First Report of Rhizopus oryzae Causing Rhizome Soft Rot of Ovate-leaf Atractylodes in South Korea.

Abstract

Ovate-leaf Atractylodes (Atractylodes ovata) (OLA) is cultivated across South Korea to fulfil domestic need as a herbal medicine. In June 2021, the rhizomes with soft rot were observed in a commercial farmer field in Mungyeong, South Korea with 25% disease incidence. Initially, watery, black and soft lesion were observed on the rhizomes. Later, abundant aerial white mycelia covered the entire rhizome resulting in rot and death. Affected rhizomes were washed thoroughly using tap water, surface sterilized with 0.5% NaOCl and rinsed with sterile distilled water. Small pieces (2 to 5 mm2) of inner diseased tissues were cut, placed on potato dextrose agar (PDA) and incubated on at 25°C in the dark. Twelve morphologically similar pure fungal isolates were obtained by hyphal tipping twice on fresh PDA. Rhizoid, sporangiophores, sporangium, and sporangiospores were observed. Rhizoid and sporangiophores were nonseptate. Sporangia were globose while columella were globose or subglobose. Sporangiospores were numerous, varied in shape and size, subglobose, or oval, and measured 9.8 ± 1.5 (6.1 to 13.5) × 7.8 ± 1.1 (5.7 to 10.1) μm (n = 50). Morphological characteristics of the isolates were analogous to Rhizopus oryzae (Kwon et al. 2012, 2015). Sequences of the internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (EF-1α) gene were analyzed for molecular identification. The primer pairs ITS1F/ITS4 and MEF10/MEF4 were used to amplify ITS and EF-1α respectively (Abe et al. 2007) and sequenced (Macrogen, Inc., Seoul, Korea). The obtained sequences were lodged with accession numbers LC705530 to LC705537. The ITS and EF-1α sequences were ≥ 99.5 % and ≥ 98.5% homologous with Rhizopus oryzae (strain CBS 381.52), respectively. In the maximum likelihood phylogenetic tree based on combined ITS and EF-1α sequences, the isolates formed a distinct clade with R. oryzae references isolates. Pathogenicity tests were conducted on healthy rhizomes with isolate R4. A conidial suspension (1× 106 conidia/mL) was sprayed on the surface of fifteen surface-sterilized rhizomes while fifteen rhizomes received sterile distilled water as controls. Treated rhizomes were placed in a plastic box containing wet paper tissues and incubated at 25°C in the12/12 day/light cycle. The typical rhizomes rot symptoms as seen in the field were observed on all inoculated rhizomes after 21 days of inoculation. The noninoculated rhizomes had no obvious symptoms. The causal fungus (3 isolates) was reisolated from inoculated rhizomes and identified as R. oryzae based on morphology and EF-1α sequence. This experiment was replicated twice. To the best of our knowledge this is the first report of R. oryzae infection on OLA rhizomes in South Korea. Rhizopus oryzae contained a complex of heterothallic closely related species and has been reported to cause 'Rhizopus rot' on various plants including apple, banana, mulberry, sweet potato, and tomato (Gnanesh et al. 2020; Khokhar et al. 2019; Kwon et al. 2012, 2015; Wang et al. 2017). Rhizome soft rot poses significant threat to OLA cultivation, sustainable management practices need to be adapted to control rhizome soft rot of ovate-leaf atractylodes.