First Report of Bot Canker (Diplodia corticola) in Pin Oak (Quercus palustris) in Tennessee

Diplodia corticola is a fungal pathogen contributing to oak (Quercus spp.) decline in the Mediterranean region and the United States (Félix et al. 2017; Ferreira et al. 2021). In 2021, this pathogen was detected in Tennessee (TN), causing branch dieback in Q. alba (Onufrak et al. 2022). In September 2021, a matured pin oak (Q. palustris) with wilted leaves and elongated branch cankers was observed in the State Botanical Garden of Tennessee-Knoxville, TN, U.S.A. Small sections of the phloem were sampled from canker margins of a symptomatic branch using a sterile scalpel, surface sterilized, and plated onto potato dextrose agar amended with antibiotics (PDA++) (Gazis et al. 2018). Three days later, a fungal isolate resembling D. corticola was cultured on half-strength PDA. D. corticola was characterized on half-strength PDA by fast growth, irregular margins, and dense white mycelium that turns dark grayish as the mycelium matures (Alves et al. 2004; Úrbez-Torres et al. 2010). Total genomic DNA was extracted from this isolate as described by Gazis et al. (2018), and the internal transcribed spacer (ITS), large ribosomal subunit (LSU), and transcription elongation factor 1-α (ef1-α) were amplified (Ferreira et al. 2021). Resulting PCR products were sequenced and assembled into consensus sequences using Unipro UGENE version 44.0 (Okonechnikov et al. 2012). Each consensus sequence identity was determined using BLAST on the NCBI nucleotide database, restricted to type material. The ITS (accession OQ189888), ef-1α (accession OQ201608), and LSU (accession OQ189887) sequences had a 99.6% (accession KF766156.1), 98.6% (accession XM_020275852.1), and 100% (accession KF766323.1) identity match with the D. corticola type culture CBS112549, respectively. To complete Koch’s postulates and assess potential pathogenicity on economically and ecologically relevant oaks, 10 pin (Q. palustris; caliper 15.6 ± 2.0 mm), 10 overcup (Q. lyrata; caliper 15.1 ± 2.4 mm), and 10 sawtooth (Q. acutissima; 16.1 ± 2.1 mm) oaks were acclimated in the greenhouse for 1 week prior to the experiment. Five trees of each species were then randomly inoculated at 30 cm above the soil line with a 3-mm-diameter plug of D. corticola (grown for 10 days on PDA; Sitz et al. 2017). To serve as a control, the remaining five trees for each species received a 3-mm-diameter PDA plug. Fifteen days postinoculation, seepage was observed in D. corticola-inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks. No seepage from wound sites was noted in control trees. Cankers were exposed, photographed, and then measured using ImageJ (Rasband 2012). Using a sterile scalpel, four wood chips were excised from canker margins and plated onto PDA++. We recovered D. corticola from symptomatic inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks and confirmed species identity by extracting DNA and amplifying the ITS, ef-1α, and LSU regions as described above (Ferreira et al. 2021; Gazis et al. 2018). The resulting consensus sequences matched the D. corticola type culture (CBS112549) ITS (99.0 to 99.8% identity), ef-1α (91.0 to 99.1% identity), and LSU (96.9 to 100% identity) barcoding regions. Cankers were significantly larger in D. corticola-inoculated pin (4.7 ± 1.5 cm²; P = 0.003), overcup (6.8 ± 2.9 cm²; P = 0.009), and sawtooth (5.1 ± 1.3 cm²; P = 0.001) oaks in comparison with the control trees from these groups. Based on current reports, this is the first record of D. corticola causing dieback in pin oak (Q. palustris) in TN.