Genetic and Pathogenic Characterization of Phacidiopycnis washingtonensis from Apple and Pacific Madrone from the Western United States

Phacidiopycnis washingtonensis is the cause of speck rot of apple and leaf blight of Pacific madrone in Washington State. In total, 314 isolates were collected from apple production areas in eastern Washington and Pacific madrone in western Washington. Using eight microsatellite markers designed in this study, 58 unique multilocus haplotypes were identified. Only one of the haplotypes was shared between the apple and Pacific madrone populations. Analysis of molecular variance showed no genetic differentiation between the apple and Pacific madrone populations. Genetic variation was present within each subpopulation of apple from different geographic locations. The apple population possessed higher genotypic diversity than the Pacific madrone population, suggesting that isolates from apple may represent an older population and could have been introduced into the native habitat of Pacific madrone. P. washingtonensis likely reproduces asexually because populations examined in this study were not in linkage equilibrium. In pathogenicity tests, representative isolates from apple and Pacific madrone all incited leaf blight on Pacific madrone and speck rot on apple fruit regardless of their host of origin. Overall, our findings indicate that the P. washingtonensis population in Washington State is largely asexual, with high genotypic flow and that apple, crabapple, and Pacific madrone could serve as sources of P. washingtonensis inoculum for these hosts.

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.

Injected Treatments for Management of Madrone Canker

Pacific madrone (Arbutus menziesii) has been experiencing a decline in the Puget Sound area, primarily as a result of a canker disease caused by the fungus Fusicoccum arbuti. Cultural methods such as prevention of stress and wounding are recommended to control canker diseases on trees. In addition to these, injected treatments can be used to protect valuable Pacific madrone trees in urban areas. An experiment testing injectable chemical fungicides and plant activators was performed on Pacific madrone trees inoculated with F. arbuti. There was little correlation between fungicidal activity in culture and canker reduction in the field tests. Two treatments that were effective in minimizing canker growth in inoculated madrones were Arbotect® (Syngenta Crop Protection Inc., Greensboro, NC, U.S.; a triazole fungicide) and BioSerum™ (phosphorous acid). Cankers on wound inoculations were 50% smaller than the control group and no infections occurred on surface-inoculated treatments. Increased callusing was observed on cankers on trees with these treatments and the mode of action for these chemicals is probably stimulation of plant defenses rather than fungicidal action. Phosphorous acid is recommended in addition to cultural methods that improve tree vigor for high-value madrone trees in urban landscapes; however, heavily infected trees that have lost most of their crown will probably not benefit.

Disease Progression of Phytophthora ramorum and Botryosphaeria dothidea on Pacific Madrone

Infection by Phytophthora ramorum was associated with stem and leaf lesions of Pacific madrone (Arbutus menziesii) seedlings and saplings. In addition, a common and native pathogen, Botryosphaeria dothidea, caused similar leaf and stem lesions. When exposed to natural levels of inoculum in forests infested with P. ramorum, 50 to 66% of madrone saplings used as bait died. Recovery of P. ramorum from colonized plant tissue on culture media was generally low. From initial infection, P. ramorum was not culturable from leaf tissue after a mean of 3.5 weeks or from stem tissue after a mean of 8 weeks. Generally, B. dothidea was recovered more frequently from necrotic stems and leaves than was P. ramorum. Experimental inoculations of madrone seedlings showed that leaf and stem lesion lengths were, on average, greater on tree seedlings inoculated with P. ramorum than on those inoculated with B. dothidea. P. ramorum and B. dothidea appear to coexist in stem and leaf tissue, forming a novel pathogen complex, affecting growth and reproduction of Pacific madrone.

Seasonal pattern of water depletion from soil–rock profiles in a Mediterranean climate in southwestern Oregon

Neutron probe measurements from aluminum access tubes (3.2 m deep) show that conifers and sclerophyllous shrubs deplete water from soil–rock profiles in distinctly different patterns. Measurements were taken during two growing seasons (1993 and 1994) in southwestern Oregon (U.S.A.) under 14-year-old stands of Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco), ponderosa pine (Pinusponderosa Dougl. ex Laws.), Pacific madrone (Arbutusmenziesii Pursh), and whiteleaf manzanita (Arctostaphylosviscida Parry) covers. Both conifer species showed very limited abilities to utilize water from bedrock, even during very dry periods. Ericaceous plants, on the other hand, were very efficient in taking water from the deep rock layers, using nearly all available water and removing about twice as much water as conifers of the same age. Patterns of water depletion suggest that the water resources used by these two types of plants overlap considerably, and competition will be very strong in mixed stands. The ability of ericaceous plants to utilize bedrock water that is unavailable to conifers in late summer will enhance their potential for dominance.

Root distribution of 12-year-old forests at rocky sites in southwestern Oregon: effects of rock physical properties

Distribution of root length density (in terms of centimetres of fine and large roots per cubic centimetre of soil and bedrock) was analyzed at harsh forest sites supporting 12-year-old stands of pure whiteleaf manzanita (Arctostaphylosviscida Parry), pure ponderosa pine (Pinusponderosa Dougl. ex Laws.), or mixtures of Pacific madrone (Arbutusmenziesii Pursh) and Douglas-fir (Pseudotsugamenziesii (Mirb.) Franco) in southwest Oregon. On such sites, soil (usually <50 cm deep) dries below the wilting point of plants before the middle of the growing season. The bedrock contains enough water to support a dense stand of woody plants. The study shows that about one quarter to one third of the total root length is located in the rock layer. Stepwise procedures were applied to find the best fitting multiple-regression model for relating root length density to depth, bulk density of the soil and rock, and the space penetrable by roots (SPR). Linear expressions of SPR and depth were found to be the only significant explanatory variables. Bulk density had very limited explanatory power, reflecting the high general density of the bedrock containing the water but lower density of the fine material in minuscule fissures in which roots were found.