First Report of Leaf Tip Necrosis of <i>Camellia oleifera</i> Caused by <i>Nigrospora aurantiaca</i> in China

In recent years, Camellia oleifera C.Abel. has become one of the most important woody oil trees cultivated at a large scale in Guizhou Province, China. However, the leaf diseases limited the development of C. oleifera industry. In October 2023, typical symptoms of leaf tip necrosis were observed on leaves of C. oleifera (Changlin series) at a plantation in Dongfeng Forest Farm (109°10'28'E, 26°18'41'N), Liping County, with disease incidence of 30-40% (n=220) in the observed fields (30 m × 30 m). The brown lesion started from the leaf tips, gradually extended inward then turned gray-white, curled, withered and fell off, and the boundary between the diseased and healthy parts was obvious. Symptomatic leaves (n=20) were collected and surface disinfected with 2% NaClO for 30 s, rinsed three times in sterile distilled water, air-dried and placed on potato dextrose agar (PDA) medium and incubated at 25°C for 7 d. The fungal colonies of 6 isolates were initially white, with red pigment, and later gradually turned gray, eventually turning black. Conidia were globose to sub-globose, black, smooth, single-celled, discrete and solitary, measuring 5.1 µm±1.28×4.7 µm±1.24 (n=50). The morphological features of the isolates are similar to the description of Nigrospora (Wang et al. 2017). For molecular identification, DNA of the representative isolate LP1021 was extracted using a fungal genomic DNA extraction kit (Sangon, Shanghai, China). The rDNA internal transcribed spacer (ITS), translation elongation factor 1-α (TEF-1α) and β-tubulin (TUB2) were amplified with the primers ITS1/ITS4 (White et al.1990), EF1-728F/EF1-986R (Carbone and Kohn, 1999) and Bt2a/Bt2b (Glass and Donaldson, 1995). The sequences of ITS, TEF-1α and TUB2 were deposited in GenBank as accession numbers PP499217, PP529954, and PP529955, respectively. BLAST revealed that ITS, TEF-1a and TUB2 sequences showed 100%, 98.80% and 100% similarity to those of N. aurantiaca (NR153477.1, KY019295.1 and MK388225.1), respectively. The phylogenetic tree constructed with the software MEGA X using the Neighbor-Joining algorithm (Felsenstein, 1985) indicated the isolate LP1021 separated from N. chinensis previously reported from C. oleifera in China. Based on morphological and molecular characteristics, the fungal pathogen was identified as N. aurantiaca. A representative isolate LP1021 was deposited in the Forest Protection Laboratory, Guizhou University. To further confirm the pathogenicity, a conidial suspension (1×104 spores/mL) of the isolate LP1021 was sprayed on six pots of two-year-old healthy C. oleifera seedlings in vivo. The same amounts of seedlings were sprayed with distilled water as control. All plants were placed at a constant room temperature of 25 ± 2°C. After 15 d, the inoculated leaves showed typical symptoms, similar to those observed in the field, while the control remained healthy. The re-isolated fungal culture was identical to that originally obtained using the methods mentioned above, which fulfilled Koch's postulates. To the best of our knowledge, this is the first report of N. aurantiaca causing leaf tip necrosis of C. oleifera in China, which provides an important theoretical basis for the pathogen identification and scientific control strategies of leaf tip necrosis in C. oleifera.