First report of Botryosphaeria dothidea causing leaf spot of Camellia oleifera in China.

Abstract

Camellia oleifera Abel., a small evergreen tree or shrub, is mainly distributed in central and southern China with a larger scale of 4.5 × 106 hectares (Zhu 2020). In May 2021, severe leaf spots were observed in plantation located in Shuangfeng County (27°41'36" N, 111°56'60" E), Hunan Province, China. More than 60 C. oleifera plants were surveyed with over 80% disease incidence. The symptoms on leaves were initially small brown lesions from leaf margins or tips, developing to suborbicular or irregular-shaped dark brown lesions, leading to leaves withered. A total of 60 symptomatic samples were randomly collected. Lesion margins were surface sterilized in 2% sodium hypochlorite for 1 min, rinsed with sterile distilled water for three times, dried, placed on potato dextrose agar (PDA), and incubated at 25°C in the dark for 3 days. Hyphal sections from colony edges were transferred to new PDA plates. Three isolates of Botryosphaeria dothidea were obtained. Colonies of B. dothidea were initially white gradually turning dark-gray with dense aerial mycelium after 6 days. To induce sporulation, colonies of YCB17 were transferred to synthetic nutrient-poor agar (SNA) with sterilized leaves of C. oleifera. Cultures were initially incubated at 25°C in the dark for 3 days, then alternatively exposed to 12-hours near-UV light and 12-hours white light (CHU et al. 2021). After 5 days, conidia formed on leaves were examined microscopically. The conidia were unicellular, aseptate, hyaline, and fusoid, 20.9-25.5×4.7-6.4 µm (n = 50). Morphological characteristics of the isolates matched the description of B. dothidea (Slippers et al. 2014). DNA sequence was amplified using primer pairs ITS1/ITS4 (Tang et al. 2022), EF1-728F/986R (Slippers et al. 2004), and βt2a/2b (Glass & Donaldson. 1995) respectively. The sequences of three isolates (YCB2, YCB3, YCB17) were deposited in GenBank with accession numbers ON714603, MZ613350, MZ613349 (ITS), OM328342, OM328343, OM328344 (TEF-1α), and OM328345, OM328346, OM328347 (TUB2). A blast search of sequences showed the ITS, TEF-1α, and TUB2 sequences had >99% identity with homologue sequences from B. dothidea isolates IRNHM-KZ49 (MG198191.1), CAP288 (EF638732.1) and Mu1 (MK423987.1), respectively. For pathogenicity testing, healthy leaves of 2-year-old C. oleifera plants in the greenhouse were spray-inoculated with conidial suspension (2×106 conidia/mL) from YCB17. Ten surface-sterilized and wounded leaves per plant were sprayed with 30 µL suspension. The other ten wounded leaves sprayed with sterile distilled water served as control. All plants were kept in the greenhouse with temperature at 26 ± 2°C and 50% relative humidity. After 12 days, initial symptoms were observed on more than 80% leaves inoculated with conidial suspension, whereas no symptoms were observed on the control leaves. The test was repeated three times with similar results. It was found that B. dothidea could cause leaf spot of C. oleifera. The infected leaves showed same symptom as samples. Re-isolated fungi from infected leaves were morphologically identical to B. dothidea. Botryosphaeria dothidea has been reported causing leaf spot in a wide range of hosts, but has not previously been reported causing disease on C. oleifera. To our knowledge, this is the first report of B. dothidea causing leaf spot of Camellia oleifera in China. The information on identification of this fungus may be helpful to the control and prevention of the disease.