Since the beginning of the twentieth century oak decline has been documented in central and eastern hardwood forests of the United States as a stress-mediated disease (Oak et al. 2016). Opportunistic canker pathogens, including Diplodia corticola, D. quercivora, D. sapinea, and Botryosphaeria dothidea have been associated with crown dieback of declining oak trees in several mid-Atlantic states (Ferreira et al. 2021). On 02 August 2022, a survey was conducted at two natural hardwood sites in Fredrick and Shenandoah Counties, Virginia that exhibited symptoms of decline (Fig. 1A). At both sites, mature Quercus montana trees were observed with bole and branch cankers, bleeding and sooty lesions, and discolored sapwood. Pycnidia were present on the margin of seven branch cankers from three trees that were felled, with hyaline, elliptical to oblong conidia 19.0 - 26.8 × 8.5 - 11.2 µm (n = 40) in size (Fig. 1C and D). Six cultures were derived from single spores that were placed on PDA medium and incubated for 10 days in the dark at 22 ± 2°C. Additionally, a 4-mm piece of necrotic tissue was selected from the margin of each of the seven cankers, disinfected with 2.5% NaOCl, again with 70% ethanol, and air-dried before being placed on half-strength acidified PDA medium (pH 4.8) and incubated in the dark at 22 ± 2°C. After 5 days, seven colonies from each canker assayed were transferred to full-strength PDA plates and incubated for 10 days in the dark at 22 ± 2°C. Colonies derived from spores and the necrotic wood were morphologically identical, with white, aerial, floccose mycelium that turned dark gray to olivaceous after five days (Fig. 1B). DNA was individually extracted from four, 10-day-old cultures (two from spores and two from wood). Mycelia was harvested with a sterile pin and extracted using a Qiagen DNeasy Plant Pro Kit (Germantown, MD) according to the manufacturer's instructions. A segment of the internal transcribed spacer (ITS), large subunit rRNA (LSU), and translation elongation factor 1-α (tef1) loci were amplified using ITS4/ITS5 (White et al. 1990), LR5/LROR (Vilgalys and Hester 1990), and EF1-728F/EF1-986R (Carbone and Kohn 1999) primer sets, respectively. The PCR amplicons were purified with ExoSap-IT (Affymetrix, Santa Clara, CA) and sequenced at Eurofins (Louisville, KY). The nucleotide sequences were analyzed using Geneious 11.1.5 software (Biomatters, Auckland, NZ). The resulting ITS sequences from the four isolates were identical. A 544-bp, 1131-bp, and 273-bp segment of the ITS, LSU, and tef1 loci from isolate GS22-DSB-17 was deposited into the GenBank database (accessions OQ597712, OQ597714, and OR754429, respectively). A Genbank BLAST analysis revealed that the ITS and tef1 fragments shared 510/516 (99%) and 271/273 (99%) nucleotides with the D. quercivora ex-type BL8 (JX894205/JX894229). Koch's postulates were fulfilled by inoculating five healthy, containerized Q. montana trees (average stem caliper 6.5 cm) with D. qercivora isolate GS22-DSB-17, while five plants were used as controls. After disinfecting the bark with 70% ethanol, a 0.5 mm section of the bark was removed 13 cm above the soil line with a sterile scalpel, and a 0.5 mm agar plug taken from the edge of a 10-day-old PDA culture was placed in the wound with the mycelium facing the cambial tissue, sealed with Parafilm, and maintained at 22 ± 6°C. The same procedure was performed on the control plants using sterile PDA plugs. After six weeks the bark was carefully removed, and all five stems treated with D. quercivora had necrotic lesions with a mean canker linear growth ([length+width]/2) of 15.6 mm from the edge of the wound, which was significantly larger (P = 0.001) than the controls (2.3 mm; Fig. 1E-M). Necrotic stem tissue was sampled as previously described, and the isolate recovered was confirmed as D. quercivora based on morphology and 100% ITS sequence homology to isolate GS22-DSB-17. D. quercivora was not recovered from the control plants. In the United States, D. quercivora has been isolated from declining white oak trees in Maryland, Massachusetts, West Virginia, and Florida (Dreaden et al. 2014; Ferreira et al. 2021; Haines et al. 2019). More surveys are needed to understand the host range and distribution of D. quercivora in the United States, as well as the environmental and site factors that impact oak health and predispose trees to infection from opportunistic cankering pathogens.
Keywords: Causal Agent; Crop Type; Epidemiology; Fungi; Pathogen detection; Pathogen diversity; Subject Areas; Trees; disease development and spread; forest.