First Report of Laurel Wilt Disease Caused by Raffaelea lauricola on Sassafras in Virginia

Laurel wilt is a lethal vascular disease affecting native Lauraceae in North America. The causal fungus, Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva and its symbiont, redbay ambrosia beetle, Xyleborus glabratus Eichhoff are native to Asia (Fraedrich et al. 2008, Harrington et al. 2008). Since their introduction near Savannah, Georgia in 2002 (Fraedrich et al. 2008), laurel wilt has spread rapidly, resulting in extensive mortality of native redbay (Persea borbonia [L.] Spreng.) [Hughes et al. 2017] and is a threat to other native Lauraceae, such as sassafras (Sassafras albidum [Nutt.] Nees) (Bates et al. 2013) and northern spicebush (Lindera benzoin [L.] Blume) [Olatinwo et al. 2021]. In June 2021 a sassafras sapling showing wilt and dieback was observed along a roadside in Scott County, Virginia, which borders a laurel wilt-positive Tennessee county (Loyd et al. 2020). The trunk (approximately 5 cm diameter) was submitted to the Virginia Tech Plant Clinic. Although beetle holes were observed, X. glabratus was not found. Discolored sapwood chips were excised and plated on malt extract agar amended with cycloheximide (200 ppm) and streptomycin (100 ppm) [CSMA]. A fungus was consistently recovered and the morphology of conidiophores and conidia, and presence of blastoconidia and mucoid growth, aligned with the description of R. lauricola (Harrington et al. 2008). Two R. lauricola-specific primer sets (Dreaden et al. 2014) were used to amplify DNA extracted from a representative isolate (0248-2021) and confirm R. lauricola. For further confirmation, the LSU region of the rDNA was sequenced (Lloyd et al. 2020). The sequence of the isolate (GenBank accession no. OL583842) showed 100% identity (573/573 bp) to R. lauricola ex-type strain sequence, CBS 121567 (accession no. MH877762) (Harrington et al. 2008, Vu et al. 2018). The isolate was also confirmed by the National Identification Services by sequencing. To confirm pathogenicity, 15 sassafras seedlings (height = 60-100 cm, diameter = 8-10 mm) were inoculated with a conidial suspension harvested from 10-day CSMA cultures of 0248-2021, as follows: two 0.4 mm diameter holes were drilled 10 cm above the soil line at a 45° angle on opposite sides of the stem, leaving at least 3 cm between holes. Ten µl of the conidial suspension (5 x 107/ml) was transferred into each hole and sealed with parafilm. Two sassafras seedlings were inoculated with sterile water. Seedlings were maintained with 12 h photoperiod at 27° ± 2° C. Off-color foliage and loss of turgor were observed 10 days post-inoculation on conidia-inoculated seedlings; at two weeks, these were completely wilted and had sapwood discoloration. Water-inoculated plants showed no symptoms. Sapwood from 15 cm above the inoculation point was excised from 0248-2021-inoculated plants (n=2) and water-inoculated plants (n=1) and plated on CSMA. R. lauricola was recovered from symptomatic plants, but not from water-inoculated plants. The identity of the recovered fungus was confirmed with two species-specific primers sets (Dreaden et al. 2014). It is likely that laurel wilt is more prevalent in the area of the roadside find. Both sassafras and northern spicebush are widespread in Virginia and their range extends into the northeastern US and lower Canada. Laurel wilt poses a serious threat to these species and their ecosystems. For example, spicebush and sassafras are primary hosts of the native spicebush swallowtail butterfly (Papilio troilus L.) [Nitao et al. 1991].

Raffaelea lauricola (Laurel wilt).

Laurel wilt is responsible for the death of hundreds of millions of redbay (Persea borbonia sensu lato) trees throughout the southeastern USA, and the disease is also having significant effects on other species such as sassafras (Sassafras albidum) in natural ecosystems and avocado (Persea americana) in commercial production areas of south Florida. Laurel wilt is caused by the pathogen Raffaelea lauricola, a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus. Thus far, the disease is confined to members of the Lauraceae that are native to the USA, or native to such places as the Caribbean, Central America and Europe and grown in the USA. The beetle and fungus are native to Asia and were likely introduced with untreated solid wood packing material at Port Wentworth, Georgia in the early 2000s. Since that time laurel wilt has spread rapidly in the coastal plains of the southeastern USA, spreading north into central North Carolina, as far west as Texas, and reaching the southernmost counties of Florida. Current models suggest that X. glabratus can tolerate temperature conditions that occur throughout much of the eastern USA, and so the disease threatens sassafras throughout much of this region. The disease poses a threat to lauraceous species indigenous to other areas of the Americas as well as Europe and Africa.

Raffaelea lauricola (laurel wilt).

Laurel wilt is responsible for the death of hundreds of millions of redbay (Persea borbonia sensu lato) trees throughout the southeastern USA, and the disease is also having significant effects on other species such as sassafras (Sassafras albidum) in natural ecosystems and avocado (Persea americana) in commercial production areas of south Florida. Laurel wilt is caused by the pathogen Raffaelea lauricola, a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus. Thus far, the disease is confined to members of the Lauraceae that are native to the USA, or native to such places as the Caribbean, Central America and Europe and grown in the USA. The beetle and fungus are native to Asia and were likely introduced with untreated solid wood packing material at Port Wentworth, Georgia in the early 2000s. Since that time laurel wilt has spread rapidly in the coastal plains of the southeastern USA, spreading north into central North Carolina, as far west as Texas, and reaching the southernmost counties of Florida. Current models suggest that X. glabratus can tolerate temperature conditions that occur throughout much of the eastern USA, and so the disease threatens sassafras throughout much of this region. The disease poses a threat to lauraceous species indigenous to other areas of the Americas as well as Europe and Africa.

Laurel Wilt: Current and Potential Impacts and Possibilities for Prevention and Management

In recent years, outbreaks of nonnative invasive insects and pathogens have caused significant levels of tree mortality and disturbance in various forest ecosystems throughout the United States. Laurel wilt, caused by the pathogen Raffaelea lauricola (T.C. Harr., Fraedrich and Aghayeva) and the primary vector, the redbay ambrosia beetle (Xyleborus glabratus Eichhoff), is a nonnative pest-disease complex first reported in the southeastern United States in 2002. Since then, it has spread across eleven southeastern states to date, killing hundreds of millions of trees in the plant family Lauraceae. Here, we examine the impacts of laurel wilt on selected vulnerable Lauraceae in the United States and discuss management methods for limiting geographic expansion and reducing impact. Although about 13 species belonging to the Lauraceae are indigenous to the United States, the highly susceptible members of the family to laurel wilt are the large tree species including redbay (Persea borbonia (L.) Spreng) and sassafras (Sassafras albidum (Nutt.) Nees), with a significant economic impact on the commercial production of avocado (Persea americana Mill.), an important species native to Central America grown in the United States. Preventing new introductions and mitigating the impact of previously introduced nonnative species are critically important to decelerate losses of forest habitat, genetic diversity, and overall ecosystem value.

Use of Semiochemicals for the Management of the Redbay Ambrosia Beetle

This review highlights current advances in the management of the redbay ambrosia beetle, Xyleborus glabratus, a primary vector of the pathogenic fungus, Raffaelea lauricola, that causes laurel wilt. Laurel wilt has a detrimental effect on forest ecosystems of southeastern USA, with hundreds of millions of Lauraceae deaths. Currently, preventive measures mostly focus on infected-tree removal to potentially reduce local beetle populations and/or use of preventative fungicide applications in urban trees. Use of semiochemicals may offer an opportunity for the management of X. glabratus. Research on attractants has led to the development of α-copaene lures that are now the accepted standards for X. glabratus sampling. Research conducted on repellents first included methyl salicylate and verbenone and attained significant reduction in the number of X. glabratus captured on redbay and swamp bay trees treated with verbenone. However, the death rate of trees protected with verbenone, while lower compared to untreated trees, is still high. This work underscores the necessity of developing new control methods, including the integration of repellents and attractants into a single push-pull system.

Comparison of Trap Designs for Detection of Euwallacea nr. fornicatus and Other Scolytinae (Coleoptera: Curculionidae) That Vector Fungal Pathogens of Avocado Trees in Florida

Laurel wilt and Fusarium dieback are vascular diseases caused by fungal symbionts of invasive ambrosia beetles (Coleoptera: Curculionidae: Scolytinae). Both diseases threaten avocado trees in Florida. Redbay ambrosia beetle, Xyleborus glabratus, is the primary vector of the laurel wilt pathogen, Raffaelea lauricola, but in recent years this symbiont has been transferred laterally to at least nine other species of ambrosia beetle, which now comprise a community of secondary vectors. Dieback disease, caused by Fusarium spp. fungi, is spread by shot hole borers in the Euwallacea fornicatus species complex. In this study, we conducted field tests in Florida avocado groves to compare efficacy of four trap designs for detection of Scolytinae. Treatments included an 8-funnel Lindgren trap, black 3-vane flight interception trap, green 3-vane interception trap, white sticky panel trap, and an unbaited sticky panel (control). In two tests targeting E. nr. fornicatus and X. glabratus, traps were baited with a two-component lure (α-copaene and quercivorol). In a test targeting other species, traps were baited with a low-release ethanol lure. For E. nr. fornicatus, sticky panels and black interception traps captured significantly more beetles than Lindgren traps; captures with green traps were intermediate. With ethanol-baited traps, 20 species of bark/ambrosia beetle were detected. Trap efficacy varied by species, but in general, sticky traps captured the highest number of beetles. Results indicate that sticky panel traps are more effective for monitoring ambrosia beetles than Lindgren funnel traps, the current standard, and may provide an economical alternative for pest detection in avocado groves.

Genetic Analyses of the Laurel Wilt Pathogen, Raffaelea lauricola, in Asia Provide Clues on the Source of the Clone that is Responsible for the Current USA Epidemic

Laurel wilt is caused by the fungus Raffaelea lauricola T.C. Harr., Fraedrich and Aghayeva, a nutritional symbiont of its vector the redbay ambrosia beetle, Xyleborus glabratus Eichhoff. Both are native to Asia but appeared in Georgia in the early 2000s. Laurel wilt has since spread to much of the southeastern United States killing >300 million host trees in the Lauraceae plant family. The aims of this research were to elucidate the genetic structure of populations of R. lauricola, to examine its reproductive strategy, and determine how often the pathogen had been introduced to the USA. A panel of 12 simple sequence repeat (SSR) markers identified 15 multilocus genotypes (MLGs) in a collection of 59 isolates from the USA (34 isolates), Myanmar (18), Taiwan (6) and Japan (1). Limited diversity in the USA isolates and the presence of one MAT idiotype (mating type locus) indicated that R. lauricola was probably introduced into the country a single time. MLG diversity was far greater in Asia than the USA. Only three closely related MLGs were detected in the USA, the most prevalent of which (30 of 34 isolates) was also found in Taiwan. Although more work is needed, the present results suggest that a Taiwanese origin is possible for the population of R. lauricola in the USA. Isolates of R. lauricola from Myanmar were distinct from those from Japan, Taiwan and the USA. Although both MAT idiotypes were present in Myanmar and Taiwan, only the population from Taiwan had the genetic structure of a sexually reproducing population.

An Acaromyces Species Associated with Bark Beetles from Southern Pine Has Inhibitory Properties Against Raffaelea lauricola, the Causal Pathogen of Laurel Wilt Disease of Redbay

Laurel wilt is a destructive disease of redbay (Persea borbonia) and other species in the laurel family (Lauraceae). It is caused by Raffaelea lauricola, a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus (Coleoptera: Curculionidae), cointroduced into the United States around 2002. During assessments of fungi associated with bark beetles from loblolly pine, an unknown fungus was isolated that appeared to have broad-spectrum antifungal activities. In this study, we identified the unknown fungus and determined the inhibitory effect of its secondary metabolites on R. lauricola. DNA analysis identified the fungus as Acaromyces ingoldii (GenBank accession no. EU770231). Secondary metabolites produced by the A. ingoldii completely inhibited R. lauricola mycelial growth on potato dextrose agar (PDA) plates preinoculated with A. ingoldii and reduced R. lauricola growth significantly on malt extract agar plates preinoculated with A. ingoldii. R. lauricola isolates inoculated on PDA plates 7 days after A. ingoldii were completely inhibited with no growth or spore germination. Direct evaluation of A. ingoldii crude extract on R. lauricola spores in a multi-well culture plate assay showed inhibition of spore germination at 10% and higher concentrations. Secondary metabolites from A. ingoldii could be potentially useful in managing the future spread of laurel wilt.

Utility of essential oils for development of host-based lures for Xyleborus glabratus (Coleoptera: Curculionidae: Scolytinae), vector of laurel wilt

Redbay ambrosia beetle, Xyleborus glabratus, is native to Southeast Asia, but subsequent to introduction in Georgia in 2002, it has become a serious invasive pest in the USA, now established in nine southeastern states. Females vector Raffaelea lauricola, the fungus that causes laurel wilt, a lethal vascular disease of trees in the family Lauraceae. Laurel wilt has caused extensive mortality in native Persea species, including redbay (P. borbonia), swampbay (P. palustris), and silkbay (P. humilis). Avocado (P. americana) is now impacted in Florida, and with continued spread, laurel wilt has potential to affect avocado and native Lauraceae in California, Mexico, and throughout the American tropics. Effective lures for detection and control of X. glabratus are critical to slow the spread of laurel wilt. No pheromones are known for this species; primary attractants are volatile terpenoids emitted from host Lauraceae. This report provides a concise summary of the chemical ecology of X. glabratus, highlighting research to identify kairomones used by females for host location. It summarizes development of essential oil lures for pest detection, including discussions of the initial use of phoebe and manuka oil lures, the current cubeb oil lure, and a newly-developed distilled oil lure enriched in (-)-α-copaene.