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First Report of Glomerella cingulata f. sp. camelliae Causing Anthracnose on Aquilaria sinensis in China

Liu, H. N., Liu, J. A., Zhou, G. Y., Hu, W. L.
Plant disease 2019 v.103 no.4 pp. 766
Aquilaria sinensis, DNA primers, Glomerella cingulata, Oriental traditional medicine, actin, anthracnose, appressoria, chitin, color, conidia, control methods, culture media, endangered species, ethanol, financial economics, fungi, genes, glyceraldehyde 3-phosphate, hyphae, internal transcribed spacers, leaves, pathogenicity, polymerase chain reaction, ribosomal DNA, storage temperature, streptomycin, tissues, trees, tubulin, China
Aquilaria sinensis (Lour.) Spreng is an endangered species of tree that produces agarwood, used in high-quality incense and Chinese medicine. In September 2017, anthracnose spots were first observed on the mature leaves of samples of A. sinensis. Further investigation showed that about 35% of such trees were affected by anthracnose from a sample taken from Huangzhuling Forest Farm (19°30′13.22″N, 110°11′21.23″E) in Tunchang County, Hainan Province, China. Anthracnose causes plant leaves to fall earlier than usual, and anthracnose spots can be seen on the leaves. These initially appear as small brown spots, later taking on various shapes, often circular, oval, or an irregular circle. Symptomatic leaf tissue samples of 3 × 5 mm were cut and their surfaces disinfected in 75% ethanol solution for 30 to 60 s. They were then rinsed three times in sterilized water. These aseptic excised tissues were then plated on potato dextrose agar (PDA) medium amended with 25 µg of streptomycin per liter. The PDA plates were incubated at 25°C in darkness for 5 days. Pure cultures of colonies were then obtained using the single-spore method. These were also cultured on PDA, and all samples were stored at 4°C. The central part of each colony became gray-green, forming the hyphae with irregular margins. These colonies extended over time, after 7 days, with their peripheral colors becoming lighter than that seen in the center. After 15 days, a large number of light-red conidia formed on the colony. These conidia were smooth and aseptate. The size of each conidium was 9.4 to 16.2 × 3.5 to 6.4 μm (average = 13.1 ± 1.3 × 5.1 ± 0.9 μm, n = 100). Globose, terete, or spindly, appressoria were also seen and were 5.6 to 9.1 × 4.7 to 8.6 μm (average = 7.2 ± 0.8 × 6.7 ± 0.7 μm, n = 100) in size. To further identify the fungus HN76 (a representative isolate collected from Hainan in 2017), the rDNA internal transcribed spacer (ITS) region, partial sequence of glyceraldehyde-3-phosphate dehydrogenase-like (GAPDH) gene, chitin synthase-1 (CHS-1) gene, actin (ACT) gene, and β-tubulin (TUB2) gene (Álvarez et al. 2014) were amplified using the primer pairs ITS4/ITS5, GDF1/GDR1, CHS-1-79F/CHS-1-354R, ACT-512F/ACT-783R, and Bt2a/Bt2b, respectively. The sequences were deposited in GenBank (accession nos. MG984763, MG984764, MG984765, MG984766, and MG984767). The results showed 100% identification of all five sequences to the ex-epitype culture of Glomerella cingulata f. sp. camelliae (Weir et al. 2012). Koch’s postulates were also verified by inoculating freshly detached leaves. To test pathogenicity, a conidial suspension of 1 × 10⁵ spores/ml collected from isolate HN76 was sprayed onto 20 punctured leaves and 20 unwounded leaves. To puncture the leaves, an area of cuticle on one side of the midrib of each selected leaf was lightly scratched with a sterile hypodermic needle prior to inoculation. As a control, distilled water was sprayed onto an equal number of punctured and unwounded leaves. All inoculated and control plants were incubated in moist chambers at 25°C. Gray-black lesions appeared on the inoculated punctured leaves within 7 days, whereas the unwounded inoculated plants and the control leaves remained symptomless. To our knowledge, this is the first report of G. cingulata f. sp. camelliae causing anthracnose on A. sinensis in China. The disease has caused considerable economic loss, so control measures should be implemented at affected sites.