Thousand cankers disease threatens Juglans nigra (Eastern Black Walnut) in urban and natural landscapes. Incidence and severity of thousand cankers disease is higher in the host’s introduced range in the western United States. We hypothesized that these differences are driven partly by geographical variation in the host phytobiome due to its roles in host stress tolerance, nutrient acquisition, and defense. To evaluate the role of the phytobiome in mediating thousand cankers disease, we characterized the J. nigra phytobiome of diseased and healthy trees in portions of its native (Indiana and Tennessee) and introduced (Washington) ranges. Grafted clones present in each state and open-pollinated populations were sampled. DNA was extracted from soil and branch (caulosphere) tissues and internal transcribed spacer and 16s regions were sequenced for characterization of fungal and bacterial communities. We found that microbial communities in the caulosphere and soil differ between native and introduced ranges of J. nigra and harbor different mutualistic and pathogenic microorganisms. Additionally, caulosphere microbial communities were more species rich and diverse in the native range of J. nigra, suggesting greater levels of functional redundancy and multifunctionality in the native-range phytobiome compared with the introduced range. We also found higher network complexity in the caulosphere of trees in the introduced range and evidence for two alternative stable community states associated with diseased and healthy trees. Our results provide support for the hypothesis that geographical variation in thousand cankers disease incidence and severity is partially driven by differences in the phytobiome of J. nigra in its introduced and native ranges. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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Diplodia corticola is a fungal pathogen causing oak dieback in Quercus (oak) spp. in parts of North America, northern Africa, and Europe (Ferreira et al., 2021; Smahi et al., 2017; Tsopelas et al., 2018). In August 2021, a single mature white oak (Q. alba) exhibiting wilt symptoms, vascular discoloration, and interveinal chlorosis was observed in Cove Lake State Park in Campbell County, Tennessee, U.S.A. Small sections of phloem tissues were cut from the margins of discolored vasculature of a single wilt symptomatic branch with a sterile scalpel and surface sterilized following Parra et al. (2020). Surface sterilized wood chips were plated onto potato dextrose agar amended with antibiotics (PDA++) following Gazis et al. (2018). Three days after plating, we recovered a single fungal isolate from wood chips that when grown in ½ PDA resembled D. corticola, having irregular margins and white aerial mycelia that progressively turned greyish-black 15 days after sub-culturing (Alves et al., 2004). Total genomic DNA was extracted from the isolate following Gazis et al. (2018). The internal transcribed spacer (ITS) was then amplified using the ITS1 and ITS4 primers and the subsequent PCR product was sequenced. Resulting reads were assembled into a consensus sequence and identity was assigned using BLAST on the NCBI nucleotide database. The assembled sequence (accession OM716006) had a 100% identity match with D. corticola type culture CBS 112549 (accession NR_111152). To complete Koch’s postulates and identify potential host range, 5 red oaks (Q. rubra; 2-3 yrs old; caliper 14.7 ± 2 mm) and 5 white oaks (Q. alba; 2-3 yrs old; caliper 22.8 ± 2.3 mm) were inoculated with D. corticola (isolate DC_2.5). Trees were inoculated 15 cm above the soil line in a greenhouse with a 3 mm diameter plug of a 10-day old culture of D. corticola grown on PDA following Sitz et al. (2017). As a negative control, 5 red and 5 white oaks were inoculated with a 3 mm diameter plug of PDA. For each species, trees were sampled when seepage was observed from D. corticola inoculated sites (15 days post-inoculation for red and white oaks). At time of sampling, bark adjacent to inoculation sites on each tree was removed and cankers were photographed. Using a sterile scalpel, four wood chips were cut from canker margins and placed onto PDA++. For all trees, canker areas were measured using ImageJ software (Rasband, 2012). Recovered isolate identities were confirmed by extracting total genomic DNA as described above (Gazis et al. 2018) and PCR amplification of the ITS, large ribosomal subunit (LSU), and elongation factor 1-α (ef1-α) following (Ferreira et al., 2021). Diplodia corticola was reisolated from wood chips of D. corticola inoculated red (5/5 trees) and white (5/5 trees) oaks and ITS (accession OM716954), LSU (accession OM716955), and ef1-α (accession OM752198) sequences matched D. corticola type culture 112549 ITS (100% identity), LSU (99.76%-100% identity; accession KF766323), and ef1-α (98%-98.9% identity; accession XM_020275852). All D. corticola inoculated trees exhibited seepage from inoculation sites with streaking present in vasculature. Cankers were significantly larger in D. corticola inoculated red (2.34 ± 1.36 cm; P=0.042) and white (2.96 ± 0.52 cm; P=0.00029) oaks compared to agar inoculated trees. To the best of our knowledge, this is the first report of D. corticola causing decline of oaks in Tennessee.

To preserve wetland ecosystem function, federal and state agencies have developed assessment procedures to better manage remaining wetland areas. Currently, wetland assessments do not consider microorganisms when determining wetland quality. This is notable, because fungi are often the primary decomposers of organic material and thus important players in nutrient cycling. The objective of this study is to quantify how wetland quality, as measured using the Ohio Rapid Assessment Method (ORAM), relates to fungal community composition. We sampled soils from six depressional emergent marshes in Ohio belonging to each of the three ORAM quality categories, assessed soil physicochemical properties, and recovered fungal DNA. We then determined if wetland quality as expressed by the ORAM reflects soil health. Our results indicate that ORAM scoring methodology significantly explains differences in fungal community composition between wetlands. We also found that soil physicochemical properties not currently included in the ORAM are strong drivers of fungal community composition, particularly bulk density, pH, soil organic matter, and soil moisture. Overall, our results suggest fungal community composition reflects wetland quality as assessed by the ORAM, and that the ORAM and potentially other wetland assessments could better capture the soil environment by including easily measured soil physicochemical properties.