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.

A Field-Portable Diagnostic Approach Confirms Laurel Wilt Disease Diagnosis in Minutes Instead of Days

Background: Laurel wilt disease has caused the extensive mortality of lauraceous species in the southeastern United States. The causal agent is an invasive fungus, Raffaelea lauricola, which is a symbiont of the beetle Xyleborus glabratus and causes a rapid, fatal vascular wilt. Early diagnosis of laurel wilt is imperative for efficient disease management. The current diagnostic process, however, is slow due to the lengthy laboratory procedures required to confirm pathogen presence. Methods: We tested the robustness and field-portability of a recently developed, species-specific, loop-mediated isothermal amplification (LAMP) assay for R. lauricola, with the overall goal of eliminating the need for a laboratory confirmation of the diagnosis. We tested the robustness of the assay using benchtop equipment with naturally infected samples. We then tested the assay directly in the field using a portable device. Results: The assay successfully detected R. lauricola directly from symptomatic wood tissue using crude DNA extracts. Furthermore, the assay readily allowed users to distinguish between symptoms caused by R. lauricola infection and similar symptoms caused by other agents. In-field, we assayed wood samples from symptomatic redbay (Persea borbonia [L.] Spreng) and sassafras (Sassafras albidum [Nutt.] Nees) across the Southeast and successfully detected R. lauricola-infected trees in less than an hour. Conclusion: Results of this study confirmed that the field-deployable LAMP assay is robust and can rapidly and accurately detect R. lauricola in infected trees directly on-site. LAMP technology is well suited for in-field implementation, and these results serve as an incentive for further development and use of this technology in the field of forest pathology.

Unique Attributes of the Laurel Wilt Fungal Pathogen, Raffaelea lauricola, as Revealed by Metabolic Profiling

Raffaelea lauricola is the causative agent of laurel wilt, a devastating disease of lauraceous trees. R. lauricola is also an obligate nutritional symbiont of several ambrosia beetle species who act as vectors for the pathogen. Here, we sought to establish the baseline “phenome” of R. lauricola with knowledge concerning its metabolic capability, expanding our understanding of how these processes are impacted by environmental and host nutrients. Phenotypic screening using a microarray of over one thousand compounds was used to generate a detailed profile of R. lauricola substrate utilization and chemical sensitivity. These data revealed (i) relatively restricted carbon utilization, (ii) broad sulfur and phosphate utilization, and (iii) pH and osmotic sensitivities that could be rescued by specific compounds. Additional growth profiling on fatty acids revealed toxicity on C10 substrates and lower, with robust growth on C12–C18 fatty acids. Conditions for lipid droplet (LD) visualization and LD dynamics were examined using a series of lipid dyes. These data provide unique insights regarding R. lauricola metabolism and physiology, and identify distinct patterns of substrate usage and sensitivity which likely reflect important aspects of the host-microbe interface and can be exploited for the development of strategies for mitigating the spread of laurel wilt.

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.

First Report of Laurel Wilt Disease Caused by Raffaelea lauricola on Spicebush in Louisiana

In the past two decades, laurel wilt disease has significantly affected members of the Lauraceae in the southeast United States, causing widespread mortality of native redbay (Persea borbonia (L.) Spreng), and incidence of infections in avocado (Persea americana Mill.), sassafras (Sassafras albidum L.) and swamp bay (Persea palustris [Raf.] Sarg.) (Fraedrich et al., 2008, 2015, Olatinwo, et al. 2019). Laurel wilt is a vascular disease caused by Raffaelea lauricola (T.C. Harr., Fraedrich & Aghayeva), a fungus vectored by a non-native ambrosia beetle Xyleborus glabratus Eichhoff (Fraedrich et al. 2008). In August 2020, we investigated the mortality of a spicebush shrub (Lindera benzoin L.) (3.8 cm diameter at root collar, two m height) located ca. 17 mi northeast of Colfax, Grant Parish, Louisiana (31.750263° N, -92.643694° W). Evaluation of the dead shrub revealed brown, persistent foliage, and black vascular discoloration of the sapwood, typical symptoms of laurel wilt (Fig. S1). Although, beetle holes were observed on the sapwood, no beetle was found in galleries at the time. In the laboratory, a fungus consistently isolated from surface-sterilized sapwood tissues plated on potato dextrose agar (PDA) was identified as R. lauricola based on the morphological characteristics of the isolate (i.e., mucoid growth, conidiophores, and oblong/ovoid shape conidia [Harrington et al. 2008]). The fungal isolate was denoted as SB1. The identity of the fungus was confirmed by positive PCR amplification of the large subunit ribosomal RNA gene region using species-specific primers; rab-lsu-rl_F: CCCTCGCGGCGTATTATAG and rab-lsu-rl_R: GCGGGGCTCCTACTCAAA (Olatinwo, unpublished). The sequence of the isolate SB1 (GenBank Accession no. MW207371) showed 100% homology to the R. lauricola strain CBS 127349 sequence (GenBank Accession no. MH877933). The pathogenicity of SB1 on spicebush was evaluated on four healthy shrubs (average: 1 m height and 40 mm in diameter) at the location from which the original detection was made. Stems of two spicebush shrubs were inoculated with SB1 agar plugs from a 14-day old culture on PDA, while plain PDA plugs were used on the remaining two shrubs as non-inoculated controls. Agar plugs were placed in 5 mm (0.2 in) diameter hole punched on the bark with cork-borer as described by Mayfield et al (2008). After six weeks, the R. lauricola inoculated shrubs were wilted with noticeable blackened tissue discoloration in the sapwood, while the control trees remained healthy (Fig. S2). Raffaelea lauricola was re-isolated from tissue of the two inoculated, symptomatic shrubs, but not from the control trees. The sequence of the re-isolated R. lauricola isolate, denoted as SB3 (GenBank Accession no. MW207372), showed 100% homology to the R. lauricola strain CBS 127349 and isolate SB1. This first documentation of laurel wilt on spicebush in Louisiana is significant because, spicebush berries, leaves, and twigs are food sources for forest animals, birds, and insects including whitetail deer and spicebush swallowtail (Papilio troilus L.). Since its first report on sassafras in 2014 (Fraedrich et al. 2015), laurel wilt has spread across Louisiana on sassafras and swamp bay (Olatinwo et al. 2019) and has been confirmed in14 parishes. This report shows the relentless nature of the disease, as the pathogen moves from one vulnerable host to the next, expanding into new locations and threatening forest ecosystems across the southern United States.

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.

Rapid Detection of Raffaelea lauricola Directly from Host Plant and Beetle Vector Tissues Using Loop-Mediated Isothermal Amplification

Since its introduction in 2002, laurel wilt disease has devastated indigenous lauraceous species in the southeastern United States. The causal agent is a fungal pathogen, Raffaelea lauricola, which, after being introduced into the xylem of trees by its vector beetle, Xyleborus glabratus, results in a fatal vascular wilt. Rapid detection and accurate diagnosis of infections is paramount to the successful implementation of disease management strategies. Current management operations to prevent the spread of laurel wilt disease are largely delayed by time-consuming laboratory procedures to confirm the diagnosis. In order to greatly speed up the operations, we developed a loop-mediated isothermal amplification (LAMP) species-specific assay that targets the β-tubulin gene region of R. lauricola, and allows for the rapid detection of the pathogen directly from host plant and beetle tissues. The assay is capable of amplifying as little as 0.5 pg of fungal DNA and as few as 50 conidia. The assay is also capable of detecting R. lauricola directly from wood tissue of artificially inoculated redbay saplings as early as 10 and 12 days postinoculation, when testing high-quality and crude DNA extracts, respectively. Finally, crude DNA extracts of individual adult female X. glabratus beetles were assayed and the pathogen was detected from all specimens. This assay greatly reduces the time required to confirm a laurel wilt diagnosis and, because LAMP technology is well suited to provide point-of-care testing, it has the potential to expedite and facilitate implementation of management operations in response to disease outbreaks.