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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.53 No.3 pp.135-140
DOI : https://doi.org/10.14397/jals.2019.53.3.135

First Report of Kiwifruit Brown Leaf Spot Caused by Fusarium tricinctum in South Korea

Min-jung Kim1, Youngho Kwon2, Youn-Sig Kwak1,2,3*
1Department of Plant Medicine, Gyeongsang National University, Jinju, 52828, Korea
2Dvision of Applied Life Science (BK21plus), Gyeongsang National University, Jinju, 52828, Korea
3Institute of Agriculture & Life Science, Gyeongsang National University, Jinju, 52828, Korea
Corresponding author: Youn-Sig Kwak Tel: +82-55-772-1922 Fax: +82-55-772-1929 E-mail: kwak@gnu.ac.kr
October 17, 2018 November 23, 2018 January 2, 2019

Abstract


Kiwifruit (Actinidia chinensis Planch.) is an economically important horticultural crop. This study reports a new pathogen of brown leaf spot disease in kiwifruit. Brown leaf spot symptoms were observed on the leaves of cv. Redvita in Jeju, South Korea. The leaf spot diseases appeared as extensive, angular, brown, yellow, white or fused color spots on the leaves. Fungi were isolated and identified as Fusarium tricinctum (Corda) Sacc. based on their morphological and molecular characteristics. Pathogenicity tests were conducted with mycelium and the conidia inoculation. The pathogen was re-isolated from the artificially developed symptoms, and identified as F. tricinctum as Koch’s postulates were confirmed. To our knowledge, this is the first report of the brown leaf spot caused by F. tricinctum on kiwifruit.



초록


    Rural Development Administration
    PJ010904 Ministry for Food, Agriculture, Forestry and Fisheries
    315004-5

    Introduction

    Kiwifruit (Actinidia chinensis Planch.) is dioecious vines and separates into male and female individual (Nieuwenhuizen et al., 2010). It is widely cultivated in New-Zealand, Italy, Korea and other countries. Kiwifruit is known as ‘the king of fruits’ because of its conspicuously high vitamin C content, nutritional compositions of minerals and other health-beneficial metabolites. According to Rural Development Administration (RDA) In Korea, kiwifruit has been cultivated from 1987 and the cultivated quantity significantly increased within a short period of time from 100 tons/ year in 1987 to about 23,000 tons/year in 2011, respectively. Thereafter, breeding researches for the new cultivars are progressing actively to complement the shortcomings of existing cultivars, typically cv. Hayward or cv. Hort16A (Kwack et al., 2017). Kiwifruit is also considered as a relatively disease-free crop in many countries. However, a number of fungal and bacterial diseases have been reported (Clover et al., 2003). Generally, fungal leaf spot diseases are ‘Brown leaf blight’, ‘Grayish brown ring spot’, ‘Silvering gray leaf blight’ and ‘Dark brown ring spot’ (Koh et al., 2008). Pathogens destroy leaf tissues and necrotic areas are change in color to reddish, brown, gray or black with irregular shape of spots. Fungal pathogens, such as Alternaria alternata, Pestalotiopsis menezesiana, Glomerella cingulata, Colletotrichum sp., Diaporthe sp. (Phomopsis sp.), Phoma sp., Epicoccum sp. have been reported as leaf spot pathogens (Hawthorne et al., 1982;Jeong et al., 2008). Pathogen spores are carried by the wind or rain to young leaves in the spring and the spores penetrate leaves and, as the temperature increases, small spots appear on the leaves (Peres et al., 2005). In general, Fusarium tricinctum is known as a causal pathogen of Fusarium head blight (FHB) disease in small grain cereals, root rot disease on alfalfa (Grove et al., 1970;Nielsen et al., 2011;Cong et al., 2016). Brown leaf spot disease caused by F. tricinctum has not been reported yet. In this study, we report that F. tricinctum causes brown leaf spot disease in kiwifruit.

    Materials and Methods

    1 Disease symptom and pathogen isolation

    In May 2016, brown leaf spot diseases of kiwifruit (cv. Redvita) were observed with around 2% disease incidence at an orchard in Jeju, South Korea (33°27' 01.5"N 126°26'24.0"E). Rectangular shapes (0.5 cm× 0.5 cm) of symptomatic leaf tissues were surfacesterilized in 70% ethanol for 30 sec, then 1% sodium hypochlorite (NaOCl) for 30 sec and rinsed five times in sterilized distilled water for 1 min. Sterilized specimens were plated on Water Agar (WA: agar 20 g/L) and incubated at 27℃ for 5 days. Grown-out mycelia were transferred on Potato Dextrose Agar (PDA: potato dextrose 15 g, agar 20 g/L) and incubated at 27℃ for 7 days.

    2 Identification of the pathogen

    Among isolates, two of them were sequenced using Internal Transcribed Spacer (ITS), β-Tubulin (BT) and Transcription Elongation Factor (TEF-1α) gene. Genomic DNA was extracted using modified CTAB DNA extraction method (Porebski et al., 1997). Mixture of PCR reaction contained the following: 10x reaction buffer 2 μL, 10 mM dNTP 1 μL, 10 pmol of each primer 1 μL, 5 units of Taq polymerase, and 100 ng of genomic DNA and fill up with PCR grade H2O as final volume 20 μL. PCR primers and programs were standardized as Table 1 (Bakan et al., 2002;Bellemain et al., 2010;Arif et al., 2012) and PCR was carried out by a gradient thermal cycler (Bibby Scientific Ltd, Staffordshire, U.K.). The amplicons were purified using AccuPrep PCR purification kit (Bioneer, Korea) as manufacturer’s protocol. The purified PCR amplicons were sequenced at Solgent (Daegeon, South Korea). Morphologically characteristics were observed under a microscopy (Olympus, German). Hypha was scratched with 1 μL of sterile water, put on the slide glass and coated with the glass coverslip. Spore shapes were identified under the microscope at a magnification of 400 x.

    3 Pathogenicity test

    The pathogenicity test was conducted in two independent methods with 12-week-old wounded and non-wounded leaves of kiwifruit cv. Redvita (Kwon et al., 2016). The first pathogenicity test was performed with 6-mm diameter mycelial plugs of the isolated fungus and the second test was performed with 10 μL of macroconidial suspension (6.2 x105 conidia/mL) of the fungus cultivated on Tryptone Yeast Extract Agar (TYA: tryptone 6 g, yeast extract 3 g, agar 10 g/L, pH 7.2±0.2) (Turner et al., 1988). All the leaves were stabbed 5 times at five points using a syringe and the pathogenesis tests performed as three replications. Untreated leaves were inoculated mock with sterile PDA plugs for plug inoculation and 10 μL of sterile water for macroconidia inoculation. Each treatment was then sealed to maintain the high humidity chamber and incubated at room temperature under the laboratory conditions (Jia et al., 2003). Disease developments were monitored daily and the fungus was re-isolated from the diseased lesions to confirm Koch’s postulates.

    Results and Discussion

    In May 2016, brown leaf spot disease was observed on kiwifruit (cv. Redvita) with 1% incidence in Jeju, South Korea (33°27'01.5"N 126°26'24.0"E). The isolated fungi produced dense reddish mycelium with white peripheries on PDA plate (Fig. 1A). Total 12 fungal isolates were isolated and the fungi produced dense reddish mycelium with white peripheries on PDA plate (Fig. 1B). Morphological characteristics were observed under a microscopy (Olympus, German). Macroconidial suspension (10 μL) was put on the slide glass and coated with the coverslip. Macroconidia were thick, bluntly pointed and fusiform after two weeks of incubation. Their size ranged from 3 to 5 μm wide and from 20 to 40 μm long and the number of septa ranged usually one or three in number (Fig. 1C). Macroconidia morphological characteristics were consistent with F. tricinctum as Table 2 (Harron et al., 2010; Cong et al., 2016).

    ITS sequences showed 99% similarity with Fusarium tricinctum (GenBank Accession No. KY458788, KU 350730 and EF311092). β-Tubulin sequences also showed 98% similarity with F. tricinctum (GenBank Accession No. AF405451 and AF405457). The TEF-1 α sequences showed no matched sequences in the NCBI. Sequences of two isolates were deposited in GenBank (Accession No. MF322526 for ITS and MH447985 for β-Tubulin). Phylogenetic trees also showed the isolates were grouped closely with F. tricinctum (Fig. 2). Among the isolates, the type specimen F. tricinctum was deposited in KACC (#48295). F. tricinctum has been reported as resulting the Fusarium head blight (FHB) in small grain cereals and the cattle disease producing the mycotoxin. Otherwise, Fusarium spp. such as F. oxysporum and F. solani are generally soil-borne pathogens are causing wilt, root rot and replant disease (Mazzola & Manici, 2012).

    Disease symptoms appeared as brown angular spots by artificial inoculation and average the lesion diameter was approximately 5-10 mm. The leaf lesions were confirmed not on mock-inoculated leaves and nonwounded leaves but on wounded leaves (Fig. 1D, E, F, G). Re-isolated fungi were also showed identically morphological and molecular characteristics as the parent isolates. The pathogens from the brown leaf spot disease symptoms were identified using the ITS and β-tubulin sequencing data in the same way to fulfill the Koch’s postulates. Therefore, F. tricinctum clearly infects the leaf through wound and it can cause the brown leaf spot disease in kiwifruit.

    To our knowledge, kiwifruit brown leaf spot disease caused by F. tricinctum is the first report worldwide.

    Acknowledgment

    This research was supported by the ‘Cooperative Research Program for Agriculture Science & Technology Development (PJ010904)’ from the RDA and by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (Project No. 315004-5).

    Figure

    JALS-53-3-135_F1.gif

    Brown leaf spot disease symptoms on kiwifruit leaves and morphological characteristics of Fusarium tricinctum. (A), Disease symptom of brown leaf spot on kiwifruit; (B), colony morphology of F. tricinctum on PDA plate; (C), macroconidia of F. tricinctum. Bar indicates 10 μm.; Disease symptoms by F. tricinctum on kiwifruit leaves as mock inoculation (D), non-wounded inoculation (E), wounded spore inoculation (F) and wounded plug inoculation (G). Bar indicates 5 cm.

    JALS-53-3-135_F2.gif

    Phylogenetic identification of Fusarium tricinctum funbio 30 and 31 based on ITS (A) and β-tubulin (B). The bootstrap values calculated from 1,000 replicates and the evolutionary analyses were conducted using the Neighbor-Joining method in MEGA7. Botrytis cinerea and Coniophora puteana which were arbitrarily selected as out-grouped.

    Table

    PCR primers used in this study

    Comparison of morphological characteristics of the isolates from kiwifruit with described Fusarium tricinctum

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