LAUSR

In recent years, postharvest rot diseases of kiwifruit (Actinidia sp.) have caused severe damage in China (Li et al. 2017). Infected fruit were obtained from a commercial farm (30°98′N) after 4 months cold storage. Nineteen fruits (21.1%) out of one package showed rot symptoms with dark-brown, sour-smelling lesions. The lesions began as small pale yellow or light brown spots and then enlarged rapidly and formed dark or dark brown lesions. The margins between symptomatic and healthy tissues (4 × 4 mm) were cut from five rotted fruits, surface disinfested in 1% NaClO and 70% ethanol solution, washed, dried, plated on potato dextrose agar (PDA) containing 50 mg/liter of streptomycin sulfate, and incubated at 25°C for 3 days. Hyphal tips were transferred to PDA to obtain pure cultures. After 7 days, a total of 20 fungal isolates were obtained, including 16 previously reported Botrytis spp. (Xue et al. 2017), Penicillium spp. (Prodromou et al. 2018), and Alternaria spp. (Li et al. 2017), and four unknown isolates (DJY16A1-4, DJY16A1-5, DJY16A5-1, and DJY16A5-2). Strains DJY16A1-4 and DJY16A1-5 were incubated at 25°C for 7 days on PDA, wherein abundant white, fluffy aerial mycelium were grown on the dish. After 25 days, colonies produced black, spherical, or bluntly conical pycnidia. The conidia were hyaline, unicellular, ellipsoidal or fusiform in shape, and 6.52 × 3.43 µm in size. Similarly, incubated DJY16A5-1 and DJY16A5-2 formed white to light brown aerial mycelial mats with gray concentric rings. Pycnidia formed after 26 days, and α-conidia of the isolates were similar to those of the DJY16A1-4 and DJY16A1-5. β-Conidia were filiform or hamate, 23.9 × 1.2 µm in size. To identify these four isolates to species, the internal transcribed spacer (ITS), β-tubulin (BT), and translation elongation factor-1 alpha regions (TEF-1α) were amplified using specific primer pairs (You et al. 2015) and sequenced. By BLASTn analysis, DJY16A1-4 and DJY16A1-5 were 98% homologous to Diaporthe passiflorae CBS 132527 (NR_120155.1) based on ITS sequences (MH595928 or MH595929), 99 or 98% to D. passiflorae CBS 132527 (KY435674.1) based on BT sequences (MH621348 or MH621349), and 91% to D. passiflorae CBS 132527 (KY435633.1) based on the TEF-1α sequences (MH621352 or MH621353). DJY16A5-1 and DJY16A5-2 were 99% homologous to D. nobilis strain JL1 (KT163359.1) based on ITS sequences (MH595930 or MH595931), 97% to D. nobilis strain JL1(KX016113.1) based on BT sequences (MH621350 or MH621351), and 99% to D. nobilis strain 3JW-01S3 (KJ623308.1) based on TEF-1α sequences (MH621354 and MH621355). A phylogenetic tree was constructed using the method of maximum parsimony (MEGA7) with a combined dataset of ITS, BT, and TEF-1α sequences. Molecular phylogenetic analysis confirmed that DJY16A1-4 and DJY16A1-5 were D. passiflorae, and DJY16A5-1 and DJY16A5-2 were D. nobilis. The pathogenicity of the isolates was tested on the fruits of cultivars Xuxiang (A. deliciosa) and Zespri SunGold kiwifruit (A. chinensis). Ripe, healthy fruit were surface disinfected with 1% NaClO solution, rinsed in sterile distilled water, and dried. The middle of the fruit was stabbed with a sterile needle. Five wounded or unwounded fruits were inoculated with a 5-mm-diameter PDA plug with actively growing mycelium for individual isolates. Five wounded and five unwounded fruits were treated with sterile PDA plugs to serve as controls. Inoculated fruit were kept in sterilized transparent plastic boxes for 7 days at 25°C with 12-h light and 12-h dark. The wound-inoculated fruits produced the same symptoms as the rotted fruits collected initially from cold storage. Fruit rot was not observed on control fruit. The test was performed twice, and the fungi were reisolated and identified as either D. passiflorae or D. nobilis morphologically. D. lithocarpus was recently reported to cause kiwifruit rot (Li et al. 2016). But, to our knowledge, this is the first report of D. passiflorae and D. nobilis causing the postharvest rot in China.