First Report of <i>Botryosphaeria dothidea</i> Causing Canker of <i>Calocedrus decurrens</i> in Tennessee

Incense cedar (<i>Calocedrus decurrens</i>) is a conifer native to the western United States, valued for its low maintenance and aesthetic appeal in ornamental landscapes. In May 2024, field and container grown incense cedar exhibited canker and dieback in a USDA research plot at the Nursery Research Center, McMinnville, TN (Fig. 1a). Disease incidence was 60% of a total 100 plants. For isolation of the causal agent, small pieces (∼0.3 mm²) of the branch and stem cankers were excised, surface sterilized with 1% sodium hypochlorite for 1 minute, followed by 70% ethanol for 30 seconds, and washed twice with sterile water. The pieces were placed on potato dextrose agar (PDA) and incubated at 25°C under 12-hour light/dark conditions. Colonies initially appeared white, and after four to six days, they gradually turned dark gray with dense aerial mycelia (Fig. 1b). Conidia produced within pycnidia after 3 weeks were hyaline, fusiform, and aseptate, with dimensions of 21.1 to 25.8 μm (avg. 24.35 ± 1.049 μm) in length and 5.7 to 8.2 μm (avg. 6.1 ± 0.687 μm) in width (<i>n</i> = 50) (Fig. 1c). Pathogen identity was confirmed by extracting total genomic DNA using the DNeasy PowerLyzer Microbial Kit from 7-day-old pure cultures (isolates FBG7412 and FBG7413). The primer pairs ITS1/ITS4 (White et al. 1990), T1F/Bt2b (Glass and Donaldson 1995; O'Donnell and Cigelnik 1997), and EF1-728F/EF2 (Carbone and Kohn 1999) were used to amplify the ribosomal internal transcribed spacer (<i>ITS</i>), and nuclear beta-tubulin (<i>TUB</i>) and translation elongation factors 1-α (<i>EF1-α</i>) genetic markers, respectively. The sequences of the two isolates (FBG7412 and FBG7413) were deposited in GenBank with accession numbers PQ482605 and PQ482606 (<i>ITS</i>); PQ493369 and PQ493370 (<i>TUB</i>); and PQ493366 and PQ493367 (<i>EF1-α</i>), respectively. GenBank BLAST search of sequences using the core nt database showed 100% identity (100% coverage) of <i>ITS</i>, <i>TUB</i>, and <i>EF1-α</i> sequences to <i>Botryosphaeria dothidea</i> isolates IS2116-1 (OR958722), MEND-F-0379 (OQ994765), and IRNBS19 (MN633962), respectively. Phylogenetic analysis of concatenated <i>ITS</i>, <i>TUB</i>, and <i>EF1-α</i> sequences confirmed the pathogen as <i>B. dothidea</i> (Fig. 2). Two same two fungal isolates FBG7412 and FBG7413 were used to inoculate incense cedar cuttings grown in 3.8L pots with each plant measuring 12-15 cm in height. The inoculum was prepared by growing each isolate on PDA for one week at 25°C. For inoculation, a thin slice of bark (4 mm²) was removed from the stem of each cutting, and a 4-mm-diameter plug colonized by the fungal isolate was placed on the wound which was then covered with Parafilm. Negative controls were mock inoculated with sterile PDA plugs. There were five replications for each isolate and control. The study was conducted in a greenhouse maintained at 23-25°C and 70% relative humidity, with a 16-hour photoperiod. Six weeks post-inoculation, the branches inoculated with <i>B. dothidea</i> exhibited browning and dieback while the controls remained green and healthy (Fig. 1d-f). A dark, necrotic lesion that extended up and down from the inoculation point gradually spread to kill the main stem. Fungal isolate (FBG7703) with the same morphology and DNA sequences as FBG7412 and FBG7413 were recovered from the symptomatic stems of the inoculated plants. Control plants remained symptomless, and <i>B. dothidea</i> was not isolated from them. <i>B. dothidea</i> was previously isolated from incense cedar in California (Ma and Michailides 2002). To our knowledge, this is the first report of <i>B. dothidea</i> causing canker disease in incense cedar in Tennessee. Accurately identifying this pathogen as the causal agent is important for developing effective and timely management strategies.