First Report of Root and Crown Rot on Ginkgo Caused by Calonectria pacifica in Tennessee and the United States

Ginkgo (Ginkgo biloba L.; family Ginkgoaceae) is a large deciduous tree valued for medicinal and ornamental use. In October 2025, 3-year-old field-grown ginkgo ‘Presidential Gold’ plants exhibited crown and root rot symptoms. Symptoms appeared as sunken lesions localized around the crown region, while the roots showed brown to black discoloration. Disease incidence was 25% of 100 plants, and severity was 40% for the affected root area. Symptomatic crown and root tissues were surface sterilized with 1% sodium hypochlorite for 1 min, followed by 70% ethanol for 30 s, and washed twice with sterile water. Then, small sections (around 0.3 mm ² ) of the diseased tissue were plated on potato dextrose agar (PDA) and malt extract agar (MEA) and incubated at 25 ±1 ºC under an 8-h photoperiod. Colonies appeared orange to reddish-brown after 5 to 7 days on PDA and MEA (Fig. 1a). Macroconidiophores consisted of a stipe, a suite of penicillate fertile branches, a stipe extension, and a terminal vesicle (Fig. 1b). The septate stipe extension (145 x 6 µm, n = 50) terminated in a sphaeropedunculate vesicle (10 µm, n = 50) (Fig 1c). The conidiogenous apparatus (72.55 x 58.22 µm, n = 50) consisted of primary branches aseptate (12.19 x 4.88 µm, n = 50); secondary branches aseptate (10.58 x 3.06 µm, n = 50); tertiary branches aseptate (9.20 x 3.41 µm, n = 50), each terminal branch producing 2-4 phialides (Fig. 1d). The uniseptate macroconidia (40.00 × 4.50 µm, n = 50) were hyaline, cylindrical, rounded at both ends, and straight (Fig. 1e). Based on morphological characteristics, the isolates were preliminarily identified as Calonectria pacifica (Liu et al. 2022). Genomic DNA was extracted from 7-day-old isolates FBG9264 and FBG9265 using the DNeasy PowerLyzer Microbial Kit (Qiagen). The genetic markers for the histone H3 (his3), translation elongation factor 1-alpha (tef1), and β-tubulin (tub2) were amplified using the primer pairs CYLH3F/CYLH3R (Crous et al. 2004), EF1-728F/EF2 (Carbone and Kohn 1999; O’Donnell et al. 1998), and T1F/CYLTUBR (O’Donnell and Cigelnik 1997; Crous et al. 2004). The sequences (GenBank accession nos. PZ224109 and PZ224110 for his3; PZ224111 and PZ224112 for tef1; PZ224113 and PZ224114 for tub2) were 99.71%, 100%, and 100% identical to the his3, tef1, and tub2 genetic markers of C. pacifica reference sequences in GenBank (his3, OQ303095; tef1, GQ267320; tub2, OQ261095). A phylogenetic tree constructed using the concatenated sequence of his3, tef1, and tub2 of C. pacifica and other closely related taxa retrieved from GenBank further confirmed the isolates as C. pacifica (Fig. 2). To fulfill Koch’s postulate, pathogenicity tests were performed on 1-year-old ginkgo ‘Presidential Gold’ seedlings grown in 1-gal containers (3.8-L). Ginkgo seedlings (five plants per isolate) were drench-inoculated (200 mL/plant) with a conidial suspension (1 x 10 ⁵ conidia/mL), while sterile water was used as a control. Four weeks post-inoculation, the inoculated plants showed root rot symptoms, while the controls remained healthy (Fig 3). The fungus was reisolated and identified as C. pacifica based on morphology and sequencing. Calonectria pacifica has been reported as a pathogen of Camellia oleifera (Pei et al. 2021). To our knowledge, this is the first report of C. pacifica causing root and crown rot in ginkgo in Tennessee and the United States. Identification of this pathogen as the causal agent is important in designing and implementing effective management practices to mitigate this threat to ginkgo production.