First Report of Diplodia mutila Associated with Southwestern White Pine Cankers and Dieback in Arizona

Southwestern white pine (Pinus strobiformis, SWWP) is native to the Southwestern United States and Mexico, providing numerous ecological and economic benefits (Looney and Waring 2013, Shirk et al. 2018). Symptoms of novel branch and bole cankers, resinosis on branches and bole, and crown dieback were first observed on SWWP in 2019 on the Apache-Sitgreaves National Forest in Central Arizona. Similar symptoms were then observed in the Coronado National Forest, a disjunct, distant location in Southern Arizona, indicating the pathogen is likely widespread. Isolation from live branches with cankers yielded fluffy fungal colonies with rapid growth and pigmentation. On potato dextrose agar (PDA) at 25°C, the fungal colonies exhibited white to gray, with moderate aerial mycelium that turned dark olive green. The hyphae were hyaline, septate, smooth-walled, and branched. The culture formed pycnidia after 2 weeks on sterile pine needles placed on the PDA surface. Conidia were ovoid, one-septate, and had a size range of 15.7 to 28.2 × 10.5 to 16.8 µm (n = 20). Genomic DNA was extracted from two representative isolates. Partial DNA sequences of the internal transcribed spacer (ITS-rDNA) and β-tubulin gene (TUB) were amplified and sequenced with the primer sets ITS1/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The DNA sequences were deposited in the GenBank database (PZ014346, PZ014347, PZ069386, PZ069387). BLASTn analyses of ITS sequences PZ014346 and PZ014347 revealed ≥ 99% identity to NR_144906, a sequence of D. mutila strain CBS 112553 and with PX589421, the sequence of D. mutila causing canker and dieback in service tree (Cormus domestica) in France (Fernandez-Conradi 2025). The TUB sequences were found to have a 99.22% identity to MT592511 (D. mutila CBS:125.37) and 98.96% identity to ON054939, the sequence of a D. mutila strain causing wood necrosis in European hazelnut (Corylus avellana) in Chile (Moya-Elizondo et al. 2023). A pathogenicity test was conducted using two isolates by stem inoculation on twenty 2-year-old SWWP seedlings maintained in a greenhouse at 18 to 26°C. A mycelial plug (4 to 5 mm 2 ) taken from the margin of an actively growing colony on PDA was put in a shallow wound (~4 mm) made by a scalpel on the stem of each seedling. Parafilm was used to seal the stem wound and removed after 2 weeks. Three weeks after inoculation, the seedlings displayed wilting of new shoots, dark brown-to-black discoloration, and resinosis. The two isolates produced similar-sized lesions, with an average lesion length of 2.9 cm (2.2 to 3.1 cm) on the stem. Twenty control seedlings with sterile PDA plugs placed on stem wound remained asymptomatic. The pathogen was successfully re-isolated from inoculated diseased stems and molecularly confirmed as D. mutila, but not from control plants, thus fulfilling Koch's postulates. This is the first report of D. mutila on SWWP in the United States, representing a new dieback and possible mortality agent for this native tree species. The symptoms caused by D. mutila may be confused with the invasive disease white pine blister rust (WPBR), which also infects SWWP in the region. Knowledge of D. mutila will aid in accurate tracking of both WPBR and D. mutila. This first report contributes to our knowledge of the damaging agents of SWWP.