A Survey of Phytophthora spp. Associated with Abies in U.S. Christmas Tree Farms.

Multiple Phytophthora spp. are known to cause Phytophthora root rot (PRR) on Abies spp. Varying host susceptibilities and regional differences in Phytophthora community compositions complicate disease avoidance and management for Christmas tree growers and conifer nurseries. A survey of 32 Christmas tree farms in seven U.S. states representing four major Christmas tree production regions was conducted in 2012 to 2013 to characterize current distributions of root-rotting Phytophthora spp. and assess host responses to disease. Symptomatic tissues from lesion margins on roots and crowns were plated on selective medium to recover Phytophthora Cultures were identified by DNA sequencing of the nuclear internal transcribed spacer and mitochondrial cox I regions. Phytophthora cambivora was the prominent PRR species in western Washington and Oregon, while P. cinnamomi was prevalent in California and North Carolina. In the eastern United States, the undescribed P. taxon kelmania was isolated frequently and was capable of killing fir species considered tolerant of PRR. Completion of Koch's postulates confirmed pathogenicity of 12 previously unpublished Phytophthora-fir associations that were observed during this survey. Information regarding regional pathogen species prevalence and relative host susceptibilities will facilitate disease prevention by improving site management strategies for Phytophthora suppression.

First Report of a New Leaf Blight Caused by Phacidiopycnis washingtonensis on Pacific Madrone in Western Washington and Oregon.

In recent years, a leaf blight disease, consisting of browned, desiccated leaves occurring mainly in the lower parts of the canopy, has been observed during wet springs on Pacific madrone (Arbutus menziesii) in western Washington and Oregon. In May 2009 and 2011, severe outbreaks occurred and symptomatic leaves from madrones growing in the region were sampled to determine the causal agent. Two symptoms, leaf necrosis or blotching along the edges and tips of the leaves, and leaf spot, were observed. Small segments of diseased tissue were cut from the leaves, surface-disinfected, rinsed, and plated on malt extract agar. Fifty percent of the leaf blotch and 30% of leaf spot samples yielded a fungus that was fast-growing (20 mm diameter in 4 days at 25°C) and produced colonies that were a pale gray with dark gray reverse and a felty texture. On potato dextrose agar (PDA), pycnidia formed and exuded conidia in peach-colored droplets after 2 weeks under room temperature and light conditions. Pycnidia were spherical and 12.5 to 39.8 µm, average 24.2 µm in diameter. Conidia were hyaline, ovoid, and 5.8 to 8.5 × 3.1 to 4.7 µm (average 7.0 × 3.7 µm). The fungus was identified as Phacidiopycnis washingtonensis based on its morphology (1). To confirm the identity, the internal transcribed spacer (ITS) region of the rDNA was amplified with ITS1/ITS4 primers (2) and sequenced (GenBank Accession Nos. JQ743784 to 86). BLAST analysis showed 100% nucleotide identity with those of P. washingtonensis in GenBank (AY608648). The fungus was also isolated from lesions on green shoots and the petiole and leaf blade of dead attached leaves. To test pathogenicity, 3-year-old Pacific madrone seedlings (three for each isolate) were inoculated with five isolates of the fungus and maintained in the greenhouse (25°C); the experiment was conducted twice. Five leaves from each tree were cold injured (-50°C) at a marked 5 × 5 mm2 area with a commercial aerosol tissue freezing product prior to inoculation and five leaves were not cold injured. A 5-mm-diameter mycelial plug cut from the margin of 6-day-old PDA culture was applied to the marked areas on the upper leaf surface. The inoculated area was covered with moist cheese cloth and wrapped with Parafilm. Leaves treated with blank PDA plugs served as control. Leaves were enclosed in plastic bags to maintain moisture for the first 15 h post inoculation and cheese cloths were removed after 15 days. All cold-injured inoculated leaves showed symptoms of blight starting at 2 weeks after inoculation, and no symptoms appeared on the controls. On non-cold injured inoculated leaves, only one isolate caused symptoms (80% of all leaves). The fungus was re-isolated from diseased leaves. These results suggest that P. washingtonensis is able to cause foliar blight on Pacific madrone when leaves are subjected to cold stress. Increased disease severity on madrone observed in spring 2011 in Washington and Oregon may have been due to predisposition of foliage to extreme cold in November 2010 and February 2011. This fungus has previously been reported to cause a postharvest fruit rot disease on apple fruit and a canker and twig dieback disease of apple and crabapple trees in WA (1). To our knowledge, this is the first report of P. washingtonensis causing a leaf blight disease on Pacific madrone in North America. References: (1) C. L. Xiao et al. Mycologia 97:464, 2005. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Phytophthora ramorum Infecting California Red Fir in California.

In May 2005, branches originating from five separate whorls below the terminal on a single California red fir (Abies magnifica) in a mixed grand fir (Abies grandis) and Douglas-fir (Pseudotsuga menziesii) Christmas tree plantation near Los Gatos, CA displayed wilting and dieback of new shoot growth. Brown dieback, delineated by needle loss, extended 6 to 8 cm into 1-year-old and sometimes 2-year-old growth. The ~7-year-old, 1-m tall tree was located near the edge of the plantation, beneath an overstory of California bay laurel (Umbellularia californica) trees that were infected with Phytophthora ramorum. Isolations from dieback margins onto corn meal agar amended with ampicillin, rifamycin, and pimaricin (CARP) yielded hyphae and large, dark brown chlamydospores that were morphologically consistent with P. ramorum (1). Microsatellite analysis confirmed that isolates were of the NA1 lineage of P. ramorum. Isolates were deposited in the Washington State University Puyallup Phytophthora Master Collection. Dormant bareroot California red fir seedlings were obtained from the USDA Forest Service Placerville Nursery (Camino, CA) in February 2006 and planted in SC-10 super cell cones (Stuewe & Sons, Inc., Tangent, OR) in a standard greenhouse potting mix. Seedlings (average height 11 cm) were then forced to initiate bud break and new shoot elongation (0.5 to 1.5 cm) in a greenhouse at 21°C. Eight unwounded seedlings were inoculated with a zoospore suspension (4.185 × 105 zoospores/ml of sterile water) produced from 3- to 4-week-old V8 juice agar cultures of isolate WSU#106-0021 using an artist's airbrush powered by Badger Propel canned propellant. Eight control seedlings were sprayed with water alone. Seedlings were placed in plastic tubs with ~2.5 cm of warm water in the bottom to provide humidity. A plastic bag supported by a wire frame was used to cover each tub. Tubs were placed in a biocontainment unit at 15 to 16°C under 24 h of fluorescent light. The plastic was removed after 5 days and seedlings were left under the same conditions. Seven days after inoculation, 25 to 100% (average 68%) of the new shoots on each of the eight inoculated seedlings were wilted and 100% of these seedlings exhibited dark brown dieback into the 1-year-old stems (range of 1.0 to 2.3 cm, average 1.6 cm). Tissues from shoots and dieback edges were plated onto CARP media. All of these attempts resulted in successful isolation of P. ramorum, and cultures exhibited the same hyphal morphology and chlamydospore characteristics when compared with the isolate tested. Control plants did not develop symptoms. This trial completes Koch's postulates to establish California red fir as a host of Phytophthora ramorum. To our knowledge, this site contains the only reported infection of California red fir by P. ramorum. The potential for infection within its native range is unknown. Reference: (1) S. Werres et al. Mycol. Res. 105:1155, 2001.

Spread of Heterobasidion annosum in Christmas Tree Plantations of the United States Pacific Northwest.

ABSTRACT The population structure of Heterobasidion annosum in the Pacific Northwest (PNW) Christmas tree plantations was estimated at two spatial scales to assess the relative importance of primary and secondary infection, colonization, and spread of the pathogen. Ninety-three isolates from single trees in 27 discrete mortality pockets and 104 isolates from 12 individual root systems of noble and Fraser fir trees were sampled near Mossyrock, Washington. Isolates were genotyped using somatic compatibility assays and microsatellite markers to determine the spatial scale at which dispersal of single genotypes (genets) was occurring. All isolates sampled from different trees in discrete mortality pockets had distinct genotypes, whereas the root systems of single trees were dominated by one or two genotypes. These results suggest that infection of PNW Christmas trees results from frequent primary infection events of adjacent stumps and localized secondary spread within root systems rather than clonal spread of the pathogen between adjacent trees. We hypothesize that mortality pockets may be due to availability of infection courts and/or variation in inoculum levels during selective harvesting of patches of mature trees.

Recovery Frequency of Phytophthora ramorum and Other Phytophthora spp. in the Soil Profile of Ornamental Retail Nurseries.

We tested the hypothesis that inoculum of the aboveground exotic plant pathogen Phytophthora ramorum would be limited to the organic layer (top layer of plant debris) of soils at infested retail nurseries located outside of the area where the pathogen has become established in the landscape. To test this hypothesis and compare inoculum levels of P. ramorum with levels of other Phytophthora spp. in the soil profile, soil cores were collected and sampled from three Washington State retail nurseries at which the soil had previously tested positive for P. ramorum. Phytophthora was isolated from soil using rhododendron leaves as bait, and pure cultures were obtained and stored on V8 juice agar. Isolates were identified to species using a combination of DNA sequencing of the internal transcribed spacer (ITS) region of rDNA, real-time polymerase chain reaction (PCR) diagnostic testing, and culture morphology. Recovery frequencies were tabulated and compared by species at the organic layer, 0 to 5 cm, 5 to 10 cm, and 10 to 15 cm depth classes. The three most common Phytophthora spp. recovered from the soil cores were P. citricola (32%), P. drechsleri (32%), and P. ramorum (27%). P. citricola and P. drechsleri were more evenly distributed throughout the soil profile, whereas P. ramorum was primarily recovered from the organic and 0 to 5 cm depth class (86% of recoveries). P. ramorum was not detected below 10 cm.

Adhesion of Nongerminated Botrytis cinerea Conidia to Several Substrata.

Conidia of the plant pathogenic fungus Botrytis cinerea adhered to tomato cuticle and to certain other substrata immediately upon hydration. This immediate adhesion occurred with both living and nonliving conidia. Adhesion was not consistently influenced by several lectins, sugars, or salts or by protease treatment, but it was strongly inhibited by ionic or nonionic detergents. With glass and oxidized polyethylene, substrata whose surface hydrophobicities could be conveniently varied, there was a direct relationship between water contact angle and percent adhesion. Immediate adhesion did not involve specific conidial attachment structures, although the surfaces of attached conidia were altered by contact with a substratum. Freshly harvested conidia were very hydrophobic, with more than 97% partitioning into the organic layer when subjected to a phase distribution test. Percent adhesion of germinated conidia was larger than that of nongerminated conidia. Evidence suggests that immediate adhesion of conidia of B. cinerea depends, at least in part, on hydrophobic interactions between the conidia and substratum.