First report of Bot Canker (Diplodia corticola) in Pin Oak (Quercus palustris) in Tennessee.

Diplodia corticola is a fungal pathogen contributing to oak (Quercus spp.) decline in the Mediterranean and US (Félix et al., 2017; Ferreira et al., 2021). In 2021, this pathogen was detected in Tennessee (TN) causing branch dieback in Q. alba (Onufrak et al., 2022). In September 2021, a matured pin oak (Q. palustris) with wilted leaves and elongated branch cankers was observed in the State Botanical Garden of Tennessee-Knoxville (TN, US). Small sections of the phloem were sampled from canker margins of a symptomatic branch using a sterile scalpel, surface sterilized, and plated onto potato dextrose agar amended with antibiotics (PDA++) (Gazis et al. 2018). Three days later, a fungal isolate resembling D. corticola was cultured on ½ PDA. Diplodia corticola is characterized on half-strength PDA by fast growth, irregular margins, and dense white mycelium that turns dark, grayish as the mycelium matures (Úrbez-Torres et al., 2010; Alves et al., 2004). Total genomic DNA was extracted from this isolate following Gazis et al. (2018), and the internal transcribed spacer (ITS), large ribosomal subunit (LSU), and transcription elongation factor 1-α (ef1-α) were amplified (Ferreira et al. 2021). Resulting PCR products were sequenced and assembled into consensus sequences using Unipro UGENE v. 44.0 (Okonechnikov et al., 2012). Each consensus sequence identity was determined using BLAST on the NCBI nucleotide database, restricted to type material. The ITS (accession OQ189888), ef-1α (accession OQ201608), and LSU (accession OQ189887) sequences had a 99.6% (accession KF766156.1), 98.6% (accession XM_020275852.1), and 100% (accession KF766323.1) identity match with D. corticola type culture CBS112549, respectively. To complete Koch's postulates and assess potential pathogenicity on economically and ecologically relevant oaks, 10 pin (Q. palustris; caliper 15.6 ± 2.0 mm), 10 overcup (Q. lyrata; caliper 15.1 ± 2.4 mm), and 10 sawtooth (Q. acutissima; 16.1 ± 2.1 mm) oaks were acclimated in the greenhouse for 1 week prior to the experiment. Five trees of each species were then randomly inoculated at 30 cm above the soil line with a 3 mm diameter plug of D. corticola (grown for 10 days on PDA; Sitz et al. 2017). To serve as a control, the remaining 5 trees for each species received a 3 mm diameter PDA plug. Fifteen days post-inoculation, seepage was observed in D. corticola-inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks. No seepage from wound sites was noted in control trees. Cankers were exposed, photographed, and then measured using ImageJ (Rasband, 2012). Using a sterile scalpel, four wood chips were excised from canker margins and plated onto PDA++. We recovered D. corticola from symptomatic inoculated pin (5/5 trees), overcup (4/5 trees), and sawtooth (4/5 trees) oaks and confirmed species identity by extracting DNA and amplifying the ITS, ef-1α, and LSU regions as described above (Gazis et al., 2018; Ferreira et al., 2021). The resulting consensus sequences matched the D. corticola type culture (CBS112549) ITS (99.0%-99.8% identity), ef-1α (91.0%-99.1% identity), and LSU (96.9%-100% identity) barcoding regions. Cankers were significantly larger in D. corticola-inoculated pin (4.7 ± 1.5 cm2; P = 0.003), overcup (6.8 ± 2.9 cm2; P = 0.009), and sawtooth (5.1 ± 1.3 cm2; P = 0.001) oaks in comparison to the control trees from these groups. Based on current reports, this is the first record of D. corticola causing dieback in pin oak (Q. palustris) in TN.

First report of leaf anthracnose on the Whorled Sunflower, Helianthus verticillatus, caused by Colletotrichum fioriniae in the United States.

Whorled sunflower, Helianthus verticillatus Small, is an endangered (U.S. Fish and Wildlife Service 2014) perennial sunflower species indigenous to the southern United States (Matthews et al. 2002; Ellis et al. 2008). Helianthus verticillatus has a showy yellow floral display in the Fall that attracts a diversity of insect visitors (Strange et al. 2020). Its hardiness in the landscape and late-season blooming makes it a potential ornamental (Trigiano et al. 2021). In June 2021, anthracnose-like lesions were observed on mature leaves collected from potted H. verticillatus plants grown in the nursery compound at the University of Tennessee, Knoxville, TN, USA. Irregularly shaped leaf spots with 1‒2 mm tan centers were observed on mature leaves, which later expanded to 3‒5 mm, and became dark brown- to- black surrounded by chlorotic halos (Fig.1). Lesions from three infected leaves were excised from a single potted plant, trimmed to 1.5-cm squares with green borders, and surface-sterilized (Trigiano et al. 2018). Tissues were placed onto potato dextrose agar (PDA), amended with 100 mg/ml of each streptomycin sulfate and chlorotetracycline, and incubated at 21 °C in the dark until axenic cultures were obtained. Initially, appressed white- to- pale gray mycelia were formed that turned light pinkish-orange with age (Fig. 2A). Conidia (Fig. 2B-C) were single-celled, hyaline, and cylindrical- to- fusiform with acute ends, and were similar to Colletotrichum fioriniae (Damm et al. 2012). Conidia measured 8.9 ± 1.3 µm long and 3.3 ± 0.6 µm wide (N=40). Genomic DNA was isolated with a Phire Direct Plant PCR kit (Thermo FisherScientific, Waltham, MA). The partial beta-tubulin (TUB2) gene, chitin synthase 1 (CHS-1) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and the internal transcribed spacer (ITS) region of ribosomal DNA were amplified with primers T1/BT2B, CHS-354R/CHS-79F, GDF1/GDR1, and ITS1/ITS4, respectively and sequenced (Damm et al. 2012). The resulting sequences were submitted to GenBank (TUB2, ON036471; CHS-1, ON036472; GAPDH, ON036470; and ITS, ON008206). Consensus sequences had 100% identity with C. fioriniae type culture CBS 128517 accessions JQ949943 (TUB2), JQ948953 (CHS-1), JQ948622 (GAPDH), and MH865005 (ITS rDNA). Because H. verticillatus is endangered, and the scarcity of available plant material, Koch's postulates were performed using a detached leaf assay (Boggess et al. 2022). Six healthy leaves were surface-sterilized using the previously described protocol, longitudinally bisected, and placed on 1.5% water agar in three 15 × 100 mm petri dishes. Three half leaves were inoculated with sterile, 5 mm-diameter PDA plugs (controls). The remaining three leaves were inoculated with 5 mm-diameter PDA plugs of C. fioriniae and incubated as described previously. After ten days, necrotic lesions developed on leaves inoculated with C. fioriniae and were similar to the initially observed lesions on plants. Lesions did not develop on control leaves. Colletotrichum fioriniae was re-isolated from lesions using the previously described protocol. The disease does not appear to cause mortality of H. verticillatus and does not require control measures but does reduce the aesthetic value of the plant. To the best of our knowledge, this is the first report of C. fioriniae infecting H. verticillatus in the United States.

First report of Diplodia corticola causing dieback of white oak (Quercus alba) in Tennessee.

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.