First Report of Laurel Wilt Caused by Raffaelea lauricola on Bay Laurel (Laurus nobilis) in the United States.

Bay laurel (Laurus nobilis L.) is an economically important evergreen tree of the family Lauraceae. It is native to Asia Minor and the Balkans and was introduced into the United States for its ornamental and culinary uses (4). In September 2013, a 6-m-tall bay laurel in Gainesville, FL, attracted our attention because it had wilted leaves, discolored sapwood, and ambrosia beetle entrance holes, all symptoms of laurel wilt. In addition, the tree was growing close to an avocado that succumbed to the disease months earlier. In an effort to determine whether the laurel wilt pathogen (Raffaelea lauricola T.C. Harr., Fraedrich & Agaveya) was, indeed, involved in the decline of the tree of current interest, discolored sapwood was sectioned into 5-mm² pieces, surface disinfested for 30 s in a 4% sodium hypochlorite solution, and plated onto CSMA media (1,2). Within 7 to 14 days, cream-colored, adpressed fungal growth typical of R. lauricola grew from the sapwood pieces (2). DNA was extracted from an isolate of a single conidium (PL1634) and a portion of the 18S rRNA gene was PCR-amplified with primers NS1/NS4, resulting in a 1,021-bp amplicon (GenBank Accession No. KF913344.1), with a BLASTn search revealing 100% homology to several R. lauricola isolates (3). To confirm pathogenicity, six bay laurel seedlings (0.5 m) and a silk bay (0.65 m) (Persea humilis, susceptible control) were wounded twice with a 0.5-mm-diameter drill bit. Then, 30 μl of a spore suspension of PL1634 (1.38 × 10⁵ condia/plant) were introduced into the xylem by pipette and the wounds were wrapped in Parafilm (1). Negative controls consisted of a mock-inoculated (water) and non-inoculated bay laurel plus a mock-inoculated silk bay. Plants were placed in a growth chamber set to a 16/8 h (25/22°C) diurnal light/temperature cycle. After 60 days, all fungal-inoculated plants were completely wilted with dead leaves and subsequent necrosis of stems, while mock- and non-inoculated controls remained asymptomatic. Sapwood dissection revealed xylem discoloration similar to the original infected tree, and fungi morphologically similar to PL1634 were recovered from all inoculated plants upon isolation on CSMA media. Mock- and non-inoculated controls lacked vascular discoloration and fungal growth on media. In order to determine if the redbay ambrosia beetle, Xyleborus glabratus Eichoff (laurel wilt vector) could successfully reproduce in this host, symptomatic branches (7 cm in diameter) of L. nobilis with external evidence of ambrosia beetle attack (frass "toothpicks") were placed in a plastic rearing box within a growth chamber (25°C). Within 4 weeks of incubation, dozens of immature and mature X. glabratus beetles emerged. This is the first record of Koch's postulates being completed for R. lauricola on L. nobilis and the ability of X. glabratus to infest and breed in its stems. This information may be of importance in the event of an introduction of X. glabratus and its fungal associate to Mediterranean areas where bay laurel is either growing wild or being cultivated as valuable commercial crop. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) T. C. Harrington et al. Mycotaxon 104:399, 2008. (3) M. A. Innis et al., eds. PCR Protocols: A Guide to Methods and Applications. Academic Press. San Diego, CA, 1990. (4) A. O. Sari et al. New Forest 31:403, 2006.