First Report of Crown and Root Rot on Eastern Redbud (Cercis canadensis) Caused by Calonectria cylindrospora in Tennessee.

Abstract

Eastern redbuds (Cercis canadensis) are the important trees in Tennessee nurseries, known for their vibrant spring blooms, and heart-shaped foliage (Kidwell-Slak and Pooler 2018). In May 2023, container-grown eastern redbuds exhibited crown and root rot symptoms. Disease incidence was 50% of 100 plants and severity was 40% for the affected root area. Symptoms appeared as sunken lesions localized around the crown region, while the roots showed dark brown to black discoloration. To isolate the causal agent, tissues from the symptomatic crown and roots were excised and surface sterilized with 1% sodium hypochlorite for 1 minute, followed by 70% ethanol for 30 seconds, and washed twice with sterile water. About 0.3 mm² sections of the diseased tissues were placed on potato dextrose agar (PDA) and malt extract agar (MEA) and incubated at 25°C under 12 hours light/dark conditions. Colonies appeared orange to red-brown after 4 to 6 days on both PDA and MEA. Microscopic observations were conducted using two weeks old cultures grown on MEA. The conidiogenous apparatus (avg. 90 μm × 60 μm, n=50) consisted of penicillate conidiophore-bearing branches. The septate stipe extension (avg. 145 μm × 6 μm, n=50) terminated in a sphaeropedunculate vesicle (avg. 10 μm, n=50). The uniseptate macroconidia (avg. 45 μm × 4.5 μm, n=50) were hyaline, cylindrical, rounded at both ends and straight. The observed morphological traits aligned with Calonectria cylindrospora described by Liu et al. (2020). Pathogen identity was confirmed by sequencing specific genetic markers amplified from genomic DNA extracted using DNeasy PowerLyzer Microbial Kit from 7-day-old pure cultures. The primer pairs ITS1/ITS4 (White et al. 1990), T1F/T222 (O'Donnell et al. 2000), and EF1/EF2 (Carbone and Kohn 1999) were used to amplify and sequence the ribosomal internal transcribed spacer (ITS), and nuclear beta-tubulin (TUB) and translation elongation factors 1-α (EF1-α) genetic markers, respectively. The sequences of the three isolates were 99.8, 100, and 100% identical to C. cylindrospora (CBS 136425, CBS 30978, and CBS 119670) in the NCBI database, respectively. Phylogenetic analysis of concatenated sequences of ITS, TUB, and EF1-α of C. cylindrospora and other closely related taxa (Liu et al. 2020) retrieved from GenBank confirmed the identity of the pathogen as C. cylindrospora (Fig. 1). The sequences of three isolates (FBG5441, FBG5751, and FBG5752) were deposited in GenBank with accession numbers PQ482527, PQ482528, and PQ482529 (ITS), PQ493360, PQ493361, and PQ493362 (TUB), and PQ493363, PQ493364, PQ493365 (EF1-α), respectively. Three isolates were used to inoculate 1-year-old container-grown (1-gal) healthy eastern redbud plants. Conidial suspension (1.0 × 10⁵ conidia/mL) was prepared for each isolate by using 14-day-old fungal cultures grown on MEA and was applied directly to the crown of the redbud, with an additional volume poured at the root zone for absorption (200 ml per plant) while sterile water was used as control. There were six replications for each isolate and control. Six weeks post-inoculation, the inoculated plants showed crown and root rot symptoms, while the controls remained healthy. Fungal isolates identical to the original in morphology and DNA sequences were recovered from inoculated plants. C. cylindrospora is recognized as a pathogen of ornamental plants (Aiello et al. 2022). To our knowledge, this is the first report of C. cylindrospora causing crown and root rot in redbud in Tennessee. Identifying this pathogen as the causal agent is important for developing effective and timely management strategies.