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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.48 No.4 pp.27-34
DOI : https://doi.org/10.14397/jals.2014.48.4.27

Rhizopus fruit Rot Caused by Rhizopus oryzae on Strawberry

Jin-Hyeuk Kwon1, Dong-Wan Kang1, Hae-Suk Yoon1, Youn-Sig Kwak2, Jinwoo Kim3*
11Gyeongsangnam-do Agricultural Research and Extension Services, Jinju 660-360, Korea
2Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, Korea
3Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Korea
Corresponding Author: Jinwoo Kim, Tel: +82-55-772-1927, Fax: +82-55-772-1929, E-mail: jinwoo@gnu.ac.kr
April 1, 2014 July 16, 2014 August 4, 2014

Abstract

Rhizopus fruit rot was observed on strawberry fruit (Fragaria x ananassa Duchesne) grown in a glass greenhouse at the Gyeongsangnam-do Agricultural Research and Extension Services, Jinju, South Korea, from 2011 to 2013. Symptoms included water-soaking, rapid softening, and rotting. When these symptoms were left untreated, vigorous fungal growth was observed on the surface of infected fruits. Colonies on potato dextrose agar (PDA) at 30°C were white-cottony to brownish-black. Sporangia were globose, black, and 40–210 μm in diameter. Sporangiophores were light-brown and 6–22 μm in diameter. Sporangiospores were globose to oval, brownish, streaked, and 4–12 μm in length. Columellae were light brownish gray, hemispherical, and 80– 120 μm in diameter. To confirm the identity of the causal fungal pathogen, the complete internal transcribed spacer (ITS) ribosomal RNA gene region was amplified and sequenced. Based on these symptoms, mycological characteristics, pathogenicity tests on host plants, and molecular identification, the fungus was identified as Rhizopus oryzae Went & Prinsen Geerligs. This is the first report of Rhizopus fruit rot on strawberry caused by R. oryzae in Korea.


초록


    Rural Development Administration
    No.PJ009192

    I.INTRODUCTION

    Strawberry (Fragaria x ananassa Duchesne) is cultivated worldwide and consumed fresh or as prepared foods such as jam, juice, pies, ice creams, and milk shakes. World strawberry production has gradually increased from 2006 (FAOSTAT, 2014), and South Korea produced 179,000 tons on 6,435 ha in 2012 (KOSIS, 2014).

    Diseases are an important limiting factor in strawberry production and failure to maintain a high level of disease control can cause yield losses and reduce strawberry marketability. Unfortunately, many fungal diseases, including anthracnose, gray mold, powdery mildew, wilt, and angular leaf spot, infect strawberry plants and affect their quality and quantity. These diseases will become more serious in the future. In South Korea, four viruses are known to cause mottle and mosaic symptoms, and 29 fungal species are known to cause fungal infection on leaves, crown, roots, and fruit. The leaves can be infected by angular leaf spot caused by the bacterium Xanthomonas fragariae. In addition, seven nematodes are known to cause root lesions on strawberry (The Korean Society of Plant Pathology, 2009).

    These diseases are most damaging to strawberries during wet weather, when the diseased fruit are covered with a gray mass of spores. The disease is very difficult to control in wet seasons, particularly when plants are matted together. Careful watering of strawberries is required to the roots, and not the leaves, because moisture on the leaves encourages bacterial and fungal growth.

    From March to April of 2011 and 2013, a new disease suspected as Rhizopus fruit rot was observed on strawberry fruit grown in a glass greenhouse at the Gyeongsangnam-do Agricultural Research and Extension Services, Jinju, South Korea. Since R. stolonifer (syn. Rhizopus nigricans Ehrenb.) is known to cause fruit rot of strawberry fruit in South Korea (The Korean Society of Plant Pathology, 2009; Kwon et al., 2009), the identity of the causal fungal isolates had to be confirmed. The objectives of this study were to isolate the causal fungal agent of common symptoms on strawberry fruit; identify fungal isolates by studying pathogenicity, morphological, cultural, and molecular characteristics; and examine temperature growth of the fungal pathogens.

    II.Materials and Methods

    2.1.Fungal isolation and microscopic characterization

    Samples of diseased strawberry fruit were sent to the laboratory for isolation. Isolation was performed as described previously (Kwon et al., 2010; Kwon et al., 2014) by transferring pieces of diseased tissues, which were surface-disinfection with 1% sodium hypochlorite solution for 1 min, rinsed with sterilized distilled water and then air-dried. The dried samples were placed on water agar (WA) and incubated at 25°C for 2 days. Mycelial tips of the fungal isolates grown on WA were transferred to fresh potato dextrose agar (PDA).

    Freshly grown fungus was examined microscopically. Detailed microscopic examinations of a representative specimen were performed under a light microscope (Axioplan, Carl Zeiss, Jena, Germany).

    2.2.Temperature growth studies

    The mycelium growth rate of all fungal isolates was determined by propagation on PDA at different temperatures (30, 35, and 40°C).

    2.3.Pathogenicity tests

    The pathogenicity of a representative isolate (ROFA-01) was tested on strawberry fruit purchased from the Agricultural Products Wholesale Market in Jinju, which were surface-disinfected with 1% sodium hypochlorite solution for 1 min, washed three times with sterilized distilled water, and dried at room temperature. The strawberry fruit were inoculated by applying 50 μl of the spore suspension (2 × 104 conidia/ml, obtained on PDA and diluted with sterilized distilled water; SDW). The inoculated fruit were placed in a plastic box (29 × 22 × 15 cm) with a lid to prevent the inoculum from drying out at 25°C for 24 h. Next, the plastic box containing the inoculated fruit was placed on a laboratory table at room temperature for an additional 3 days. Ten strawberry fruit were used, and the same number of control fruit was treated with SDW.

    2.4.DNA extraction and PCR amplification

    Total DNA of the fungus was extracted using the Exgene Plant-Fungal SV Mini Kit (Geneall Biotechnology Co., Seoul, South Korea), following the manufacturer’s instructions. To amplify the nuclear rDNA region spanning internal transcribed spacer (ITS)1, ITS2, and the 5.8S RNA gene, primers ITS1 (5’-TCC GTA GGT GAA CCT GCG G-3’) and ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) were used (White et al., 1990). The polymerase chain reaction (PCR) mixture contained 5 units of Taq polymerase (TaKaRa, Tokyo, Japan), 1× PCR buffer, 0.2 mM of each dNTP, 10 nM of each primer, and 5 μl of diluted fungal genomic DNA with the total volume adjusted to 50 μl with sterile water. The following thermal cycling conditions were performed in an Astec PC 802 (Astec, Fukuoka, Japan): an initial denaturing step at 98°C for 2 min, followed by 30 cycles of 30 s at 98°C, 30 s at 55°C for annealing, and 30 s at 70°C for extension, with a final extension cycle of 4 min at 72°C.

    2.5.Cloning and sequencing of the ITS rDNA region

    The PCR products were separated by electrophoresis on a 0.8% agarose gel in 1× TBE buffer at 100 V for 20 min. The PCR amplification yielded only a single visible DNA product, whose product band was excised from the ethidium bromide (EtBr)-stained gel and purified using a gel extraction kit (Geneall Biotechnology Co.) following the manufacturer’s instructions. 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 strain by heat shock. The transformed cells were selected on a Luria– Bertani (LB) agar plate supplemented with 50 μg/ml ampicillin and 25 μg/ml 5-bromo-4-chloro-3-indolyl-β -D-galactopyranoside (X-gal, Duchefa Direct, St. Louis, MO, USA). Plasmid DNA from white transformed colonies were extracted and purified using a plasmid extraction kit (Geneall Biotechnology Co.) and checked for the expected insert size by EcoRI (TaKaRa) digestion and visualization using gel electrophoresis and EtBr staining. Resulting plasmid clones (pJW108 of isolate ROFA-01 and pOR14 of isolate ROFA-02) that contained an insert of the expected size were isolated, and the insert was sequenced in both directions using the primers M13F and M13R at Macrogen Services (Daejeon, South Korea).

    2.6.Phylogenetic analysis and molecular identification

    The nucleotide sequence of the ITS rDNA region obtained from the Rhizopus fungus was compared wit h those of reported ITS sequences in the NCBI nucle otide database (Abe et al., 2006). Previously publishe d ITS sequences from R. oryzae strains were include d for reference, and Rhizomucor miehei CBS209.77A (GenBank Accession No. JN206322) was used as an outgroup. The sequences were also analyzed using the Basic Local Alignment Search Tool (BLAST) progra m (http://www.ncbi.nlm.nih.gov/). Multiple sequence al ignment of the ITS rDNA region was performed usin g ClustalW software. Phylogenetic analysis was perfor med using MEGA4.1 (http://www.megasoftware.net/) with the neighbor-joining method and Tajima–Nei dist ance model (Tamura et al., 2007). The tree was dra wn to scale, with branch lengths in the same units as the evolutionary distances used to infer the phylogene tic tree.

    III.Results and Discussion

    Fruit rot caused by Rhizopus spp. on the succulent tissues of vegetables, fruits, and ornamentals occurs globally. Wounded mature fruit are often affected, but undamaged or immature fruit are not attacked. The disease occurs during cultivation, packing, transport, storage, and retail display on market shelves (Agrios, 2005).

    Rhizopus fruit rot was reported as a postharvest pathogen in strawberry (Kwon et al., 2009). However, occurrence of pre-harvest disease caused by Rhizopus spp. has not been reported. From 2011 to 2013, a new disease suspected as Rhizopus fruit rot was observed on strawberry fruit grown in a glass greenhouse at the Gyeongsangnam-do Agricultural Research and Extension Services, Jinju, South Korea. Here, we report fruit rot symptoms on strawberry fruit. The infected parts of the mature strawberry fruit appeared water-soaked at first, and then softened and rotted rapidly. White mycelia grew from the primary infection site and gradually covered the fruit with tufted whisker-like gray sporangiophores and sporangia (Fig. 1A). The infected tissues disintegrated into a watery rot. The infection usually started from wounds that occurred during the latter term of growth and development.

    Ten fungal isolates were successfully isolated from the diseased strawberry fruit and a representative culture (ROFA-01) of the fungal isolates was deposited with the Korean Agricultural Culture Collection (KACC 45813), National Academy of Agricultural Science, Rural Development Administration, Suwon, South Korea. To fulfill Koch’s postulates, the representative isolate KACC 45813 was used for pathogenicity testing. The typical symptoms appeared 3 days after inoculation. The infection usually started from the inoculated sites of the strawberry fruit (Fig. 1B), and symptoms were identical with those of the naturally occurring disease. Morphological characteristics of the re-isolated fungus from the inoculated fruits were the same as those from the original isolate. Fruit rot on strawberry caused by R. oryzae has not been previously reported in Korea (The Korean Society of Plant Pathology, 2009; Farr & Rossman, 2014).

    Sporangia and sporangiophores were observed under a light microscope (Table 1). Mycelial growth was measured 30 h after inoculation on PDA. The colonies of fungus grown on PDA were initially white and cottony, and became heavily speckled with sporangia and finally became brownish-gray to blackish-gray and spread rapidly with stolons extending at various points to the substrate from rhizoids (Fig. 2A). Sporangiophores were usually straight, 6–22 μm in diameter, smooth-walled, simple or branched, non-septate, long, and arose from stolons opposite rhizoids in groups of 3–5 or more. Sporangia were globose, white at first and then turned black with many spores, and were mostly 40– 210 μm in diameter (Fig. 2B). Columellae were globose to subglobose in shape, pale-brown in color, and mostly 80–120 μm in diameter (Fig. 2C). Sporangiospores were unequal, numerous, irregular, subglobose or oval, angular with striations, and 4–12 μm in length (Fig. 2D). Rhizoids and stolons were dark-brown (Fig. 2E) and zygospores were not measured. These measurements and taxonomic characters coincided with those of R. oryzae (Lunn, 1977).

    Temperature growth studies are important to distinguish between the two Rhizopus species: R. stolonifer (syn. R. nigricans) grows at 30°C, but not at 37°C, whereas R. oryzae grows at 40°C (Schipper & Stalpers, 2003). The optimum temperature for mycelial growth was 30°C and growth was still apparent at 37°C.

    Due to the high economic value of the strawberry industry, molecular identification of this progressing fungal disease is important. To confirm the identity of the fungal isolate, the complete ITS rRNA gene region of the two isolates including ROFA-01 (KACC 45813) and ROFA-02 was amplified and sequenced. The resulting 626-bp ITS rRNA gene sequences were deposited in GenBank (Accession Nos. KJ636462 of ROFA-01 and KJ636463 of ROFA-02). The resulting sequences were analyzed using BLAST and exactly matched sequences of R. oryzae strain Bb7 (GenBank Accession No. JQ991619) isolated from China. The nucleotide sequences showed only one base substitution with sequences from R. oryzae infecting apple fruit (GenBank Accession No. HQ897687). Phylogenetic analysis was performed using MEGA4.1 software employing the neighbor-joining method and the Tajima–Nei distance model. Previously published ITS sequences from Rhizopus strains were included for reference, and Rhizomucor miehei CBS209.77A (GenBank Accession No. JN206322) was used as an outgroup. The fungal isolates isolated from strawberry were placed within a clade comprising reference strains of R. oryzae in the phylogenetic tree (Fig. 3).

    Based on mycological characteristics, molecular data and pathogenicity to the host plant, the fungus was identified as R. oryzae Went & Prinsen Geerligs (Lunn, 1977). This is the first report of R. oryzae on strawberry in Korea.

    For cultural control, field sanitation will be extremely important. Handle fruit with care at all times. It should be sure that all ripe fruit have to be removed from the field at harvest as it can serve as a site for invasion by fungus. Cultivars with thick cuticles are resistant to Rhizopus fruit rot.

    Figure

    JALS-48-27_F1.gif

    Symptoms of fruit rot on strawberry (Fragaria x ananassa) caused by Rhizopus oryzae. A: Typical symptoms with mycelia, sporangia, and sporangiospores on the surface of fruit, B: Symptom induced by artificial inoculation.

    JALS-48-27_F2.gif

    Morphological characteristics of Rhizopus oryzae isolated from strawberry (Fragaria x ananassa). A: Colony on PDA 6 days after inoculation, B: Sporangium and sporangiophore, C: Columella, D: Sporangiospores, E: Rhizoids.

    JALS-48-27_F3.gif

    Phylogenetic tree using ITS sequences showing relationships among Rhizopus oryzae isolated from strawberry (Fragaria x ananassa) and closely relative Rhizopus species. DNA sequences from the NCBI nucleotide database were aligned using ClustalW and a phylogenetic tree was constructed using MEGA4.1 with neighbor-joining method and Tajima–Nei distance model. Numbers above the branches indicate the bootstrap values. Bars indicate number of nucleotide substitutions per site. The fungal isolates infecting strawberry (Fragaria x ananassa) were marked in bold font.

    Table

    Comparison of morphological characteristics of the fungus isolated from strawberry (Fragaria x ananassa) with the previous descriptions of Rhizopus oryzae

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