First report of Diaporthe gardeniae causing branch blight of Gardenia jasminoides Ellis in Zhejiang Province, China.

Gardenia jasminoides Ellis, an important Chinese medicine, is cultivated on approximately 1,400 hectares in China. From August to October 2016, a severe disease affecting leaves, stems, and fruits of G. jasminoides, occurred in Cangnan (120°39'E, 27°48'N), Zhejiang province. Infected leaves or stems became shriveled, and in severe cases, young fruits presented red-brown or yellow necrotic lesions with numerous black spots. More than thirty diseased fruit and stem samples were collected, ten diseased fruits were surfacedisinfected (70% ethanol for 30 s, 1.5% sodium hypochlorite for 1 min) and kept at about 25℃ for 24 h with 80% humidity. The conidial suspension (105 conidia/ml) made of creamy drops secreted from the lesion black spots was spread onto PDA (Potato-Dextrose-Agar) and incubated at 25℃ in the dark for 7 d. Only one isolate (step01) was suspected to be the target pathogen, and other three isolates were Alternaria sp. The colony of step01 was white to grayish with an irregular edge on the front and a white to brown spiral grain on the back. Black pycnidia, produced after 20 d, were globose to subglobose, individual or overlapped, with an ostiole secreting a creamy conidial suspension. Alpha-conidia were aseptate, hyaline, oval to oblong with two oil balls, 7.4-15.9×2.4-4.5 µm (average 10.2×3.3 µm); beta-conidia were hyaline, aseptate, linetype, straight or slightly curled, 15.3-26.5×1.3-2.5 µm (average 20.8×1.6 µm). This isolate resembled Diaporthe sp. (Hansen and Barrett 1938). For species identification, DNA was extracted (Sangon Biotech Rapid Fungi Genomic DNA Isolation Kit - B518229)，and the internal transcribed spacer region (ITS), elongation factor (EF1-α), β-Tubulin (TUB), and histone H3 (HIS) of step01 were amplified using the primer pairs ITS1/ITS4, EF1-728F/ EF1-986R, BT-2a /BT-2b and CYLH3F/CYLH3R, respectively (Udayanga et al 2014, Huang et al. 2015).These sequences were submitted to GenBank as KY797655 (ITS), MF158048 (EF1- α), MF158049 (TUB), and MF158050 (HIS). In comparison with the other sequence of Diaporthe sp. using MEGA7.0 (maximum likelihood, bootstrap replications=1,000), step01 showed 100% identity with D. gardeniae. Based on their morphology and molecular identification, step01 was identified as D. gardeniae (syn. Phomopsis gardeniae). Pathogenicity was tested on three one-year-old G. jasminoides plants by stem inoculation. Two or three stems per plant were inoculated by binding a mycelial plug (5 ×12 mm), covered by humid cotton and plastic film, to the tender stem. A total of two plants were treated. Plants were kept at about 25℃ for 4 weeks. Control plants were inoculated with PDA plugs. Leaf blight started from the apex, extended to the stalk, and the leaves finally fell off. Three months after inoculation, symptoms developed on the underlying leaves, the stem was withered with black spots, a pattern like that observed in the field. No symptoms appeared in the control leaves. Five identical colonies were re-isolated from symptomatic tissues and identified again as D. gardeniae, fulfilling the Koch's postulates. Several fungi are associated with canker, leaf spot, and fruit rot in Gardenia throughout China, including Pestalotiopsis sp. (Huang et al. 2006), Botryosphaeria dothidea (Dong et al. 2016), and Phoma sp. (Luo et al. 2016). To the best of our knowledge, this is the first report of D. gardenia infecting G. jasminoides Ellis in Zhejiang Province, China.