferred to a growth chamber (23°C, 12-h diurnal cycle) where the plastic was removed from the lower branches after leaves had dried. A single control tree was treated similarly with uncolonized V8 plugs, followed by a water spray. Leaf spots were visible 4 days later, with inoculated leaves turning necrotic and abscising after 3 weeks. Cankers from inoculated branchlets measured from 12 to 60 mm long after 60 days. Phytophthora ramorum was isolated from the margin of every inoculated canker and leaf spot. No P. ramorum was isolated from the control tree. To our knowledge, this is the first report of P. ramorum on L. confertus, in the world. Natural inoculum presumably came from infected Umbellularia californica trees located less than 800 m west of the trees in Sausalito. This detection will further limit the planting choices of arborists and landscapers in P. ramorum infected locations.
