LAUSR

The North American beech leaf disease (BLD) nematode, Litylenchus crenatae mccannii Handoo, Li, Kantor, Bauchan, McCann, Gabriel, Yu, Reed, Koch, Martin and Burke, 2020, is recognized as a newly emergent nematode species that causes BLD in beech trees (Fagus spp.) in North America (Carta et al. 2020; Kantor et al. 2022a). Since the first report of BLD on American beech (Fagus grandifolia Ehrh) within the Lake County, located at the north-eastern corner of the state of Ohio in 2012 (Carta et al. 2020), the disease has rapidly spread to other US states and a province in Canada (Erwing et al. 2018; Carta et al 2020; Marra and LaMondia 2020; Reed et al 2020; Kantor et al. 2022b). Currently, besides Ohio, this nematode has been reported in Pennsylvania, New York, Connecticut, Massachusetts, Maine, Rhode Island, New Jersey, West Virginia, and Virginia, as well as Ontario, Canada. Different life stages of L. crenatae mccannii were isolated from symptomatic American beech leaves from an isolated natural maple-beech stand in rural Saint Clair Cty., Michigan, US; presenting typical symptoms of beech leaf disease, i.e., swelling and darkening of interveinal leaf tissues. Samples were taken to the Forest Pathology Laboratory at Michigan State University where L. crenatae mccannii presence was confirmed in the leaves after which samples were sent to the Mycology and Nematology Genetic Diversity and Biology Laboratory (USDA-ARS) in Beltsville, Maryland for official confirmation. Nematodes were identified based on morphology and sequence analysis of the internal transcribed spacer (ITS), and the D2D3 region of the 28S large subunit ribosomal DNA. To validate the morphological identification two different ribosomal DNA loci were amplified, sequenced and the phylogenetic relationships were generated. The amplification yielded fragments of 784 and 741 bp flanked by the ITS (GenBank accession no. OP689654) and D2D3 (GenBank accession no. OP689710) primers, respectively. The sequences obtained for the specimens collected in Michigan revealed 100% similarity to L. crenatae mccannii sequences obtained from specimens collected from other geographical areas in the US, and therefore validating the morphological analyses as well. The ITS sequence shared a 99.75% similarity with the subspecies L. crenatae (GenBank accession no. LC383724.1), and 90.53% similarity to L. coprosma Zhao, Davies, Alexander and Riley, 2011 (GU727548.1). While the D2D3 sequences of both L. crenatae subspecies revealed a 100% similarity (versus LC383725.1), they revealed 95.35% similarity to L. coprosma (KY679564.1). Since the first confirmed detection of BLD in June 2022 in St. Clair Cty, BLD has been reported in Oakland and Wayne Ctys (7 reports total across the three counties), suggesting BLD spread in the SE of Michigan. BLD confirmation was based on either physical symptoms (leaf banding), and/or the presence of the beech leaf nematode by morphological or molecular confirmation. The presence of the beech leaf nematode in symptomatic leaves follow the results obtained by Carta et al. (2020) after inoculation of beech seedlings with L. crenatae mccannii. Based on both morphological and molecular analyses the specimens collected in the state of Michigan were identified as L. crenatae mccannii. To our knowledge, this is the first report of this species in conjunction with symptomatic F. grandifolia leaves in this state.