Mycobiology Research Article
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Mycobiology Research Article Phylogenetic Placement and Morphological Characterization of Sclerotium rolfsii (Teleomorph: Athelia rolfsii) Associated with Blight Disease of Ipomoea batatas in Korea Narayan Chandra Paul, Eom-Ji Hwang, Sang-Sik Nam, Hyeong-Un Lee, Joon-Seol Lee, Gyeong-Dan Yu, Yong-Gu Kang, Kyeong-Bo Lee, San Go and Jung-Wook Yang* Bioenergy Crop Research Institute, National Institute of Crop Science, Rural Development Administration, Muan 58545, Korea Abstract In this study, we aimed to characterize fungal samples from necrotic lesions on collar regions observed in different sweetpotato growing regions during 2015 and 2016 in Korea. Sclerotia appeared on the root zone soil surface, and white dense mycelia were observed. At the later stages of infection, mother roots quickly rotted, and large areas of the plants were destroyed. The disease occurrence was monitored at 45 and 84 farms, and 11.8% and 6.8% of the land areas were found to be infected in 2015 and 2016, respectively. Fungi were isolated from disease samples, and 36 strains were preserved. Based on the cultural and morphological characteristics of colonies, the isolates resembled the reference strain of Sclerotium rolfsii. Representative strains were identified as S. rolfsii (teleomorph: Athelia rolfsii) based on phylogenetic analysis of the internal transcribed spacer and large subunit genes along with morphological observations. To test the pathogenicity, sweetpotato storage roots were inoculated with different S. rolfsii strains. ‘Yulmi’ variety displayed the highest disease incidence, whereas ‘Pungwonmi’ resulted in the least. These findings suggested that morphological characteristics and molecular phylogenetic analysis were useful for identification of S. rolfsii. Keywords Fungal morphology, Ipomoea batatas, Molecular phylogeny, Pathogenicity, Sclerotium rolfsii Sweetpotato (Ipomoea batatas) is a common plant of the parts of Asia, including the Philippines, India, Korea, Convovulaceae family and is ranked as the seventh most Taiwan, some eastern islands of Indonesia (Bali and Irian important food crop in terms of production; in developing Jaya) and Papua New Guinea [3, 4]. In Korea, the sweetpotato countries, sweetpotatoes rank fifth in economic value is widely cultivated and consumed, and the total area of production, sixth in dry matter production, seventh in energy cultivation reached as much as 22,000 ha of land in 2015, production, and ninth in protein production and have which was 27% higher than that in 2000 [5]. many applications as foods, feeds, and industrial products Numerous fungal diseases have been reported on [1, 2]. Roughly 80% of the world’s sweetpotatoes are grown sweetpotatoes from different regions worldwide. Sclerotial in Asia. In addition to China and Vietnam, sweetpotatoes blight is a major fungal disease caused by Sclerotium rolfsii play important roles in the rural economy in many other during the early cultivation period and has been observed in plant beds, mature plants as circular spots, and tubers as a post-harvest disease. S. rolfsii Sacc. (teleomorph Athelia Mycobiology 2017 September, 45(3): 129-138 rolfsii [Curzi] C. C. Tu. & Kimbr.) is a serious disease-causing https://doi.org/10.5941/MYCO.2017.45.3.129 fungal pathogen that affects diverse crops grown around pISSN 1229-8093 • eISSN 2092-9323 © The Korean Society of Mycology the world, particularly tropical and subtropical countries [6]. Because of its broad host range, S. rolfsii is considered *Corresponding author E-mail: [email protected] one of the most destructive pathogens worldwide; indeed, about 500 plant species from 100 families, including tomatoes, Received April 13, 2017 Revised June 14, 2017 potatoes, chili peppers, carrots, cabbage, sweetpotatoes, Accepted June 26, 2017 common beans, and ground nuts [7, 8], are affected by this pathogen. The fungus generally infects the collar region or This is an Open Access article distributed under the terms of the stems near the soil surface and spreads over the whole Creative Commons Attribution Non-Commercial License (http:// plant or fruits and remains in soil surface [9, 10]. S. rolfsii creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, produces sclerotia on hosts, survives and overwinters for a provided the original work is properly cited. long time and infects the same host crop or other nearby 129 130 Paul et al. crops [7, 11]. Various molecular approaches have been employed to identify S. rolfsii and other fungal populations [12-15]. The internal transcribed spacer (ITS) is common genomic target used to measure fungal diversity at the molecular level [6]. The level of divergence between species or strains or within the species examining by ITS rDNA sequences is low; however, this method shows improved sensitivity at the genus level [16]. The ITS and large subunit (LSU) show slight phylogenic differences among species that are closely related to S. rolfsii (e.g., S. coffeicola and S. delphinii) [17]. However, no studies have performed molecular analysis along with morphological characterization of S. rolfsii in sweetpotatoes in Korea. Therefore, in the present study, we aimed to evaluate disease occurrence on sweetpotatoes by S. rolfsii, analyze the molecular data by ITS and LSU sequences and morphological characteristics of the pathogen, and test Fig. 1. Symptoms of sclerotial blight disease observed in their pathogenicity on different variety. different farmers’ field in Korea during 2015 and 2016. Sudden wilting and deaths of seedlings (A), appearance of MATERIALS AND METHODS mycelia on the collar region of seedlings (B), aerial mycelia, mycelial mat along with sclerotia observed on rootzone soil (C, D). Disease symptom and incidence. Initially sudden wilting of the stalks appeared in plant beds followed by deaths (Fig. 1A). In high humid conditions the disease of affected plants, and ‘N’ is the total number of evaluated spreads and infection was initiated near the collar regions plants. and soon after infections, necrotic lesions were observed along with aerial cottony, coarse, white mycelia (Fig. 1B Pathogen isolation and culture maintenance. Leaves, and 1C). Mycelium covers the soil surface and produced roots, stems, and storage roots affected by Sclerotium blight mycelial mats. After that sclerotia appeared large in numbers disease were collected and were transported to the laboratory. on the mycelial mats, mats and sclerotia were found on the Samples were washed under running tap water and dried surface layer of the newly grown storage roots. Then, disease in a laminar air flow chamber. After surface sterilization [19], incidence was measured as the proportion of a plant samples were cut into small pieces, dried, and transferred to community that is diseased. Field surveys were conducted potato dextrose agar (PDA) supplemented with rifampicin in farmers’ fields in 6 different locations of Yeoju, Nonsan, to stop bacterial growth. PDA petri dishes were then Iksan, Gimje, Yeongam, and Haenam (Table 1). During incubated at 25oC for 3–7 days. Plates were checked on a the survey of disease incidence, we inspected 45 and 84 regular basis by the naked eye for hyphal growth. Hyphae farmlands in 2015 and 2016, respectively, and monitored were then transferred to PDA, and pure cultures of the presence of disease and the affect land area. After Sclerotium-like fungi were prepared. A total of 36 strains obtaining data from different locations, we calculated the were obtained after confirming pure cultures by growing disease incidence by the following formula [18]. on PDA several times. Fungi were then assigned identification numbers (Table 2), maintained in PDA slant tubes and ni I ()% = ----- × 100 20% glycerol stock solution, and deposited in the culture N collection of the Sweetpotato Laboratory, Bioenergy Crop Where, ‘I’ is the disease incidence, ‘ni’ is the total number Research Institute, Rural Development Administration (RDA), Table 1. Sclerotial blight disease incidence of sweetpotato in different locations in Korea during 2015 and 2016 Farm surveyed Disease incidence (%) Province Location 2015 2016 2015 2016 Gyeong-gi Yeoju 06 08 11.7 10.6 Chungnam Nonsan 081508.8 07.0 Jeonbuk Iksan 071420.003.9 Gimje 12 11 07.9 03.2 Jeonnam Yeongam 031201.7 05.8 Haenam 09 24 20.8 10.8 Total 45 84 11.8 06.8 Sclerotium rolfsii Associated with Ipomoea batatas in Korea 131 Table 2. Isolates collected from different sweetpotato growing regions in Korea during 2015 and 2016 Accession No. Species Strain number Host Location ITS LSU Athelia sp. SPL15001 Stem, Sweetpotato Yeoju, Korea KY446394 KY446368 Athelia sp. SPL15002 Stem, Sweetpotato Iksan, Korea KY446393 KY446369 Athelia sp. SPL15004 Root zone soil, Sweetpotato Haenam, Korea KY446392 KY446370 Athelia sp. SPL15005 Storage roots, sweetpotato Haenam, Korea KY446391 KY446371 Athelia sp. SPL15009 Storage roots, sweetpotato Gimje, Korea KY446389 KY446372 Athelia sp. SPL15011 Storage roots, sweetpotato Yeongam, Korea KY446388 KY446373 Athelia sp. SPL16001 Stem, Sweetpotato Haenam, Korea KY446387 KY446374 Athelia sp. SPL16005 Stem, Sweetpotato Haenam, Korea KY446386 KY446375 Athelia sp. SPL16006 Root zone soil, Sweetpotato Haenam, Korea KY446385 KY446376 Athelia sp. SPL16011 Storage roots, sweetpotato Yeongam, Korea KY446384 KY446377 Athelia sp. SPL16020 Stem, Sweetpotato Gimje, Korea KY446383 KY446378 Athelia sp. SPL16023 Stem, Sweetpotato Nonsan, Korea KY446382 KY446379 Athelia sp. SPL16057