Report of Phoma herbarum Causing Leaf Spot Disease of Camellia sinensis in China

The tea plant, Camellia sinensis (L.) O. Kuntze, is a crucial commercial crop in China. In September 2016, a new spot disease was observed on the leaves of a mature (age: 10 years) tea plant in Chizhou, Anhui Province, China. The symptoms began with brown lesions with irregular margins that expanded to 10 mm. Brown to gray color changes in the centers of the mature lesions with dark brown edges were noticed. The disease incidence reached 20% of tea plants in 100 ha of tea fields. The infected parts (junction of diseased and healthy region) were cut into small pieces (5 to 7 mm²) and subsequently underwent surface sterilization (Chen et al. 2016). Thereafter, the pieces were blotted dry on sterilized filter paper and placed on water agar plates, which were incubated in the dark at 28°C until fungal hyphae started to grow from the pieces. The single-hyphal tip was then transferred onto potato dextrose agar plates for further analysis. After 7 to 10 days, the purified colony produced yellowish green colonies with sparse aerial hyphae, and yellow with scattered black dots appeared on the back. Pycnidia were black, globose, nonostiole, 27.5 to 112.1 μm (mean, 62.0 µm; n = 50) in diameter, and they contained smooth ovoid conidia with round ends 3.9 to 8.1 µm in length (mean, 5.6 µm; n = 50) by 2.2 to 3.9 µm in width (mean, 3.1 µm; n = 50). Chlamydospores and swollen hyphal bodies were present. On oatmeal agar, the colonies were gray with sparse aerial hyphae; yellowish green occurred on the reverse side. The colonies turned purplish pink with the addition of 1% NaOH. The same colonial characters were noticed on malt extract agar, and yellow with dark brown was noticed on the dorsal side. From the morphological data, the isolate was identified as Phoma sp. (Boerema et al. 2004). To identify the fungal isolate, DNA was extracted (Fungal DNA extraction kit, Biomiga, China), amplified, and sequenced using universal primers (ITS1/ITS4) (White et al. 1990) and the D1/D2 region of 28S rDNA (LR0R/TW13) (Hamayun et al. 2009). BLAST analysis of GenBank data (National Center for Biotechnology Information) showed 100 and 99% similarity with Phoma herbarum for KU204761 and LT966056, respectively. To confirm its pathogenicity, ten 2-year-old tea plants were selected, and upper and middle leaves were injured on the upper surface through acupuncture (Gilbert and Webb 2007) with slight modifications (two needle pricks per leaf, using a sterile needle). A conidial suspension (10⁶/ml) was spread over the wounds, and sterile water was applied to the wounded control leaves. After inoculation, each plant was covered by a plastic bag containing moist cotton wool for 10 days at 28°C to maintain high relative humidity (85 to 90%). At 12 to 15 days after inoculation, lesions appeared on the wounded leaves, with symptoms similar to those previously described, whereas the wounded control leaves and leaves without puncture remained healthy. The experiment was performed twice, and the same fungus was isolated both times from the infected leaves. P. herbarum causes leaf spot disease in other plants, such as Elaeis guineensis and Vetiveria zizanioides in China, Bituminaria bituminosa in Australia, and Salvia nemorosa in Italy. To our knowledge, this is the first report of P. herbarum causing leaf spot disease on tea plant leaves in China, which has a strong influence on tea plant cultivation.