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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.49 No.6 pp.163-172
DOI : https://doi.org/10.14397/jals.2015.49.6.163

Morphological and Molecular Identification of Powdery Mildew caused by Neoerysiphe galeopsidis on Stachys sieboldii Miq.

Jin-Hyeuk Kwon1, Dong-Wan Kang1, Jinwoo Kim2,3*
1Gyeongsangnam-do Agricultural Research and Extension Services, Jinju 52733, Korea
2Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Korea
3Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Korea
Corresponding author: Jinwoo Kim Tel: +82-55-772-1927 Fax: +82-55-772-1929 jinwoo@gnu.ac.kr
January 7, 2015 December 17, 2015 December 17, 2015

Abstract

From August to October 2013, a powdery mildew was found on Japanese artichoke(Stachys sieboldii) in Jinju, Korea. White colonies were present on leaves and petioles. Severely infected lesions were discolored, being brown. In the present study, the morphological features of anamorphic and teleomorphic Korean specimens were studied. To complete the identification, the sequence of the internal transcribed spacer region of the ribosomal RNA gene(ITS rRNA) was analyzed. Based on morphological characteristics, including anamorphic and teleomorphic features, as well as analysis of the ITS rRNA gene region, the fungus associated with the symptoms was identified as Neoerysiphe galeopsidis. Although the host ranges of powdery mildew caused by N. galeopsidis has been previously recorded, no full description or illustration of fungal symptoms and signs on Japanese artichoke has yet appeared. To the best of our knowledge, this is the first full description of powdery mildew disease on Japanese artichoke featuring molecular identification, morphological features, symptoms, and signs in Korea.


초록


    Rural Development Administrationhttp://dx.doi.org/10.13039/501100003627$$Project No.PJ01082304

    Introduction

    Powdery mildew is a fungal disease that affects a wide range of plants. Powdery mildew diseases are caused by many different species of fungi in the order Erysiphales. Powdery mildew occurs and spreads well in environments of high humidity with moderate temperatures(Huang et al., 2000; Siebold & Tiedemann, 2012). From August to October 2013, a powdery mildew disease was observed on the leaves of Japanese artichoke(Stachys sieboldii) in Jinju, Korea. Japanese artichoke is a perennial herbaceous plant of the family Lamiaceae, and the rhizome thereof can be eaten as a root vegetable(Wikipedia Foundation).

    Powdery mildew fungi cause serious diseases on a wide variety of crops, including cereal plants, vegetables, fruit plants, and ornamental plants. Powdery mildew fungi are biotrophic parasites and invade only epidermal cells of host plants using the haustoria(Huckelhoven, 2005). The genus Neoerysiphe of powdery mildew fungi includes six teleomorphs: N. chelones, N. cumminsiana, N. galii, N. rubiae, N. geranii, and N. galeopsidis; as well as three anamorphs: Oidium baccharidis, O. aloysiae, and O. maquii(Liu et al., 2005; Takamatsu et al., 2008). Each species has a different distribution. N. galeopsidis, parasitizing many Lamiaceae as the main host family, but also a few species of Acanthaceae, Bignoniaceae, Dipsacaceae, and Malvaceae, is nearly circumglobal, being found in Europe, Asia, Africa, North America, and New Zealand(Amano, 1986; Braun, 1987; Braun & Takamatsu, 2000; Mori et al., 2000; Liu et al., 2005; Takamatsu et al., 2008).

    Japanese artichoke, which has a nutty artichoke-like flavor, is used as a vegetable, in salads, and as a garnish. In Japanese cuisine, the Japanese artichoke is primarily pickled. In particular, the tuber is a part of Osechi, and is cooked to celebrate the Japanese New Year. In French cuisine, the cooked tuber is often served alongside Japanese-styled dishes. A powdery mildew disease was observed on Japanese artichoke plants in Jinju, Korea. Powdery mildew on Japanese artichoke plants, caused by Erysiphe, has not previously been reported in Korea(Shin, 2000; Farr & Rossman, 2014). In addition, no full description or illustration of symptoms, signs, and anamorphic and teleomorphic features; no detailed information on molecular identification and phylogenetic analysis of such a fungal species, have yet been recorded from Korea.

    In the present study, we explored the morphological features of anamorphic and teleomorphic Korean specimens, and obtained the complete sequence of internal transcribed spacer region of the ribosomal RNA gene(ITS rRNA) of the powdery mildew fungus infecting Japanese artichoke plants. Based on morphological characteristics and analysis of the ITS rRNA gene sequence, the fungus associated with the symptoms was identified as Neoerysiphe galeopsidis. To the best of our knowledge, this is the first full description of powdery mildew disease on Japanese artichoke plants, featuring molecular identification, symptoms, and signs. It is important to be aware of powdery mildew because the disease may significantly reduce Japanese artichoke production.

    Materials and Methods

    1.Sample sources

    Conidial and cleistothecial specimens of powdery mildews were collected from the farmer's field (35°1 3′47.15″N, 128°08′15″E) in Jinju, Korea, in 2013. Fungal DNA was extracted from fresh materials as soon as possible. The remaining specimens were dried and kept in the Mycological Herbarium of Gyeonsang National University.

    2.Morphological characteristics

    Detailed microscopic examination of sample specimens(anamorphs and teleomorphs) was performed under a light microscopy (Axioplan; Carl Zeiss, Jena, Germany; 400× magnification).

    3.Molecular identification

    3.1.DNA extraction and PCR amplification

    Fungal DNA extraction and polymerase chain reaction(PCR) were performed using Phire® Plant Direct PCR Kit(Finnzymes, Espoo, Finland) following the manufacturer's instructions. Briefly, conidia were added to 50μL of Dilution Buffer(Finnzymes) in a 1.5-mL microcentrifuge tube and incubated at room temperature for 30min. The extract was mixed vigorously and centrifuged at 10,000×g for 1min. The supernatant(0.5μL) served as a template for PCR.

    To amplify the nuclear rRNA gene region spanning the ITS1, ITS2, and 5.8S RNA regions, primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′ -TCCTCCGCTTATTGATATGC-3′) were used (White et al., 1990). PCR amplification was performed in a 20 μL reaction volume following the manufacturer's instructions. PCR was performed using an Astec PC 802 thermal cycler(Astec, Fukuoka, Japan) running the following thermal profile: 98°C for 2min; followed by 30cycles of 98°C for 30s, 55°C for 30s, 70°C for 30s; and a final extension step of 72°C for 4min to refill gaps in PCR products. Amplified products were separated via electrophoresis on a 0.8%(w/v) agarose gel in 1 × TBE buffer at 100V for 20min. PCR amplification yielded only a single visible DNA product. The DNA product band was excised from an ethidium bromide-stained gel and purified using a gel extraction kit(GeneAll Biotechnology Co., Seoul, Korea) following the manufacturer's instructions.

    3.2.Cloning and sequencing of the ITS rRNA gene region

    Purified PCR products were ligated into the pGEM-T Easy Vector(Promega, Madison, WI, USA) following the manufacturer's instructions. The ligated products were transformed into competent cells of Escherichia coli DH5α, via heat shock. Transformed cells were selected on Luria Bertani(LB) agar plates supplemented with 50μg/mL ampicillin and 25μg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal, Duchefa). Plasmids from white transformants were extracted and purified using a plasmid extraction kit(GeneAll) following by the manufacturer's instructions, and the expected insert size was verified via EcoRI(TaKaRa, Tokyo, Japan) digestion and visualization using gel electrophoresis and ethidium bromide staining. One resulting plasmid clone(pITS21) containing an insert of the expected size was isolated and the insert DNA was sequenced in both directions, using the M13F and M13R primers, at Macrogen Services(Daejeon, South Korea). The resulting 592-bp ITS rRNA gene sequence was deposited in GenBank(Accession no. KP347576).

    4.Phylogenetic analysis

    The host plants, fungal species, locations of collections, and accession numbers for the nucleotide sequence database(GenBank) are shown in Table 1. The nucleotide sequence of the ITS rRNA gene region of the powdery mildew fungus was compared with those of reported reference ITS sequences. The sequences were additionally analyzed using the Basic Local Alignment Search Tool(BLAST) (http://www.ncbi.nlm. nih.gov/). Multiple sequence alignment of the ITS rDNA region was performed using ClustalW to analyze single nucleotide polymorphisms. Phylogenetic analysis was performed using MEGA 4.1(http://www.megasoftware.net/) using a neighbor-joining method, and the Tajima-Nei distance model(Tamura et al., 2007).

    Results and Discussion

    1.Symptoms of powdery mildew infections on Japanese artichokes

    From August to October 2013, fresh leaves of Japanese artichokes with powdery mildews were collected in Jinju, Korea. Infected plants exhibited white powdery spots on leaves and stems(Fig. 1). The lower leaves were the most affected, but mildew was also evident on the aboveground parts of the plant. As the disease progressed, white circularto- irregular patches consisting of epiphytic mycelia and conidia became larger and denser on leaves and petioles, and the mildew spread both up and down the entire length of the plant. When the disease developed further, whole regions of the plants were covered with fungal mats featuring white anamorphic mycelia and blackish teleomorphic structures.

    2.Morphological characteristics

    The morphological features of the powdery mildew fungus were examined and photographed under light microscopy(Carl Zeiss, Göttingen, Germany). Conidiophores, 110–150 × 10–15μm in dimensions, arose from the upper regions of hyphae and formed conidial chains with crenate edges(Fig. 1b). Conidia, 25–45 × 11–18μm in dimensions, formed chains, and were mostly oval with distinct fibrosin bodies(Fig. 1c-e). Chasmothecia, produced in autumn, were 100–150μm in diameter, subglobose, and dark brown in color(Fig. 1fh). The appendages were somewhat brownish in the basal parts of chasmothecia, and were simple, hypha-like, and interwoven with the hyphae of the surrounding mildew colony(Fig. 1j). Totals of 7–12 asci were present in each chasmothecium, and the asci were oblong, or ellipsoid-to-obpyriform, and irregular in outline, being 45–55 × 20–30μm in dimensions(Fig. 1k). These measurements and taxonomic characteristics are in full agreement with previous data on Neoerysiphe species(Braun, 1987; Liu et al., 2005; Takamatsu et al., 2008).

    3.Molecular identification

    To confirm the identity of the causal fungus, the complete nucleotide sequences of the ITS rRNA gene region, ITS1, the 5.8S rRNA gene, and ITS2, were amplified and verified. The resulting 592-bp sequence was deposited in GenBank under accession no. KP347576. The sequences were analyzed using BLAST and exhibited high-level identities to those of the powdery mildew fungus N. galeopsidis deposited in the NCBI GenBank database. Our ITS rRNA gene sequence was 99% similar to sequences from N. galeopsidis infecting Marrubium praecox in the Ukraine(Heluta et al., 2010); Chelonopsis moschata in Japan(Mori et al., 2000); and Acanthus spinosus(Cook et al., 2006b), Catalpa bignonioides, and Lamium album in the UK(Cook et al., 2006a) (GenBank accession nos. AB498943, AB022370, DQ350136, DQ359698, and DQ359697, respectively). Therefore, sequence analysis confirmed that the pathogen was N. galeopsidis.

    4.Phylogenetic analysis

    To generate a phylogenetic tree, the ITS rRNA gene regions of powdery mildew fungi were analyzed using the approach of Heluta et al.(2010). According to recent studies, the Neoerysiphe analyzed in the present study can be divided into three large clades (A–C) clearly defined on the basis of geographical distribution and host plants. Clade A has only single species, N. galeopsidis, the hosts of which are mostly among the Lamiaceae, but included the Acanthaceae and Bignoniaceae. Clades B and C are larger. Clade B hosts include Asteraceae, Verbenaceae, and Elaeocarpaceae of North and South America and Japan. Clade C hosts include Asteraceae, Geraniaceae, and Rubiaceae of Eurasia, including northern part of Israel and the southern part of the Ukraine(Takamatsu & Matsuda, 2004; Heluta et al., 2010). The 29 ITS sequences of Neoerysiphe species(Table 1), including sequences newly determined in the present study, were aligned; Arthrocladiella mougeotii served as an outgroup taxon. The 29 ITS sequences of Neoerysiphe species could be divided into three large clades(A, B, and C), consistent with the grouping of powdery mildew fungi(Takamatsu & Matsuda, 2004; Heluta et al., 2010). In the phylogenetic tree, the powdery mildew fungus isolate infecting Japanese artichokes was placed in Clade A(Fig. 2). The most comprehensive phylogenetic tree possible was constructed.

    Figure 3 compares the nucleotide sequences of ITS rRNA gene regions(18S rRNA gene, partial sequence; internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2, complete sequence; and 28S rRNA gene, partial sequence) of 11 N. galeopsidis isolates of powdery mildew fungi of Clade A. The nucleotide sequence of the ITS rDNA region was highly conserved among such fungi.

    Based on morphological characteristics and comparisons of nucleotide sequences of the ITS rRNA gene region, the causal agent of mildew was identified as N. galeopsidis. To the best of our knowledge, this is the first report of powdery mildew disease on Japanese artichokes in Korea. Powdery mildews on such plants are of potential practical importance because Japanese artichoke cultivation has increased in Korea. It is important to be aware of powdery mildew, and to manage the disease appropriately, as the condition could significantly reduce Japanese artichoke production.

    Figure

    JALS-49-163_F1.gif

    Symptoms of powdery mildew infections of Japanese artichokes and morphological characteristics of the pathogenic Neoerysiphe galeopsidis. a: Typical symptoms occurred on leaves and stems; b: conidiophore; c: conidia; d, e: germinated conidia; f: a close-up view of a leaf infection of powdery mildew showing the superficial mycelia; g: chasmothecia under reflected light; h: a chasmothecium; i: peridial cells; j: chasmothecial appendages; k: asci. Scale bars: b-e, I-k= 20μm; g= 200μm; h= 50μm.

    JALS-49-163_F2.gif

    Phylogenetic analysis of the nucleotide sequences of ITS rRNA gene regions from 29 Neoerysiphe sequences including the powdery mildew fungus infecting Japanese artichokes (bold), and Arthrocladiella (used as an outgroup taxon). The numbers above the branches indicate bootstrap values. Bars indicate the numbers of nucleotide substitutions per site. Phylogenetic analyses divided the fungi into three main clades: A, B, and C, following Heluta et al.(2010).

    JALS-49-163_F3.gif

    Comparison of the nucleotide sequences of ITS rRNA gene regions(18S rRNA gene, partial sequence; internal transcribed spacer 1, 5.8S rRNA gene, and internal transcribed spacer 2, complete sequence; and 28S rRNA gene, partial sequence) of Neoerysiphe galeopsidis of powdery mildew fungi of Clade A(GenBank accession nos.). The alignment was drawn using CLUSTAL X. Asterisks indicate positions with single, fully-conserved bases.

    Table

    Host plants, fungal species, locations of collections, and clades of Neoerysiphe material and DNA database accession numbers

    1The nucleotide sequence data will appear in the GenBank database under the respective accession number.

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