First Report of Leaf Spot Blight of Camellia oleifera Caused by Diaporthe mahothocarpus in China

Abstract

Camellia oleifera Abel. is one of the native and important natural edible oil species in China. The cultivation of C. oleifera has vigorously increased in Guizhou Province in recent years. From June to August 2022, a severe leaf spot blight was observed on C. oleifera in Longli Plantation, with an incidence of 53.5% (n=200), which caused severe defoliation, negatively affected plant growth, and led to significant economic losses. Pale yellow and sub-circular leaf spots of 2-5 mm in diameter first appeared in the margin. The center of the spots then turned grey, and the edges turned brown. The symptomatic leaves gradually developed symptoms of blight with some brown acervular conidiomata, died, and fell off, with many deep black spots on the leaves (Fig. 1A-B). The fungal isolates GZU-Y2 and GZU-Y3 were obtained from the infected leaves of five-year-old symptomatic C. oleifera trees using the tissue isolation method, and a voucher specimen was deposited in the Forest Protection Laboratory, Guizhou University. Cultures grown on potato dextrose agar medium (PDA) were incubated at 28℃, 16L/8D. A round cream-like colony was formed on PDA, with a white surface, while the back gradually turned brown (Fig. 1C-E). The aerial hypha grew vigorously with an initial milky white color before turning grayish white. At 10 days after incubation, the pycnidia were dark brown to black and spherical, with a diameter of 563.3 μm (500 to 700) (n=20). The alpha conidia were unicellular, hyaline, aseptate, oval or fusiform and measured 6.1 μm (4.1 to 8.0) × 2.6 μm (1.9 to 3.6) (n=50). However, no beta conidia were observed (Fig. 1I). For further identification, total DNA from the pure culture was extracted using a DNA extraction kit (Sangon, Shanghai, China), and the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF-1α), and beta-tubulin (TUB2) were amplified by PCR using the primers ITS1/ITS4 (White et al., 1990), EF1-728F/EF1-986R (Carbone and Kohn, 1999) and Bt2a/Bt2b (Glass and Donaldson, 1995), respectively, and sequenced for a BLASTn analysis and phylogenetic tree construction. The sequences of ITS, TEF-1α and TUB2 were deposited in GenBank as accession numbers OQ168242 (99.25%), OQ689451 (99.71%) and OQ689453 (100.00%) for GZU-Y2 and OQ674554 (99.25%), OQ689452 (99.71%) and OQ689454 (100.00%) for GZU-Y3, respectively. A phylogenetic tree (Fig. 2) was constructed with the software MEGA X using the Neighbor-Joining algorithm (Felsenstein, 1985). Based on its morphological and molecular characteristics, the pathogen was identified as Diaporthe mahothocarpus, one of the synonyms of D. eres and the teleomorph of Phomopsis mahothocarpi (Gao et al., 2014 and 2015; Chaisiri et al., 2021). A pathogenicity test was conducted by spraying spore suspensions (2 × 107 spores/mL) of isolate GZU-Y2 on the leaves of 20 pots of annual C. oleifera seedlings in vivo. The same number of control seedings were sprayed with sterile water. The seedlings were placed at a constant room temperature of 28°C, with the inoculation points wrapped in Parafilm for 5 d to retain moisture. After 10 d, typical symptoms appeared on the inoculated leaves (Fig. 1F-H), and the re-isolated fungal culture was identical in morphology and ITS sequence to that originally obtained, fulfilling Koch’s postulates. To our knowledge, this is the first report of D. mahothocarpus causing leaf spot blight of C. oleifera in China. In our future work, we tend to study the green prevention and control of this disease.