First Report of Leaf Spot Disease Caused by Colletotrichum tropicale on Ficus binnendijkii var. variegata in China

Abstract

In late January 2019, a population of Ficus binnendijkii var. variegata, used as a horticultural cultivar throughout tropical and subtropical regions, was found with leaf spot disease at a 96% infection rate, causing a loss of their ornamental value. The F. binnendijkii var. variegata plants were growing in a park near a creek in Fuzhou, Fujian, China (26°01′53″N, 119°14′42″E). Lesions initially appeared along the margin of the leaves and were subcircular or irregularly shaped, brown to black, water-soaked, and sunken. The black lesions enlarged and coalesced into large necrotic areas. Later, the middle parts of the lesions became grayish white and often had irregular fine wrinkles. To isolate the pathogen, leaf sections (5 × 5 mm) from symptomatic plants were surface sterilized with 75% alcohol for 1 min and 2% NaClO for 30 s, rinsed four times in sterile distilled water, and then incubated on potato dextrose agar (PDA) plates amended with 50 μg/ml of ampicillin at 25°C in darkness. Pure cultures were obtained by monosporic isolation; the colony of a representative isolate, FJ-A5, growing on PDA was initially grayish white and appeared slightly cottony. After 10 to 14 days, the cultures turned gray to grayish-black with orange conidial masses. The conidia were hyaline, aseptate, fusiform with both ends rounded or obtuse at one end, and were in the range of 15.7 to 18.2 × 4.6 to 6.4 μm (n = 50). The appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and were in the range of 6.9 to 9.4 × 5.3 to 6.6 μm (n = 50). These morphological characteristics are consistent with species in the Colletotrichum genus (Prihastuti et al. 2009). Genomic DNA was extracted from the representative isolate FJ-A5, and the internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1), manganese superoxide dismutase (SOD2), calmodulin (CAL), and actin (ACT) genes were amplified with the primers described in Weir et al. (2012). The obtained sequences showed 97 to 100% similarity with those from C. tropicale accessions in GenBank. The sequences from this isolate were deposited in GenBank under the following accession numbers: ITS, MK790630; ACT, MK796811; CAL, MK796812; CHS-1, MK796813; GAPDH, MK796814; and SOD2, MK796815. A neighbor-joining phylogenetic tree was generated by combining all sequenced loci in MEGA7. The isolate FJ-A5 clustered in the C. tropicale clade with 99% bootstrap support. To confirm pathogenicity, 10 detached healthy leaves and five 1-year-old F. binnendijkii var. variegata plants were inoculated by excising 5-mm mycelial plugs from a 7-day-old colony grown on PDA and placing them on the adaxial surfaces of leaves. As a control treatment, five additional detached leaves and potted seedlings were inoculated with 5-mm PDA plugs without mycelia. All plants were covered with clear polyethylene bags and incubated in a growth chamber at 25°C, 70% relative humidity, and a 12-h light/dark cycle, and disease development was monitored daily. The experiment was performed twice. Seven days after inoculation, the symptoms were similar to those on the original infected plants, whereas the control leaves remained asymptomatic. The same fungus was reisolated from the lesions, confirming Koch’s postulates. To our knowledge, this is the first report of C. tropicale associated with leaf spot disease on F. binnendijkii var. variegata in China. This study provides the foundation to further investigate the biology, epidemiology, and management of this disease.