Nigrospora oryzae Causing Black Leaf Spot Disease of Hibiscus mutabilis in China.

Abstract

Cotton rose (Hibiscus mutabilis Linn.) is a deciduous shrub native to China. It has been widely cultivated in many provinces in China for its ornamental and ecological value (Shang et al., 2020). In May 2017, an unknown leaf spot symptom was first observed on H. mutabilis at the Chengdu Campus of Sichuan Agricultural University (30°42'31″ N, 103°51'28″ E). The disease occurred from May to September with approximately 81% incidence by field sample survey of 300 plants in Chengdu Greenway. The symptoms at first appeared as irregular black spots on the leaves. Then the lesions grew and coalesced into large, black necrotic areas, which later produced leaf chlorosis and abscission (Fig. 1-A). This disease seriously reduced the ornamental value of H. mutabilis. Forty diseased lesions (4 × 5 mm) were surface sterilized with 75% alcohol for 60 s and 3% NaClO for 45 s, rinsed three times in sterile water, placed onto potato dextrose agar (PDA), and then incubated in a dark at 25°C. From the 7 obtained isolates, 4 isolates exhibited the morphology described as Nigrospora oryzae (Hao et al., 2020). The fungus produced initially circular white colonies, and then the centers turned dark gray or black with age on the PDA. Hyphae were smooth, branched, septate, hyaline, or pale brown. Conidia (N = 100 spores) were abundant, and were solitary, dark-brown to black, smooth, aseptate, and measured 11 to 15 μm in diameter (Fig. 1). DNA was extracted from the fungal colonies using a DNeasyTM Plant Mini Kit (Qiagen). The internally transcribed spacer (ITS), β-tubulin gene (TUB), and translation elongation factor 1-alpha (TEF1) were amplified with primers ITS1/ITS4 (White et al., 1990), BT2A/BT2B (Glass and Donaldson 1995), and EF1-728F/EF1-986R (O'Donnell et al., 1998; Carbone and Kohn 1999), respectively. BLAST results indicated that the ITS, TUB, and TEF1 sequences (GenBank accession Nos. MN515070, MN733956, and MN635723, respectively) had 99% identity with N. oryzae sequences (GenBank accession Nos. KX986031, KY019553, and KY019358). The result was confirmed by multilocus phylogenetic analysis (Fig. 2). The morphological characteristics and molecular analyses of the isolate matched the description of N. oryzae. To confirm pathogenicity, Koch's postulates were fulfilled under controlle conditions. The seedlings of 20 two-year-old potted H. mutabilis plants were inoculated by spraying conidial suspension at the concentration of 1 × 106 conidia/ml on both sides of leaves. Sterilized distilled water (20 seedlings) were used as negative controls. The experiment was performed three times. All plants were incubated at 25°C ± 2°C under a 16 h/8 h photoperiod and 70%-75% relative humidity (RH) after inoculation, and observed daily for disease development. Two weeks later, the inoculated plants showed the same symptoms as the original diseased plants and the controls remained asymptomatic. The pathogen N. oryzae was re-isolated from all ioculated plants, and the culture and fungus characteristics were the same as those of the original isolate. But N. oryzae was not isolated from the control plants. The results indicated that N. oryzae is a causal agent of the disease. N. oryzae was reported as a leaf pathogen on cotton (Zhang et al., 2012), but this is the first report of N. oryzae causing leaf black spot on H. mutabilis in the world. The identification could provide relevant information for adopting appropriate management strategies to control the disease.