First Report of Colletotrichum nymphaeae Causing Anthracnose on Camellia oleifera in China

Abstract

Camellia oleifera (tea-oil tree) is native to China and is cultivated in many parts due to its high-quality cooking oil (Liu et al. 2017; Ma et al. 2011). In February 2017, anthracnose lesions were found on leaves of C. oleifera planted in Guizhou province, China. The disease incidence was 20 to 30% in plantations with over 50 acres (20.2 ha) of tea-oil trees. The necrotic lesions on green leaves were surrounded by a ring of brown tissue and gradually turned gray. Three single-spore isolates of Colletotrichum-like fungus (GZGYYC6, GZGYYC7, and GZGYYC13) were isolated and cultured on potato dextrose agar (PDA) from surface-disinfested (75% ethanol and 0.5% v/v NaOCl) samples. Colonies of these cultures on PDA were gray, cottony in surface view, and pale gray to pale orange in reverse. The average mycelial growth rate was 9.3 mm per day (n = 5). Setae and sclerotia were absent. Conidia were smooth, hyaline, tapered to the base, rounded at the apex, and 6.5 to 19.6 × 2.0 to 4.8 μm (n = 200). These morphological characteristics correspond to those of Colletotrichum nymphaeae (Damm et al. 2012). Three single-spore isolates were used for molecular identification: internal transcribed spacer (ITS) and partial actin (ACT), β-tubulin 2 (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genomic DNA regions were amplified by PCR and sequenced using the ITS5/ITS4 and ACT-512F/ACT-783R, T1/Bt2b, and GDF/GDR primer pairs, respectively (Weir et al. 2012). Gene sequences were deposited in GenBank (MF426212 to MF426214; MF426217 to MF426219; MF426232 to MF426234; MF426237 to MF426239). BLASTn analyses revealed that DNA sequences of our strains at the ITS, GAPDH, ACT, and TUB2 loci showed 99.8, 99.5, 100, and 100% sequence identity, respectively, to their corresponding loci in strains CBS129945 (GenBank accession no. MH865694), CBS126507 (JQ948533), Col-680 (MH717707), and BRIP62666 (KU221376) of C. nymphaeae. All four gene sequences were compared with representative sequences from type specimens or ex-type of Colletotrichum (Damm et al. 2012), and a phylogenetic tree was generated via the neighbor-joining method with 1,000 bootstrap replications using MEGA7 (Kumar et al. 2016). The tree placed the three isolates in the same group as C. nymphaeae (bootstrap 100%). Both morphological and molecular characteristics identified our isolates as C. nymphaeae. A pathogenicity assay was performed on three plants. Conidial suspension (10⁶ conidia/ml) of the representative isolate GZGYYC6 was dropped on three nonwounded and three wounded leaves, three wounded stems, and four wounded fruits per plant. Control leaves and stems were treated with distilled water. After inoculation, plants were kept under greenhouse conditions at 28 ± 2°C and 90% relative humidity on a 12-h fluorescent light/dark regimen. Anthracnose-like symptoms were observed 5 days postinoculation. The control plant tissues remained healthy. C. nymphaeae was reisolated on PDA from lesions, and the morphological features were consistent with GZGYYC6 isolate, confirming Koch’s postulates. To our knowledge, this is the first report of C. nymphaeae infection on C. oleifera in China. These findings will help in developing better preventive measures in accordance with the emergence of the new pathogen.