First Report of Laurel Wilt Caused by Raffaelea lauricola on Sassafras albidum in Tennessee and Kentucky

Laurel wilt is a vascular wilt disease caused by the fungus Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva, an ambrosial symbiont of the redbay ambrosia beetle, Xyleborus glabratus Eichhoff (Fraedrich et al. 2008). All members of the Lauraceae native to North America are susceptible. Since its discovery in 2004, laurel wilt has resulted in the death of nearly half a billion redbay (Persea borbonia) in the Southeastern United States, and the disease has been reported as far west as Texas and north to North Carolina (Hughes et al. 2017). In July 2019, wilted and dead sassafras (Sassafras albidum) were observed in north-central Tennessee (TN) and southwestern Kentucky (KY). Hundreds of sassafras trees that ranged in size from saplings to trees up to 64 cm in diameter at breast height in six counties (Montgomery, Williamson, and Dickson Cos., TN; and Christian, Todd, and Logan Cos., KY) exhibited symptoms including foliar wilt, dieback, and black discoloration in the xylem. Discolored xylem tissue of one sample from each county of was plated on 2% malt extract agar (MEA), with or without cycloheximide (200 µl/liter) and streptomycin (100 µl/liter). Mucoid colonies with conidia similar in size and shape to R. lauricola (Harrington et al. 2008) were isolated from all samples. A representative isolate (345366TN) was submitted to the USDA ARS NRRL fungal culture collection (accession no. 66901). The morphological identity of three of the isolates was confirmed by sequencing the LSU region of the rDNA with primer pair NL1 (Kurtzman and Robnett 1997) and LR3 (Vilgalys and Hester 1990). All sequences (GenBank nos. MN326699, MN326700, and MN326701) showed 100% homology with R. lauricola isolate C2339 (GenBank no. EU123077). Furthermore, all six isolates were subjected to PCR with R. lauricola species-specific primers pairs chk and/or ifw (Dreaden et al. 2014), which produced amplicons of the correct size, confirming R. lauricola. Pathogenicity tests were conducted on sassafras seedlings (mean height = 150 cm, mean diameter = 1.3 cm at groundline) in a growth chamber at 25°C and with a 15-h photoperiod. Three seedlings per isolate were inoculated with three of the isolates of R. lauricola (345366TN, SF1TN, and SF2KY) obtained from symptomatic trees in KY and TN. Inoculum was produced on MEA, and seedlings were inoculated with 50,000 spores following Fraedrich et al. (2008). An additional three seedlings were mock inoculated with sterile deionized water. After 4 weeks, seedlings inoculated with R. lauricola exhibited symptoms including foliar wilt and xylem discoloration, and the fungus was recovered from all seedlings. Mock-inoculated seedlings remained healthy. Furthermore, female X. glabratus beetles were observed in the stands of wilting sassafras in TN and KY. The southern edge of the outbreak in TN and KY is located approximately 200 miles north of the nearest counties in Alabama where laurel wilt was previously known (Bates et al. 2013) and, thus, represents another major jump in the disease spread. Based on the large area over which the disease is occurring and the condition of trees affected, it is probable that laurel wilt has been established in KY and TN for several years. Sassafras is an important ecological species in the eastern United States and a minor use hardwood. The continued spread of this disease is a threat to forest health. Land managers and forest health experts should be aware of the possibility that the beetle and disease could continue to spread into more northerly states (Formby et al. 2018), where sassafras is a more common component of forest ecosystems, and highlights the need to not move infested material to new areas.