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.

Sensitivity of Phacidiopycnis spp. Isolates from Pome Fruit to Six Pre- and Postharvest Fungicides

Phacidiopycnis washingtonensis and P. pyri cause speck rot and Phacidiopycnis rot on apple and pear, respectively. Infection occurs in the orchard and remains latent, and symptoms appear after months of storage. Decay management relies on orchard sanitation and pre- and postharvest fungicides. In a 2017 survey, speck rot accounted for 6.4% of apple decay in central Washington, whereas Phacidiopycnis rot accounted for 3.9 and 6.7% of total pear decay in Washington and Oregon, respectively. Sensitivities of baseline populations of 110 P. washingtonensis and 76 P. pyri isolates collected between 2003 and 2005 to preharvest fungicides pyraclostrobin (PYRA) and boscalid (BOSC) and to postharvest fungicides thiabendazole (TBZ), fludioxonil (FDL), pyrimethanil (PYRI), and difenoconazole (DFC) were evaluated using a mycelial growth inhibition assay. Mean effective concentrations necessary to inhibit 50% growth (EC50) of P. washingtonensis were 0.1, 0.3, 0.8, 1.8, 2.1, and 4.8 µg/ml for FDL, PYRI, TBZ, DFC, PYRA, and BOSC, respectively. Respective mean EC50 values for P. pyri were 0.2, 0.6, 1.6, 1.1, 0.4, and 1.8 µg/ml. The sensitivity of exposed P. washingtonensis and P. pyri populations collected in 2017 revealed potential shifts toward BOSC and PYRA resistance. The efficacy of the six fungicides to control isolates of each pathogen with different in vitro sensitivity levels was evaluated on apple and pear fruit. FDL, DFC, and PYRI controlled both Phacidiopycnis spp. regardless of their EC50 values after 5 months of storage at 0°C in a regular atmosphere. The consistent occurrence of Phacidiopycnis spp. will require continuous monitoring and development of disease management strategies based on fungicide phenotypes and efficacy of existing fungicides assessed herein.

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.

Infection Courts and Timing of Infection of Apple Fruit by Phacidiopycnis washingtonensis in the Orchard in Relation to Speck Rot During Storage

Phacidiopycnis washingtonensis is the cause of speck rot, a recently reported postharvest fruit rot disease of apple. The pathogen is believed to incite infections in the field, and disease symptoms become evident only during storage. To determine the timing of apple fruit infection in relation to development of speck rot in storage, ‘Red Delicious’ and ‘Fuji’ apple fruit were inoculated in the orchard with P. washingtonensis at different times during the growing season, harvested, and monitored for decay development during storage at 0°C. Fruit inoculated in both field and laboratory also were used to identify the infection courts and mode of apple fruit penetration by P. washingtonensis. In all 3 years, stem-end speck rot and calyx-end speck rot developed during cold storage on fruit inoculated during the growing season, regardless of inoculation time; and the incidence of total speck rot in storage increased as the fruit inoculation time approached harvest. On fruit floral parts, the pathogen colonized sepals at higher rates than stamens. Availability of naturally occurring necrotic tissues favored the colonization of the fungus on sepals. Histological studies indicated that infection occurred through micro-cracks on the surfaces of pedicels and sepals of the fruit, and invasion of these tissues was restricted between the cuticle and epidermis. Findings of this study will assist in the development of effective control strategies for speck rot.

Development of PCR Assays for Diagnosis and Detection of the Pathogens Phacidiopycnis washingtonensis and Sphaeropsis pyriputrescens in Apple Fruit

Speck rot caused by Phacidiopycnis washingtonensis and Sphaeropsis rot caused by Sphaeropsis pyriputrescens are two recently reported postharvest diseases of apple. Infection by these two pathogens occurs in the orchard but remains latent before harvest. Symptoms develop after harvest and are similar to those of gray mold caused by Botrytis cinerea. Accurate diagnosis of these diseases is important during the fruit inspection process, particularly in the instance of fruit destined for export. Early near-harvest detection of latent infections in apple fruit is an important step to implement relevant pre- and postharvest measures for disease control. The aim of this study was to develop polymerase chain reaction (PCR) assays for diagnosis and early detection of latent infections of apple fruit by P. washingtonensis and S. pyriputrescens. Species-specific primers based on the ribosomal DNA internal transcribed spacer region were designed for use in PCR assays. Conventional and real-time PCR assays were developed and validated using fruit inoculated with P. washingtonensis, S. pyriputrescens, or B. cinerea and compared with identifications using traditional isolation-based assays. For wound-inoculated fruit, the PCR assays consistently provided the correct identification of the pathogen used as the inoculant in 6 h of processing time, compared with 5 to 6 days using culture-based methods. Real-time PCR assays effectively detected latent infections in symptomless stem and calyx tissues of fruit that were inoculated with the pathogens in the orchard during the growing season. The PCR assays provide a rapid, accurate method for diagnosis and early detection of these diseases.