First Report of Bunch Rot Caused by Aspergillus tubingensis of Shine Muscat Grape in Korea

Abstract

Shine Muscat (Vitis vinifera L.) is an increasingly popular grape cultivar in South Korea because of the sweet and unique flavors and large berries. Fungal diseases, including bunch rot, are a continual challenge for grape growers, causing pre- and postharvest fruit decay. In September 2018, Shine Muscat plants showing bunch rot were collected from an orchard in Gimcheon, South Korea. Initial symptoms of the disease consisted of brown spots on the fruits. Whole fruit became rotten as the disease progressed and were covered by black powdery spores. The pathogens were isolated from diseased fruit tissue to potato dextrose agar (PDA). To avoid contamination, small sections of diseased tissue were cut from affected fruits, immersed in 0.5% NaOCl for 1 min, rinsed in sterile distilled water, and blotted with sterile tissue. Then, sterilized sections of infected tissue were placed on PDA plates and incubated at 25°C in the dark. Pure cultures were obtained by transferring hyphal tips to a fresh PDA plate. Two pure cultures (AS181 and AS182) were used to examine morphological characteristics. Seven-day-old colonies were initially white and then covered with dark gray conidial heads. Conidial heads were large and radiate, and vesicles were globose. Conidia were globose or subglobose (3.6 to 5.5 μm in diameter) and dark brown to black in color. The morphological characteristics of isolates were typical of Aspergillus spp. of Aspergillus section Nigri (black Aspergilli), including Aspergillus tubingensis (Mirhendi et al. 2016; Zulkifli and Zakaria 2017). The species of Aspergillus section Nigri cannot be distinguished using only morphological characteristics (Zulkifli and Zakaria 2017). Total genomic DNA was isolated from mycelium of AS181. The internal transcribed spacer (ITS) region and β-tubulin (TUB) gene were amplified using primers ITS-1F/ITS4 and Bt2a/Bt2b, respectively. PCR products were purified and sequenced by Macrogen (Seoul, South Korea) and deposited in GenBank. BLAST search analysis of the ITS sequence (LC467944) showed 602/604 (99.7%) similarity with the sequence from A. tubingensis strain CMXY25850 (GenBank accession no. MG991653). TUB (LC467945) showed 559/562 (99.5%) similarity with the sequence from A. tubingensis strain IHEM 23902 (MH614560). Phylogenetic analysis using maximum likelihood and Bayesian inference methods based on concatenated sequences of ITS and TUB showed that isolate AS181 clustered together with previously identified A. tubingensis strains (USML03, USML13, and USMJ02) (Zulkifli and Zakaria 2017). Molecular analysis using TUB sequences data is an important tool for species delineation of black Aspergillus isolates (Zulkifli and Zakaria 2017). To confirm pathogenicity, 1 × 10⁶ conidia/ml (conidial suspension of isolate AS181) was sprayed on five healthy bunches of fruit. The same number of bunches were sprayed with sterile distilled water as noninoculated controls. The treated fruit bunches were placed in a separate sterile plastic box containing moist tissue paper and incubated at 25°C in the dark. Ten days after inoculation, symptoms similar to naturally infected fruits developed on 20% of inoculated fruits, whereas control fruits remained asymptotic. One pure culture was reisolated from artificially infected fruits and identified as A. tubingensis following procedures described above. In a previous study, A. tubingensis was reported as the causal agent of vine canker disease of grapevine in Italy (Vitale et al. 2012). A. tubingensis has also been reported as the causal agent of postharvest fruit rot of Prunus salicina and leaf spot of Jatropha curcas. (Guo et al. 2017; Wang et al. 2012). To our knowledge, this is the first report of A. tubingensis casing bunch rot on V. vinifera in South Korea.