Arboriculture & Urban Forestry 47(3): May 2021 101 Table 2. Results of laboratory LAMP assays and corresponding validation via fungal isolation to detect Raffaelea lauricola from wood tissue samples collected from sites without laurel wilt disease in the southeastern US. Results are reported as number of positive assays or fungal isolations out of the total samples tested. State Arkansas Georgia Louisiana North Carolina Texas Collecting agency Arkansas Department of Agriculture, Forestry Division University of Georgia United States Forest Service North Carolina Forest Service United States Forest Service Site name Beirne Bottom Road University of Georgia Fishville Ditchbank Road The Woodlands Host species Sassafras Redbay Sassafras Redbay Redbay LAMP 0/5 0/5 0/3 0/5 0/5 Fungal isolation 0/5 0/5 0/3 0/5 0/5 no-template negative control. A result was considered positive if 2 out of 3 technical replicates amplified. Fungal Isolation To verify the presence of the pathogen in each tested sample, a portion of tissue of each sample was plated onto amended malt extract agar. Processing consisted of debarking the sample and surface sterilizing it by dipping it in 10% common bleach in water (corre- sponding to 8.25% sodium hypochlorite) for 10 sec- onds, then 70% EtOH for 10 seconds, and finally in sterile water for 10 seconds. After the sample was allowed to air dry, it was placed onto malt extract agar (VWR) amended with 200 ppm cycloheximide (Sigma- Aldrich, St. Louis, MO, USA) and 100 ppm streptomy- cin (Sigma-Aldrich)(CSMA)(Harrington and Fraedrich 2010). Plates were wrapped with parafilm (Bemis, Neenah, WI, USA) and incubated at 25 °C in an Isotemp® incubator (Model 655D, Thermo Fisher Scientific) until R. lauricola growth was observed or a minimum of 2 weeks had passed. Identification of R. lauricola was performed based on morphological features such as its characteristic mucoid growth, the presence of conidiophores, and the shape and size of its conidia (Harrington et al. 2008). Objective II: In-Field Testing of the LAMP Assay Sample Collection Redbay and sassafras samples were assayed in-field from November 2019 to March 2020 in 5 different southeastern states, including Georgia, Kentucky, North Carolina, South Carolina, and Tennessee (Table 3). As previously noted, site locating and sampling was again achieved with the assistance of local state and federal forestry agencies. At each location, testing was performed at a single site on a single day, with the exception of Georgia, where redbay trees were sampled at one site and sassafras trees at another, but the testing was still carried out on the same day. At least 6 individual potentially diseased trees were selected in each state based on visual symptoms, including leaf wilt and sapwood discoloration upon debarking. Collected samples consisted of sapwood tissue from the main stem of larger trees or entire sec- tions of stems from sprouts and saplings. DNA Extraction in the Field For in-field crude DNA extractions, we used a proto- col similar to that previously used in the laboratory, but with some adaptations to accommodate the field conditions. In particular, we wanted the protocol to be workable with minimal equipment and to avoid an open flame. Stem wood samples were acquired from visually symptomatic host trees only. For smaller diameter trees, a hand saw was used to fell trees and expose discolored sapwood, and whole stem sections were then collected. For larger diameter trees that could not be readily felled, a hatchet was used to remove bark and expose discolored sapwood, and samples were collected with a pocket knife. Tools were disin- fected with 10% bleach and 70% ethanol in between each sample. From each tree, part of the collected wood sample was processed for crude DNA extraction and LAMP analysis directly on-site, while another part was stored in a clean resealable plastic bag and transported in a cooler with ice packs back to the lab- oratory for further analyses. One to three thinly shaved, discolored pieces of sapwood (comparable in terms of weight to the 15 mg to 20 mg used for the crude extraction protocol in the laboratory) were placed into a previously prepared extraction tube ©2021 International Society of Arboriculture
May 2021
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