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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.49 No.5 pp.47-56
DOI : https://doi.org/10.14397/jals.2015.49.5.47

Characterization of Powdery Mildew Caused by Podosphaera fusca Infecting Melothria japonica(Thunb.) Maxim

Jin-Hyeuk Kwon1, Hyeong-Jin Jee2, Jinwoo Kim3,4*
1Gyeongsangnam-do Agricultural Research and Extension Services, Jinju 52733, Korea
2Organic Agriculture Division, National Academy of Agricultural Science, Rural Development Administration, Wanju-gun 55365, Korea
3Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Korea
4Institute 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 October 11, 2015 October 11, 2015

Abstract

From August to October of both 2012 and 2013, powdery mildew was observed on white-fruited creeping cucumber(Melothria japonica [Thunb.] Maxim.) at Jinju, Korea. White mycelial colonies were observed on leaves and petioles. Lesions of severe infections were discolored or brown. In the present study, the morphological features of the anamorphic and teleomorphic reproductive stages of the fungus were studied. To complete the fungal identification, the sequence of the internal transcribed spacer region of a ribosomal RNA(ITS rRNA) gene was determined. Based on morphological characteristics and analysis of the ITS rRNA gene, the fungus causing the powdery mildew symptoms was identified as Podosphaera fusca. Although the host range of powdery mildew caused by P. fusca has been previously described, no full description and illustration of the infection, including symptoms and signs on white-fruited creeping cucumber, has yet appeared. To the best of our knowledge, this is the first full description of powdery mildew on white-fruited creeping cucumber, featuring molecular identification, symptoms, and signs.


초록


    Rural Development Administration
    No.PJ01082304

    Introduction

    Powdery mildew growth is dependent on weather and the fungus spreads under conditions of elevated temperature and low humidity(Amano, 1986; Siebold & Tiedemann, 2012). From August to October, in both 2012 and 2013, powdery mildew caused by a Podosphaera species, with symptoms evident on the leaves of white-fruited creeping cucumber(Melothria japonica [Thunb.] Maxim.), was noted in Jinju, Korea. White-fruited creeping cucumber belongs to the Cucurbitaceae family and is regarded as a weed because economical importances are unveiled. Powdery mildew on weeds has potentially significant implications, as weed can function as alternative plant hosts for pathogens of economically important crops (Clarke & Akhkha, 2002).

    Powdery mildew fungi infect 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 via the haustoria(Huckelhoven, 2005). Podosphaera fusca (or xanthii) (synonym Sphaerotheca fusca [or xanthii]), known as the causal agent of powdery mildew on various cucurbit plants, is one of the most important limiting factors for cucurbit production worldwide(Perez-Garcia et al., 2009; Kousik et al., 2011). The genus Podosphaera belongs to the Erysiphaceae order(Braun & Takamatsu, 2000).

    White-fruited creeping cucumber is an annual herb native to western Asia. The herb grows in wet places, such as riverbeds, and climbs via tendrils. The small white flowers bloom from August to September. The fruits(which are inedible) are globose(about 1cm across), initially bright green, and turn pale greyish- green at maturity. Powdery mildew has been frequently observed on white-fruited creeping cucumber plants in the Jinju areas. Powdery mildew on such plants was previously observed in Korea(Shin, 2000). However, a full description(with illustrations); data on symptoms, signs, and teleomorphic features; detailed molecular identification; and phylogenetic analysis of the fungal species, are lacking.

    In the present study, we describe the morphological features of P. fusca at the anamorphic and teleomorphic reproductive stages. We also sequenced the internal transcribed spacer region of the ribosomal RNA(ITS rRNA) gene of the powdery mildew fungus infecting white-fruited creeping cucumber plants. Based on the morphological characteristics and analysis of the ITS rRNA gene sequence, the fungus associated with the observed symptoms and signs was identified as P. fusca. To the best of our knowledge, this is the first full description of powdery mildew on white-fruited creeping cucumber plants, featuring molecular identification, symptoms, and signs. We believe that this information will assist mycologists, plant pathologists, weed scientists and others in assessing the biogeography and ecology of weeds and pathogens.

    Materials and Methods

    1.Sample sources

    Conidial and cleistothecial specimens of powdery mildew were collected in Jinju, Korea, from 2012 to 2013. Fungal DNA was extracted from fresh, infected plant material. The remaining specimens were dried and stored at the Mycological Herbarium of Gyeonsang National University(MHGNU).

    2.Morphological characteristics

    Detailed microscopic examination of sample specimens of the causal fungus was performed under a light microscope(Axioplan; Carl Zeiss, Jena, Germany; 400×magnification). The fungal species was identified using the morphograph of Shin(2000).

    3.DNA extraction and PCR amplification

    Fungal DNA extraction and PCR amplification were performed using a Phire® Plant Direct PCR Kit (Finnzymes, Espoo, Finland) following the manufacturer’s instructions. Briefly, some hundreds of conidia were added to 50μL of Dilution Buffer(Finnzymes) in a 1.5-mL microcentrifuge tube and incubated at room temperature for 30min. The extracts were mixed vigorously, and centrifuged at 10,000g for 1min. Halfmicroliter amounts of supernatants served as templates for polymerase chain reaction(PCR).

    To amplify the nuclear rDNA region spanning the ITS1, ITS2, and 5.8S RNA gene, primers ITS1(5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) were used (White et al., 1990). PCR amplification was performed in 20 μL reaction volumes, following the manufacturer’s instructions, 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 fill 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 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.

    4.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. Clones 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 transformed colonies were extracted and purified using a plasmid extraction kit(GeneAll) following the manufacturer’s instructions, cut with EcoRI(TaKaRa, Tokyo, Japan), and the expected size validated by electrophoresis and ethidium bromide staining. One clone(pOR166) containing an insert of the expected size was isolated and the inserted DNA sequenced in both directions using M13F and M13R primers, by Macrogen Services(Daejeon, South Korea). The resulting 563-bp ITS rRNA gene sequence was deposited in GenBank(Accession no. KP329589).

    5.Phylogenetic analysis and molecular identification

    The nucleotide sequence of the ITS rDNA region from the powdery mildew fungus was compared with reported reference sequences using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm. nih.gov/). Multiple sequence alignment of ITS rDNA regions was conducted using ClustalW to analyze single- nucleotide polymorphisms. Phylogenetic analysis was performed using MEGA 4.1 (http://www.megasoftware.net/) software, which ran neighbor-joining analysis and computed Tajima-Nei pairwise distances(Tamura et al., 2007). A phylogenetic tree was drawn to scale, with the branch lengths in the same units used to draw the evolutionary distances employed to infer the tree.

    Results and Discussion

    1.Symptom and morphological characteristics

    From August to October, in both 2012 and 2013, fresh leaves of white-fruited creeping cucumber exhibiting powdery mildew symptoms were collected at Jinju, Korea. The symptoms were observed on plants growing in shaded areas, but not full sunlight. Disease symptoms included greyish-white circular- to-irregular patches, consisting of epiphytic mycelia and conidia, on both leaves and petioles(Fig. 1).

    As the disease progressed, leaves became covered by a powdery fungal mass(Fig. 2A), and older leaves became necrotic. When the disease was well-advanced, whole regions of plants were covered with fungal mats containing white anamorphic mycelia and blackish teleomorphic structures.

    Freshly collected fungal material was examined using standard light microscopy(Carl Zeiss, Göttingen, Germany). Conidia 24–35 × 15–20μm in diameter were present in chains, and were mostly oval, with distinct fibrosin bodies(Fig. 2B). Conidiophores 100– 250 × 10–15μm in dimensions were produced by the upper regions of hyphae and developed into conidial chains with crenate edges(Fig. 2C-D).

    Chasmothecia, produced in the autumn, were 80– 120μm in diameter, subglobose, and dark brown(Fig. 3AC). The appendages were brown at the base and paler toward the top, simple, hypha-like, and interwoven with hyphae of the surrounding mildew colony( Fig. 3D). A single thick-walled ascus was contained in a chasmothecium(Fig. 3E). An ascus contained eight ascospores, which were ellipsoidal-to-oval and 20–30 × 15–20μm in dimensions(Fig. 3E, F). These measurements and taxonomic features fit with those previously described for P. fusca(syn. P. xanthii; Braun, 1987; Shin, 2000).

    2.Molecular identification and phylogenetic analysis

    The resulting 563-bp sequence was deposited in GenBank under accession no. KP329589. The sequence was analyzed via BLAST and was similar to sequences of the powdery mildew fungus P. fusca, previously deposited in the NCBI GenBank database. A comparison of the ITS rDNA sequence revealed 100% similarity with sequences from P. fusca infecting cucurbit plants(GenBank accession nos. KJ698669 and AB774158). Therefore, sequence analysis verified the pathogen as P. fusca.

    To generate a phylogenetic tree of the ITS rRNA gene region, the sequences of ITS1, the 5.8S rRNA gene, and ITS2 of the powdery mildew fungus were analyzed as described by Takamatsu et al.(2010). Podosphaera species of the powdery mildew fungi have been previously split into two major clades: Clade 1, consisting of the Podosphaera that grow on Prunus, and subsection Magnicellulatae; and Clade 2, containing the other members of the section Podosphaera, based on sequence analysis of the ITS rRNA gene region(Takamatsu & Matsuda, 2004; Takamatsu et al., 2010).

    A total of 26 sequences, including the sequences reported in the present study, was used in our current analysis(Table 1). Cystotheca wrightii served as the outlying group, based on the data of Mori et al.(2000). The 26 ITS sequences of Podosphaera species were divided into two large clades, Clade 1 and Clade 2(Fig. 4). Podosphaera spp. growing on Prunus spp. are placed in Clade 1 and the other members of Podosphaera in Clade 2, consistent with the recognized grouping of powdery mildew fungi( Takamatsu & Matsuda, 2004; Takamatsu et al., 2008 & 2010). In the phylogenetic tree, the powdery mildew fungus infecting white-fruited creeping cucumber was placed in Clade 2, which included the powdery mildew fungus infecting cucurbit plants(Fig. 4). A comprehensive phylogenetic tree was thus constructed.

    Figure 5 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 6 P. fusca isolates of powdery mildew fungi that infect cucurbit plants in Clade 2. The nucleotide sequence of the ITS rRNA gene region was highly conserved among such fungi.

    On the basis of mycological characteristics and a comparison of ITS rDNA sequences, the causal agent of the powdery mildew was identified as P. fusca. In Korea, several cucurbitaceous hosts, including Citullus, Cucumis, Cucurbita, Lagenaria, Melothria, and Thladiantha, are infected by P. fusca(Shin, 2000; The Korean Society of Plant Pathology, 2009; Farr & Rossman, 2014). Powdery mildew on the white-fruited creeping cucumber is of potential economic importance, as the plants can function as alternative hosts for powdery mildew attacking cucurbit plants. Detection of these powdery mildew associated with diseases of weed hosts is very important to check the possibility of further spread of powdery mildew diseases to other commercial crops(Lee, 2012). Our results will assist mycologists, plant pathologists, weed scientists, and cucurbit crop growers in assessing the biogeography and ecology of weeds and pathogens.

    Figure

    JALS-49-47_F1.gif

    Powdery mildew infections of white-fruited creeping cucumber in nature, caused by Podosphaera fusca.

    JALS-49-47_F2.gif

    Anamorphic features of Podosphaera fusca on the leaves of white-fruited creeping cucumber. A: A close-up view of a leaf infection by powdery mildew; white superficial mycelia are apparent; B: conidia (bar=20μm); C-D: conidiophore(bar=20μm).

    JALS-49-47_F3.gif

    Teleomorphic features of Podosphaera fusca on the leaf of white-fruited creeping cucumber. A: Colonies of powdery mildew with chasmothecia; B: a close-up view of chasmothecia on infected leaf; C: chasmothecium in transmitted light(bar=50μm); D: chasmothecium with appendages(bar=50μm); E: chasmothecium with ascus(bar=50μm); F: ascus with ascospores(bar=20μm).

    JALS-49-47_F4.gif

    Phylogenetic analysis of 26 nucleotide sequences of the ITS rRNA gene region from Podosphaera, including the powdery mildew fungus infecting white-fruited creeping cucumber(bold), and Cystotheca(outlier taxon). The numbers above the branches indicate bootstrap values. Bars indicate numbers of nucleotide substitutions per site. The phylogenetic analyses identified the Clade 1 and 2 of Takamatsu et al.(2010).

    JALS-49-47_F5.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 Podosphaera fusca of powdery mildew fungi that infect cucurbit plants(GenBank accession nos.). The alignment was drawn using CLUSTAL X. Asterisks indicate positions with single, fully-conserved bases.

    Table

    Sources of Podosphaera material sequenced in this study, and DNA database accession numbers

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

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