First Report of Diplodia corticola Causing Stem and Branch Cankers on White Oak (Quercus alba) in Missouri, U.S.A.

An investigation of white oaks (Quercus alba) with thin crowns, branch dieback, or both was performed in Crawford and Washington counties, Missouri, during September 2015. Affected stem and branch segments were cut from each tree for examination. After debarking, isolations were performed by using a sterile scalpel to cut cubes (approximately 2 mm³) from tissues at the edges of cankers and underlying stained sapwood. Cubes were placed in quarter-strength potato dextrose agar (PDA) amended with chloramphenicol and streptomycin sulfate (100 mg/liter). A fungus frequently recovered from four of six symptomatic trees was suspected to be a Botryosphaeriaceae species based on its colony morphology, reproductive structures, and conidia (Alves et al. 2004). DNA was extracted from seven isolates, and the internal transcribed spacer (ITS) and the gene portions of nuclear ribosomal small subunit (SSU) regions were sequenced after amplification using ITS1F and ITS4 and NS1 and NS4 primers (White et al. 1990). The resulting sequences were deposited in GenBank (accession nos. MH041506 to MH041510, MH036328, and MH036329). Contigs had 100% homology with the ITS and SSU regions of multiple Diplodia corticola isolates (accession nos. KX595187.1, KT440896.1, KX463057.1, KF766239.1, and EU673206.1). Two isolates were chosen as inocula for a pathogenicity test. In a greenhouse trial, five 2-year-old seedlings were inoculated with isolate 25, and another five seedlings were inoculated with isolate 101. The fully leafed and healthy, potted seedlings were exposed to natural light. Inoculations were made by puncturing the bark of seedling stems 5 cm above the soil surface with a sterile 8-mm-diameter cork borer, peeling back the outer bark, and placing PDA bearing mycelium of either isolate into the wound. The bark was replaced and the wound wrapped with Parafilm. As a control treatment, a sterile plug of noncolonized PDA was wound inoculated 10 cm above the D. corticola inoculation site. The bark was peeled from the wounds at 8 weeks. Canker shapes were drawn on transparency film and photographed prior to measuring length and surface area with ImageJ 1.51j8 (from the National Institutes of Health). Stems were split in half and cankers measured lengthwise. Tissues were removed from the canker margin and placed in PDA for recovery of the pathogen. Vascular tissues were sampled at 0, 1, 2, and 3 cm above and below the control wound to determine if D. corticola had spread from the inoculation to the control wound. Cankers of variable sizes developed around wounds. Mean (and range) of canker areas were 4.5 cm² ± 1.4 cm² SE (2.3 to 9.1 cm²) for isolate 25 and 4.9 cm² ± 1.7 cm² SE (1.1 to 9.6 cm²) for isolate 101. The means for lengths of cankers induced by isolates 25 and 101 were 5.7 ± 0.6 and 4.6 ± 1.0 cm, respectively, when measured on stems split lengthwise. Xylem was discolored to the pith for four of five and two of five wounds inoculated with isolates 25 and 101, respectively. Cankers did not develop on wounded controls. D. corticola was recovered from canker edges from three of five seedlings for each isolate but not from sampled tissues 0, 1, 2, or 3 cm from control wounds. Hence, the pathogen did not progress far from the inoculation point. The identities of the recovered isolates were confirmed by sequencing the ITS region. This is the first report of D. corticola-caused cankers on oaks in Missouri. Earlier reports have shown that D. corticola causes cankers on Q. alba and other oak species also abundant in Missouri’s oak-dominated forests (Acimovic et al. 2016; Munck et al. 2017; Smith and Stanosz 2018). The role of D. corticola in oak dieback and mortality bears further study to understand any potential impacts on oaks that are of predominant importance in Missouri forests.