As part of the Phytophthora ramorum testing program from 2005 through 2007, a Phytophthora sp. was isolated on PARP-CMA medium (4) at the CDFA lab in Sacramento, CA, from the margin of necrotic spots and tissue suffering from dieback on Arctostaphylos sp. (manzanita), Camellia spp., Laurus nobilis (bay), Buxus sempervirens (boxwood), Rhododendron sp., Arbutus unedo (strawberry tree), and Sequoia sempervirens (coast redwood). Isolates were collected from Shasta, Contra Costa, San Diego, Solano, Santa Cruz, Alameda, Sacramento, San Joaquin, Monterey, and Los Angeles Counties. Isolates from A. unedo tissue on PARP medium produced apapillate, obovate sporangia 25 to 80 × 15 to 40 μm (48.0 × 26.9 μm average) and a few isolates produced intercalary and terminal chlamydospores at 22°C (30 to 46 μm diameter, 38.9 μm average). The internal transcribed spacer region (ITS) of rDNA was amplified from four isolates using ITS1 and ITS4 primers as described by White et al. (3) and the amplicons sequenced (GenBank Accession Nos. JQ307188 through JQ307191). BLAST analysis of the amplicons showed 99 to 100% identity with the ITS sequence of Phytophthora taxon Pgchlamydo from forest streams in Oregon (GenBank Accession No. HM004224) (1). Pathogenicity tests were performed on B. sempervirens, C. sasanqua, L. nobilis, and A. unedo. Five plants of each species were inoculated with 6-mm plugs taken from the margin of a 7- to 10-day-old culture grown on V8 juice agar. Plant leaves were wounded with a sterile pushpin and two agar plugs were covered with a freezer tube cap filled with sterile dH2O and clipped to the underside of the leaves with a sterile pin-curl clip (4). Inoculated plants were sprayed with water, covered with plastic bags, and incubated for 2 days, when bags and plugs were removed. Five leaves of each isolate plus five control plugs using V8 juice agar alone were inoculated on each plant. Plants were incubated for 12 days at 18°C (16-h photoperiod). Lesions formed on all inoculated plants, ranging in size from approx. 1 mm on B. sempervirens to 9.2 × 10.9 mm average on A. unedo. The lesions on A. unedo grew into and caused the mid-vein to blacken. The lesion sizes on camellia and bay were larger than those formed on B. sempervirens and smaller than those formed on A. unedo, with most lesions surrounded by a dark ring. Phytophthora taxon Pgchlamydo is associated with leaf lesions on rhododendron and dieback of yew in Minnesota (2). To our knowledge, this is the first report of Phytophthora taxon Pgchlamydo causing disease in camellia, bay, strawberry tree, and boxwood in California. Phytophthora taxon Pgchlamydo causes damage that is indistinguishable from the quarantine pest, P. ramorum (4). References: (1) P. W. Reeser et al. Mycologia 103:22, 2011. (2) B. W. Schwingle and R. A. Blanchette. Plant Dis. 92:642, 2008. (3) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds., Academic Press, San Diego, 1990. (4) L. E. Yakabe et al. Plant Dis. 93:883, 2009.

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.

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