Deutzia crenata Sieb. et Zucc, native to Japan, with white flowers in early summer, is a high quality ornamental shrub widely planted in China. In October 2021, a new leaf spot disease was observed on approximately 70% of the 320 D. crenata trees growing in Nanjing Botanical Garden, Jiangsu Province, China. The disease started as irregular small gray spots on the leaf of D. crenata that coalesced into larger lesions. Infected leaves turned yellow (Figure S1A) and leaves with multiple spots withered. To isolate the pathogen, leaf sections (3 to 4 mm) were excised from the lesion margin, surface sterilized in 75% alcohol for 30 s and then in 1.5% NaClO for 90 s, rinsed three times in sterilized distilled water, plated on potato dextrose agar (PDA) and incubated at 25℃in the darkness. Pure cultures were obtained by monosporic isolation. The colony of a representative isolate (L-1), growing on PDA was circular, white, and cottony, and the surface undulate and pale luteous (Figure S1B). The reverse was similar in color (Figure S1C). The conidial masses were black and appeared over PDA plates after 12 days (Figure S1D). Conidia [18.3 to 28.4×5.4 to 8.5 µm (mean 24.5×6.7 µm)] (n=35) were fusiform to ellipsoid and four-septate (one basal and one apical cell hyaline, and three brown median cells), with two to three apical appendages (Figure S1E). These characteristics were consistent with the description of Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). Three regions of the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1α), and β-tubulin (TUB) genes (GenBank Accession No. OM663738, No. OM687134 and No. OM687133, respectively) were amplified and sequenced with the primers pairs ITS1/ITS4 (Innis et al. 1990), EF1-526F/EF1-1567R (Maharachchikumbura et al. 2014) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The obtained sequences were 95.4-99.8% similar to those from Neopestalotiopsis sp. accessions in GenBank. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate L-1 clustered in the N. ellipsospora clade with 98% bootstrap support (Figure S2). To test pathogenicity, three detached healthy leaves and three one-year-old D. crenata seedlings were inoculated with 20 μL conidia suspension (1×106 spores/mL) on the left sides of leaves. The right side of each leaf was inoculation with 20 µL of sterile water as the experimental control. All plants were covered with clear polyethylene bags and incubated in a greenhouse (Institute of Botany, Jiangsu Province and Chinese Academy of Sciences) at 25℃, 80% relative humidity, and a 12-h light/dark cycle. The experiment was repeated three times. After 5 days of inoculation, leaf spots typical of those observed in the orchards were observed on the left sides of all inoculated leaves and the right sides did not have any leaf spot symptoms (Figure S1F-G). The same fungus was isolated from the diseased spots of the inoculated leaves to complete Koch,s postulates (Figure S1H). N. ellipsospora is known to cause leaf spots on Camellia sinensis and sweet potato, infects fruits of Ardisia crenata in China (Maharachchikumbura et al. 2014; Maharachchikumbura et al. 2016; Wang et al. 2019), and causes stem spots on Acanthopanax divaricatus in Korea (Yun et al. 2015). This is the first report of N. ellipsospora causing leaf spot on D. crenata in the world. The occurrence of this disease needs to be monitored, because it can reduce the ornamental value of D. crenata. This finding provides the foundation to further investigate the biology and epidemiology of this disease so that effective strategies can be developed to manage this disease.
Keywords: Causal Agent; Crop Type; Epidemiology; Fungi; Subject Areas; Trees; ornamentals; pathogen survival.