First report of boxwood blight caused by Calonectria pseudonaviculata in Kentucky

Boxwood is propagated on a small scale in Kentucky, but large numbers are imported to retail centers and landscape suppliers. In October 2014, a homeowner sample of English boxwood (Buxus sempervirens, cultivar unknown) grown in Fayette County was submitted to the University of Kentucky Plant Disease Diagnostic Laboratory owing to defoliation and twig dieback. Symptoms had spread throughout the landscape since May 2014, beginning with the introduction of new boxwood purchased from a retail center in Jefferson County, KY. Dieback symptoms spread from new plants to adjacent boxwood, and then to established plants throughout the landscape. Symptoms also included dark, circular leaf spots and elongated black/purple cankers on current season’s wood; cankers quickly girdled twigs. The causal pathogen was identified as Calonectria pseudonaviculata (syn. Cylindrocladium pseudonaviculatum and C. buxicola) by morphological and molecular techniques, as described below. Symptomatic tissue was incubated in moist chambers at room temperature under constant light. White conidial masses formed within 3 days; a secondary pathogen, Volutella sp., was also abundant on tissue. Masses of spores visually confirmed to be C. pseudonaviculata were diluted in sterile, deionized water and spread on ¼-strength PDA to help differentiate the target isolate from the Volutella sp. Germinating Calonectria spores were transferred to carnation leaf agar within 24 h. Resulting colonies were dark brown with white sporulation at the center of the colony. Conidia (n = 30) were hyaline with single septations and rounded on both ends, ranging from 44.8 to 64.4 µm (mean 57.3 µm) long and 4.2 to 5.6 µm (mean 5.3 µm) wide. Stipe extensions (n = 20) ranged from 112 to 140 µm (mean 121.5 µm). Vesicles were broadly ellipsoid (n = 20) averaging 29.7 µm long and 8.4 µm at the widest point above the middle; ends were pointed to papillate. These morphological characteristics were consistent with published reports for C. pseudonaviculata (Crous et. al. 2002; Malapi-Wight et. al. 2014). To verify pathogen identity, genomic DNA was extracted from single-spore colonies grown in potato dextrose broth, shaking at 125 rpm for 3 days. Primers Bt2a and Bt2b were used to amplify a portion of the β-tubulin gene (TUB2) and sequenced using the same primers (Glass and Donaldson 1995). Sequence fragment (GenBank Accession No. KU310967, 339 bp) was compared using a BLASTn search; results were 100% identity to several other C. pseudonaviculata isolates, including Accession Nos. KF785809.1, KF785808, and KF785811.1 (Malapi-Wight et. al. 2014). To confirm pathogenicity, one-gallon-size dwarf English boxwood Buxus sempervirens ‘Suffruticosa’ were inoculated to runoff with a 1.5 × 10⁵ conidia/ml suspension and maintained at room temperature while covered with plastic bags to retain humidity for 48 h. Inoculated plants (n = 3) developed boxwood blight symptoms within 4 to 6 days, and the pathogen was recovered using methods described above; water-inoculated control plants (n = 2) did not develop symptoms or signs. Recovered isolates were confirmed using morphological characteristics. This is the first report of C. pseudonaviculata as the causal agent of boxwood blight in Kentucky. In 2015, two reports of boxwood blight were confirmed in imported nursery stock in wholesale nurseries; morphological characteristics were used for identification. Thus, movement of nursery stock may be the largest threat of boxwood blight to Kentucky landscapes.