©2023 International Society of Arboriculture Arboriculture & Urban Forestry 49(4): July 2023 171 should be Diaporthe ambigua Nitschke. Diaporthe is a very complex genus that is still undergoing taxo- nomic elucidation. Indeed, morphologically similar isolates can be genetically distinct (i.e., cryptic spe- cies), while certain species are pathogenic in some hosts and endophytic in others (Gomes et al. 2013). Because of this complexity, molecular identification of Diaporthe species warrants the use of a multi-locus phylogenetic approach (Udayanga et al. 2012; Gomes et al. 2013; Udayanga et al. 2014). Furthermore, Udayanga et al. (2014) developed specific primers for the DNA-(apurinic or apyrimidinic site) lyase (Apn2) gene to help identify Diaporthe species. Developing tree resistance is a key strategy to pro- tect trees against established or emerging pathogens. Tools to develop resistance range from conventional breeding and use of molecular markers to compara- tive genomics and metabolomics (Naidoo et al. 2019). One way to introduce new resistance genes to the gene pool is to identify naturally resistant individuals. The Ohio State University campus in Columbus, Ohio, is home to more than 200 American sycamores (The Ohio State University Tree & Plant App 2023). In prior observations over many years, we identified a P. occidentalis individual that was consistently asymptomatic or showing significantly fewer symp- toms of anthracnose and canker diseases than conspe- cifics in the immediate vicinity, suggesting that that individual may be resistant, rather than an escape. We, therefore, proceeded to vegetatively propagate the tree of interest and protected the cultivar under the name ‘Davis’ with the objective to assess the field resistance of P. occidentalis ‘Davis’, relative to wild- type, to canker pathogens under conditions of natural disease pressure. MATERIALS AND METHODS Outdoor Trials Two trials were established on the Columbus campus of The Ohio State University on April 27, 2018, at bud break, by planting 6 4-year-old ‘Davis’ ramets, an independent member of a clone, and 6 4-year-old ramets taken from a mixture of wildtype genets (for a total of 12 ‘Davis’ and 12 wildtype ramets), under each of 2 mature American sycamores that had dis- played evident anthracnose and canker symptoms for the past several years. Before the trial began, the ramets were maintained in 3-gallon pots filled with Metro-Mix® 360 growth medium coated with one tablespoon of Osmocote® 14-14-14 slow-release fertil- izer. Plants were maintained in a greenhouse at a tem- perature of 24 °C and hand watered as needed until they reached about 1 m in height. In each trial, ramets were transplanted around the mature American syca- more tree, 1 m from the trunk, alternating between wildtype and ‘Davis’. The 2 trials (40.004129, −83.027528 and 40.005008, −83.027657) were pro- tected with fences and excluded from the Universi- ty’s routine groundskeeping activities throughout the experimental period. The trees were hand irrigated as needed for about 4 months post-transplant, until established. Incidence and severity of leaf necrosis were assessed weekly or biweekly on each plant over 2 consecutive seasons from the time symptoms first appeared in the trial (i.e., July 2018 and May 2019) until the end of August. Disease incidence was expressed as percent- age of symptomatic leaves on each plant, while dis- ease severity was expressed as percentage of total foliage area with symptoms. Photos of each leaf showing necrotic symptoms were taken and uploaded to the American Phytopathological Society (APS) Assess software (Lamari 2002) to calculate the per- cent area of necrotic tissue. Because severe dieback symptoms were observed on the wildtype ramets in late-June 2019, incidence of dieback was also evaluated every 2 weeks until the end of August 2019 by recording the percentage of symp- tomatic branches on each plant (number of branches showing dieback/total number of branches × 100). Fungal Isolation and Characterization At the end of each season (August 2018 and 2019), symptomatic tissues (i.e., necrotic leaf lesions and twig cankers) were collected from each plant and subject to pathogen isolation procedures in the labo- ratory. Leaves were surface disinfected in a 2% sodium hypochlorite (NaOCl) solution for 1 minute, then rinsed 3 times in sterile Milli-Q® water (Milli- pore Corporation, Billerica, MA, USA). Twigs were surface disinfected by uniformly spraying them with 95% ethanol followed by flaming, and then longitudi- nally sectioned using a sterile scalpel to expose the vascular tissue. Following disinfection, the transition zone between necrotic and healthy tissue of either leaf or vascular tissue was plated on potato dextrose agar (PDA)(Difco Laboratories, Sparks, MD, USA) amended with 1.5% streptomycin sulfate and 1%
July 2023
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