First report of Xylella fastidiosa subsp. multiplex infecting southern shagbark hickory (Carya carolinae-septentrionalis) in Georgia, USA.

Southern shagbark hickory (Carya carolinae-septentrionalis) is one of several deciduous trees in the family Juglandaceae and genus Carya that are native to North America. Southern shagbark hickory has a restricted distribution to the Southeast U.S.A. (USDA, 2024). During a disease survey in September, 2023, symptoms of leaf scorch were noticed on trees planted in a hickory collection at the USDA-ARS, Byron, GA. Scorch was characterized by tan to light brown, irregularly shaped necrotic lesions often starting along the leaf margins, with the necrosis spreading across the entire leaflet, resulting in curling of leaflet and in some cases affecting the whole leaf (Supplementary Fig. 1). Some defoliation of leaflets on compound leaves was noted. Symptomatic shoot terminals with compound leaves were collected, stored in a refrigerator (~4°C) and processed within three days. The epidermis was stripped from a sample of leaflet petioles and ground with a tissuelyser. DNA was extracted from the petiole sample using a Zymo Research® kit (Quick-DNA™ Fungal/Bacterial Kit, Zymo Research®, Irvine, CA) following the manufacturer's protocol. Conventional PCR was performed on the sample with positive controls (DNA of Xylella strain M12) and negative controls (water, healthy pecan) using four previously characterized Xylella fastidiosa (Xf)-specific primer sets (Francis et al., 2006; Minsavage et al. 1994; Rodrigues et al., 2003). The resulting amplicons each had the characteristic size expected for X. fastidiosa. A further two samples of DNA were extracted using a NucleoSpin Plant II kit (Machereey-Nagel, Duren, Germany) following the manufacturer's protocol. The DNA samples were tested by SYBR-green real-time PCR with primer sets Teme150fc/Teme454rg (specific to Xf subsp. fastidiosa, Xff) and Dixon454fa/Dixon1261rg (specific to Xf subsp. multiplex, Xfm) (Chen et al., 2005), yielding Ct values of 31.39 and 18.96, respectively, suggesting dominant Xfm infection. One sample (designated Cc-sR5T1) was further selected and subject to next generation sequencing (NGS) using an Illumina NovaSeq 6000 (PE150) platform. A total of 58,601,960 paired reads were generated with a mapping rate of 0.97% to Xfm M12 (NC_010513.1) and 0.95% to Xff M23 (NC010577.1) using Bowtie2 (Langmead and Salzberg, 2012), confirming the Xfm status of strain Cc-sR5T1. Read coverages on both M12 and M23 genomes were >60X. Top-5 and bottom-5 reads in the mapped read data sets were selected and used as queries for a BLAST search against the National Center for Biotechnology Information (NCBI) core-nr database. All top hits were Xf subsp. multiplex (query coverage = 97 to 100%, Percentage Identity = 99 to 100%) with one exception likely related to the region of a mobile element (Supplementary Table 1). In summary, a Xfm strain was identified in a shagbark hickory tree based on leaf scorch symptoms, PCR characteristic loci, and NGS whole genome approaches. The pathogen Xfm infects other Carya (Hilton et al. 2020), including C. illinoinensis (pecan) (Sanderlin and Heyderich-Alger, 2000), which is an economically important nut crop in the southern U.S.A. Shagbark hickory is not of high economic value, but now a possible reservoir of Xfm. Knowledge of the causal agents of the disease in Carya species is important for disease management purposes in both agricultural plantings and natural stands of trees. Furthermore, Xylella pathogens have both national and international phytosanitary ramifications.