First report of Neopestalotiopsis protearum causing seed rot on Camellia oleifera in China.

Abstract

Camellia oleifera, an evergreen small tree or shrub with high medicinal and ecological values, is mainly distributed in subtropical regions of China. Camellia oil obtained from Camellia oleifera seeds is rich in unsaturated fatty acids and unique flavors, and has become a rising high-quality edible vegetable oil in south of China (Zhuang 2008). The tea-oil tree Camellia oleifera plays important economic and ecological roles in Hunan province. During collecting trips, seeds of C. oleifera with disease symptoms have been observed in almost all oil-tea forests. In lab, the seeds can be infected by wounds and directly, however, wound infection is more rapid. In oil-tea forests, the wound of seed is often caused by external factors such as mechanical and insects. Symptomatic seeds exhibited brown rot symptoms with irregular, black spots, brown necrosis of the kernels, and accounted for 65% of the surveyed seeds (Fig. 1). Rotted seeds were surface-sterilized for 1 min in 75% ethanol, 3 min in 1% sodium hypochlorite, then rinsed for 2 min in sterile water and blotted on dry sterile filter paper. Discolored seed tissues were cut into pieces of 3 mm × 3 mm using a sterile scalpel, placed on potato dextrose agar (PDA) medium, and then incubated for 7 days at 25°C with a 12-h photoperiod. After 7 days of incubation, circular fungal colonies with dense aerial mycelium, produced black, wet spore masses. Four-septate conidia were ellipsoidal to obovoid, measuring 24 (22 to 26) × 6.5 (6 to 7) µm (n = 30). Conidia had three median cells, which were dark brown, with a single basal hyaline appendage, 4 (3.5 to 4.5) µm long, and two to four (usually three) apical hyaline appendages, 32 (27 to 35) µm long, similar to these recorded by Crous et al. (2011). Two single-spore isolates cultured on PDA medium were selected for DNA extraction. The ITS region was amplified using primers ITS5 and ITS4 (White et al. 1990). The partial translation elongation factor 1-alpha (tef1-α) gene region was amplified using primers EF1-728F (O'Donnell et al. 1998) and EF-2 (Carbone & Kohn 1999). The partial β-tubulin (tub2) was amplified using primers T1 and Bt2b (Glass & Donaldson 1995). The sequences of ITS (MW391815), tef1-α (MW398222), and tub2 (MW398223) were submitted to GenBank. BLAST analysis demonstrated that these sequences were 99%~100% similar to the sequences of ITS (MH553959), tef1-α (MH554377), and tub2 (MH554618) published for Neopestalotiopsis protearum. Phylogenetic analysis revealed that all the representative isolates recovered from symptomatic Camellia oleifera seeds showed 91% bootstrap support with Neopestalotiopsis protearum isolate in references (Fig. 2). Pathogenicity tests were conducted on 20 healthy seeds. We wounded the seeds by a sterilized needle on the middle position, and put the 5-mm-diameter agar plugs with actively grown mycelia (strain HNWC04) or pure PDA on the wound. We then covered the wounds with clean masking tape to prevent contamination and desiccation. After inoculation, the seeds were kept at 90 to 100% relative humidity at 25°C in a greenhouse for 3 weeks and monitored daily for lesion development. Twenty days after inoculation, all the seeds inoculated presented similar typical symptoms observed under natural conditions, whereas the control seeds showed no symptoms. Koch's postulates were fulfilled by reisolating the same fungus and verifying its colony and morphological characters as Neopestalotiopsis protearum. To our knowledge, this is the first report of Neopestalotiopsis protearum causing oil-tea seed rot in China.