First Report of Leaf Spot Caused by <i>Sphaerulina cornicola</i> on <i>Cornus alba</i> L. in Liaoning Province, China

Cornus alba L. (red-barked dogwood) is a widely cultivated ornamental shrub in China, valued for its aesthetic and ecological benefits (Yuan et al. 2021). During the summer of 2023, leaf spot symptoms were first observed on leaves of C. alba ‘Sibirica’ in Shenyang Botanical Garden, Liaoning Province, China, with an incidence of diseased plants at approximately 5% among 200 surveyed trees. A severe outbreak occurred the following year (August 2024) in the same garden, with the average plant disease incidence reaching 68% (range: 47% to 100%). Infected leaves early displayed minute black-brown spots that gradually developed into circular to irregular necrotic lesions, with white-gray to light brown centers bearing pycnidia, and surrounded by purple periphery. Eventually, numerous lesions coalesced and dried out, resulting in central cracking. Conidia produced within pycnidia on leaves were filiform to filiform-cylindrical, hyaline, straight or curved, tapering to a narrowly rounded or pointed apex, and moderately attenuated toward a narrowly truncate base. They were 0–5-septate, with several oil-droplets and granular material in each cell, 20.96–48.74 × 2.09–4.70 μm (avg. 35.85±9.56 × 3.97±0.58 μm, n=150). The pathogen was isolated by single-spore isolation from 20 symptomatic leaves collected from 20 plants, and cultured on potato dextrose agar at 25°C in the dark for 5 wk. Colonies were dark grayish, covered with black spore massed, reverse black, sometimes arranged in concentric rings, and formed slow growing with 0.61±0.07 mm/day (n=30). A total of 38 isolates were obtained, as the morphological and cultural characteristics of the isolates were very similar, two representative isolates, H5-1, and H6-1, were selected for further study. Molecular characterization was carried out based on translation elongation factor 1-alpha (EF1-728F/EF2), internal transcribed spacer (ITS5/ITS4), β-tubulin (T1/Beta-Sandy-R), and 28S nrDNA (LSU1Fd/LR5) genes (An et al. 2021). Sequences for both isolates were 100% identical. The four gene sequences of H5-1/H6-1 isolates were submitted to GenBank as PV844358/PV844359, PV848373/PV848374, PV844360/PV844361, and PV848375/PV848376, respectively. BLAST searches of the obtained sequences revealed 99.42% (340 bp/342 bp), 99.79% (479 bp/480 bp), 98.41% (309 bp/314 bp), and 100% (825 bp/825 bp) homology on DNA base-pair matching with those of Sphaerulina cornicola CBS102324 in GenBank (KF253557, KF251610, KF253076, and KF252115, respectively). Multilocus phylogenetic analyses of the combined four loci sequence alignment revealed that the two isolates closely clustered with Sph. cornicola (Type strain CBS102324) based on neighbor-joining (NJ) method through MEGA7 (Kumar et al. 2016). Pathogenicity tests were confirmed on leaves of living C. alba ‘Sibirica’ plants. Three healthy plants per treatment were spray-inoculated with spore suspension (106 spores/mL) of H5-1 and H6-1 isolates; controls received sterile water. Inoculated plants were covered with transparent plastic bags for 24 h to maintain &gt;90% RH at approximately 28°C in greenhouse. Typical leaf spot symptoms appeared within 7 days on inoculated leaves, while controls remained asymptomatic. The same fungus was reisolated and identified as Sph. cornicola by morphology and molecular sequencing, thus fulfilling Koch’s postulates. The experiment was independently repeated twice. Previously, Sph. cornicola has been reported on C. sanguinea in Germany, Netherlands, and USA (Verkley et al. 2013). To our knowledge, this is the first report of Sph. cornicola causing leaf spot on C. alba in China. Our findings aid in the diagnosis and management of this emerging disease.