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].

Metabolomic responses of indigenous and nonindigenous plants to deer exclosure fencing and deer herbivory in a suburban forest

Trees and shrubs in suburban forest understories can be subject to chronic herbivory from abundant white-tailed deer. An undocumented consequence of this stress may be shifts in secondary metabolite production associated with defense. We aimed to learn whether plants protected from deer exhibited different metabolomic profiles compared to those exposed to deer. We tested the indigenous species Nyssa sylvatica and Lindera benzoin and the invasive, nonindigenous species Rosa multiflora and Euonymus alatus within a suburban forest understory in New Jersey, USA, in unfenced plots and plots fenced for 5.3 years. We did untargeted metabolomics by sampling leaves from three plants of each species per 6-7 fenced and unfenced plots, conducting chloroform-methanol extractions followed by LC-MS/MS, and conducting statistical analysis on Metaboanalyst. We also scored each species for deer browse frequency over eight years, and compared their heights and percent cover between unfenced and fenced plots. The analysis identified 2,333 metabolites. The global metabolome diverged significantly between fenced and unfenced plots pooled across species, but for individual species only N. sylvatica exhibited a significant fencing effect. Nyssa sylvatica was one of the most browsed species and was the only one with both greater cover and height in fenced plots, suggesting greater susceptibility to deer browsing. The metabolites most responsible for the fenced/unfenced divergence also were affected by the species-fencing combination, with increases in certain species but decreases in others. The most significant metabolites that were upregulated in fenced plants include some involved in defense-related metabolic pathways, e.g. monoterpenoid biosynthesis. Further study of more species in multiple sites is needed to learn how common metabolomic responses to deer are among forest species, how the intensity of deer pressure influences the responses, which types of metabolites are most affected, and if there are ecological consequences at the physiological, population, and/or community levels.

Associated habitat and suitability modeling of goldenseal (Hydrastis canadensisL.) in Pennsylvania: explaining and predicting species distribution in a northern edge of range state

Goldenseal (Hydrastis canadensisL.) is a well-known perennial herb indigenous to forested areas in eastern North America. Owing to conservation concerns including wild harvesting for medicinal markets, habitat loss and degradation, and an overall patchy and often inexplicable absence in many regions, there is a need to better understand habitat factors that help determine the presence and distribution of goldenseal populations. In this study, flora and edaphic factors associated with goldenseal populations throughout Pennsylvania—a state near the northern edge of its range—were documented and analyzed to identify habitat indicators and provide possible in situ stewardship and farming (especially forest-based farming) guidance. Additionally, maximum entropy (Maxent) modeling was applied to better predict where suitable habitat might be encountered more broadly and explain species absence from regions of the state (and the northeastern states). Habitat study results identified rich, mesic, woodland sites as being suitable for goldenseal. The most prevalent overstory tree associates on such sites were tulip-poplar (Liriodendron tulipiferaL.) and sugar maple (Acer saccharumMarshall), and the most common understory associates were spicebush (Lindera benzoinL.), Virginia creeper (Parthenocissus quinquefolia(L.) Planch.), Jack-in-the-pulpit (Arisaema triphyllum(L.) Schott), mayapple (Podophyllum peltatumL.), wood fern (Drypoteris marginalis(L.) A. Gray), and rattlesnake fern (Botrypus virginianus(L.) Michx.). Loam soils were the most common textural class (average sand, silt, and clay ratio of 50:30:20) with an average pH of 6.2 and high variation in macronutrients. While such sites are widespread in the state, Maxent modeling suggested the present distribution in Pennsylvania is largely restricted by winter temperatures and bedrock type. The latter of these, in turn, is correlated in part with land use legacy (e.g., clearing for farming or livestock grazing), especially in southeast portions of the state.

Suitability of Eight Northeastern U.S. Native Shrubs as Replacements for Invasive Plants in a Difficult Landscape Site with White-tailed Deer Pressure

Nursery and landscape professionals are interested in white-tailed deer (Odocoileus virginianus)–resistant native plants to replace invasive species used in difficult landscape sites, such as parking lot islands, which are dry, nutrient-poor, and exposed to sun and heat. Eight native shrubs [creeping sand cherry (Prunus pumila var. depressa), elderberry (Sambucus canadensis), gray dogwood (Cornus racemosa), highbush blueberry (Vaccinium corymbosum), round leaf dogwood (Cornus rugosa), northern spicebush (Lindera benzoin), sweetbells (Eubotrys racemosa), and virginia rose (Rosa virginiana)] were planted in a large commuter parking lot on the University of Connecticut campus to evaluate their suitability for use in difficult landscapes. The non-native, invasive shrubs ‘Compactus’ winged euonymus (Euonymus alatus) and ‘Crimson Pygmy’ japanese barberry (Berberis thunbergii) were also planted as controls representing non-native species typically planted in such sites. Aesthetic quality ratings for sweetbells matched the controls (rating of 4.5 out of 5.0) and plants exhibited a high level of white-tailed deer resistance. Virgina rose and creeping sand cherry had similar aesthetic quality to controls, despite light grazing of plants by white-tailed deer. Elderberry was damaged by moderate white-tailed deer grazing and snow load, but plants regenerated to 485% of initial size in one growing season with white-tailed deer exclusion. Gray dogwood, round leaf dogwood, and northern spicebush exhibited the least resistance to white-tailed deer grazing. Both dogwood species had lower aesthetic quality than the controls, and round leaf dogwood had the lowest survival rate (68%) after 2 years. However, several individuals of gray dogwood, round leaf dogwood, and northern spicebush that were less heavily damaged by white-tailed deer grew into attractive shrubs after white-tailed deer exclusion. Highbush blueberry had significantly lower aesthetic quality than controls and only 75% survival after 2 years, indicating that this species is an unsuitable replacement for invasives in difficult landscape sites. This study identified the underused native shrubs sweetbells, virginia rose, and creeping sand cherry as suitable replacements for invasives in difficult landscape sites with white-tailed deer pressure.