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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.50 No.4 pp.249-253
DOI : https://doi.org/10.14397/jals.2016.50.4.249

Alternaria solani Causing Leaf Blight Disease on Aster glehni in Korea

Chang Wook Jeon1, Sung Woon Hong1, Hyunji Cho1, Youn-sig Kwak2*
1Division of Applied Life Science, Gyeongsang National University, Jinju, 52828, Korea
2Department of Plant Medicine, Institute of Agriculture & Life Science, Gyeonsang National University, Jinju, 52828, Korea
Corresponding author: Youn-sig Kwak +82-55-772-1922+82-55-772-1929kwak@gnu.ac.kr
May 3, 2016 May 23, 2016 May 30, 2016

Abstract

Aster glehni Franchet et Schmidt is a compositae plant and has been known as a native specie in Ulleung Island, Korea. It is officially recognized as a regional specialty that grows only in this region. In 2014, brown and dark spots were observed on aster leaves in a forest research field, Jinju, Korea. A causal agent was isolated from the disease symptomatic leaves and identified as fungus Alternaria solani. Fungal morphological characteristics and molecular identification with internal transcribed spacer sequences were synchronized as A. solani. The isolated fungi reproduced the same disease symptoms when the fungus was artificially inoculated on healthy aster leaves. This is the first report that A. solani caused leaf blight disease in Aster glehni in Korea.


초록


    Rural Development Administration
    PJ011800

    Introduction

    Aster glehni Franchet et Schmidt(AG) is recognized as a regional specialty that grows only in Ulleung island in Korea. AG has been used as alternative herbal medicine to treat diabetes mellitus, hypercholesterolemia, insomnia, and cardiovascular disease(Lee et al., 2013). AG grows to a height of 1-1.5m with hairs around stem. The leaves of AG, which has long petiole, are arranged in rough and sharp saw-toothed appearance regularly. Many down feathers on both side of the leaves of the AG is similar to sesame leaf shape as Homachae. Each year demand of AG is increasing and supply is not able to follow the demand with various reason such as reduce of rural labor. Open field cultured AG has higher quantity than in green-house cultivated AG. AG has strong adaptability in low-temperature for winter production, which is possible to harvest year around(Lee & Chung, 1998).

    Alternaria spp. have broad host range and commonly infect aerial parts of various host plants. In the leaf, symptoms of Alternaria infection typically start as a small, circular, brown and dark spots development(Agrios, 2006). The infection spot may grow up to 1cm or even bigger diameter and in the infection spots are usually developing gray, gray-tan, or near black in color during the disease progresses. The fungus may sporulate in these disease symptom sites, causing affine, black, velvety growth of fungus and spores to cover the affected area(Laemmlen, 2001).

    Materials & Methods

    Leaf blight disease symptome on aster plants were founded at Southern Forest Resource Research Center in Gyeongsangnam province of Korea on 20 May and 25 May 2014. The disease AG plant were showed typically symptoms such as dark brown spots on the leaves and wilted. This symptom was observed approximately 28% on cultivated area. Infected AG was transported immediately to the laboratory maintaining fresh materials.

    To isolate putative pathogens of the disease, the surface of infected leaves were soaked in 70% ethanol for 30s - 45s and put in sodium hypochlorite for 45s. The infected leaves washed in distilled water for five times to remove excessive moisture, then the leaves were put in a fume hood for 15min to dry. The dried pieces of leaves were placed on water agar(WA) and incubated at at 28°C for 4 days. To propagate fungi, hyphae on WA were transferred with a needle and incubated on potato dextrose agar(PDA) at 28°C for 7 days. The fungi color grown on PDA were dark brown to black and conidia spores developed on the media. Morphological characteristics of conidia were observed to identify and to describe the pathogen under a microscope(BX51, Olympus, Germany).

    Genomic DNA extracted from hypha of the fungi, which grown on PDA media, by CTAB method. Internal transcribed spacer(ITS) in rDNA region was amplified with ITS1(5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4(5’-TCCTCCGCTTATTGATATGC-3’) primers according to the method of White et al.(1999). Total 20ul volume consist of 2ul 10x reaction buffers, 100ng of genomic DNA, 200uM dNTP and 10pmol each primer and 0.5U Taq polymerase. The PCR condition were as follows on 94°C for 4min, 35 cycles of 94°C for 30sec, 50°C for 30sec, 72°C for 1min and a cycle of 72°C for 10min. The sequences of ITS-5.8S rDNA were deposited and blasted in the GenBank database. Molecular identification and phylogenetic analysis were performed with neighbour-joining method and Tajima–Nei distance algorithms in MEGA4 program(Tamura et al., 2007).

    Pathogenicity was tested to fulfill Koch’s postulates in laboratory condition. Incubating the causal agents on PDA at 28°C for 7days was cut by sterile a 5-mm cork borer and inoculated on to the fresh AG leaves then the leaves were incubated in sterilized humid containers at room temperature. At 25 days after treatment, the artificially inoculated leaves reproduced same symptoms, which were observed in the field. And the fungi were re-isolated based on these symptoms to compare with the original isolates.

    Results and Discussion

    In May 2014, leaves of aster showed severe blight disease symptoms and dried at Southern Forest Resource Research Center in Korea(Fig. 1c). The causal agents were isolated and maintained on PDA media at 28°C. The morphological characteristics of the fungi colonies of PDA from were spreading, hairy and branched. Initial fungi color was dark green to black and conidia spores developed on the media(Fig. 1a). The conidiophores were olive brown color in septate, short, simple, erect, flexuous form. The conidia consist of 9 to 11 transverse septa and 2 to 3 longitudinal septa with dark and muriform(Fig. 1b). Branched ellipsoid spores became to oblong with a long beak occasionally. All observed morphological characteristics were similar to Alternaria solani (Laemmlen, 2001).

    The PCR amplicon of rDNA of the causal pathogens generated 500bp product(Accession no. KP052778). The sequence data was 100% equal to A. solani in GenBank blast. In the phylogenetic analysis of other Alternaria species, the isolate was coincided with A. solani in monophyletic clade with bootstrap value(Fig. 2). Phylogenetic analysis the isolate showed the blight disease pathogen in AG plant identical to previously reported A. solani.

    Pathogenicity test performed at room temperature with saturated humid condition. The symptoms typically were wilt, circular dark brown on the AG leaf surface. And the infected symptoms on AG leaf were enlarged. After 14 days, the spots were small irregular to circular dark brown spots on leaf and enlarged spots were narrow yellow halo around each spot. Irregular lesions were coalesced with 3-5 spot to the infect leaf differ from non-inoculated leaf(Fig. 3).

    Overall, the symptoms of pathogenicity test were same as the initial appearance. Both morphological characteristic and molecular data of the isolates showed 100% identity with A. solani. To our knowledge, this is the first report of A. solani causing leaf blight on Aster in Republic of Korea.

    Acknowledgement

    This research was supported by the Next- Generation Bio Green 21 Program(PJ011800) in Korea.

    Figure

    JALS-50-4-249_F1.gif

    Morphological characteristics and the disease symptoms of Alternaria solani.

    (a) A. solani incubated on PDA for 7 days, (b) conidia form(scale bars= 50μm), (c) symptom of the blight disease in field.

    JALS-50-4-249_F2.gif

    Phylogenetic identification of Alternaria solani(KP052778) with ITS sequences.

    The numerical value on each branches represents the bootstrap values using 1000 replication to calculate the percentage. MEGA4 program inferred the phylogeny and neighbor-joining algorism calculated the phylogenetic distances. Fusarium solani and Candidatus phytoplasma served as the outrange group of fungi.

    JALS-50-4-249_F3.gif

    Symptoms of the leaf blight disease, which developed by artificial inoculation of Alternaria solani on aster leaf.

    (a) at 0 day, (b) symptom 25 days after treatment. (i) untreated, (ii) wound inoculated, (iii) inoculated without wound.

    Table

    Reference

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