text: Journal of Agriculture & Life Science Vol.53 No.1 pp.151-156
  DOI : https://doi.org/10.14397/jals.2019.53.1.151
  The Occurrence of Sooty Mold of Blueberry Caused by Cladosporium sphaerospermum in Korea
  Jin-Hyeuk Kwon1 , Kyoungmi Park1, Yeyeong Lee2, Byeongsam Kang3, Okhee Choi4, Jinwoo Kim2,3,4*
  1Gyeongsangnam-do Agricultural Research and Extension Services, Jinju, 52733, Korea
  2Department of Plant Medicine, Gyeongsang National University, Jinju, 52828, 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: +-55-772-1929 E-mail: jinwoo@gnu.ac.kr
  Received July 7, 2018 Review July 24, 2018 Accepted August 6, 2018
  In March of 2015, severe sooty mold was observed on blueberries cultivated in a farmer's greenhouses in Goseong-gun, Gyeongsangnam-do, South Korea. Typical symptoms included a black or dark brown, superficial fungal growth on the leaves, stems, and fruit of blueberry plants. The fungal colonies on PDA were flat and black. The conidia were dark green in color, globose to sub-globose in shape and 3-5×3-4 μm in size. The ramoconidia were cylindrical and 18-42×3 μm in size. The conidiophores were dark brown, cylindrical-oblong in shape and 42-138×3-4 μm in size. To confirm the identity of the fungus(F130), the 18S rDNA as well as actin and β-tubulin gene sequences were PCR-amplified and sequenced. The pathogenicity of the isolate was then explored on 2-month-old potted blueberry plants. Based on the mycological characteristics, molecular identification, and pathogenicity on host plants, this fungus was identified as Cladosporium sphaerospermum. This is the first report on sooty mold of blueberry caused by C. sphaerospermum in Korea.
  Key Words : Blueberry , Cladosporium sphaerospermum , Sooty mold
  Blueberries(Vaccinium spp.) containing anthocyanins and polyphenols are known to have their potential role in the human body. Blueberries, native to eastern Canada and southeastern United States, are settled and cultivated in Europe and various countries in the northwest pacific regions such as Japan and New Zealand(Vander Kloet, 1980;Hancock et al., 2008;Ballington, 2009). In 2014, world production of blueberries was about five hundred thousand tones, led by the United States(50% of total production) and Canada(35%)(FAOSTAT, 2017). In 2016, Canada was the largest producer of wild blueberries(AAFC, 2017). In South Korea, the culture, production, and consumption of blueberries have increased rapidly over the past 10 years with wellbeing trends(KOSTAT, 2018).
  Cladosporium-mediated disease is a very common disease that occurs in many plants worldwide and has been reported to cause major damage to leave, stems, flowers and fruits of annual plants(Agrios, 2005). There are 134 records based on the fungushost combinations in the Fungus-Host database using the criteria: Cladosporium sphaerospermum and its synonyms(C. hibisci and C. papyricola) (Farr & Rossman, 2018). Caruso & Ramsdel(1995) described the occurrence of sooty mold caused by Cladosporium spp. on blueberry leaves. Since blueberries have been introduced in Korea, sooty mold is often foundin any of the blueberry cultivation areas. However, since there is no report on the isolation and identification of causative pathogens and mycological characteristics, it is difficult to establish a method for effective control.
  In March of 2015, severe sooty mold was observed on blueberries cultivated in a farmer's greenhouse in Goseong-gun, Gyeongsangnam-do, South Korea. Samples of blueberry fruits and leaves with sooty molds were collected and brought to the laboratory for isolating and identifying pathogens. Thus, the aim of the present study was to isolate and identify sooty mold fungus from blueberries. To do these, we performed the fungal isolation, morphological characterization, molecular identification, and pathogenicity tests on a host plant. This is the first report of sooty mold on blueberries caused by Cladosporium sphaerospermum in Korea. Currently, blueberry cultivation area and production are increasing, and at this point, our research results will provide information necessary for blueberry production and cultivation in Korea.
  1. Fungal isolation
  Blueberry leaves and fruit with sooty mold were sampled from a farmer's greenhouse in Goseong-gun, Gyeongsangnam-do, South Korea. To obtain a pure isolate, the fungal structures were collected from infected fruit tissues using medical forceps and added to sterile water in an Eppendorf tube. The tube was vortexed vigorously and a 100-㎕ aliquot was spread on water agar plates containing 100 ㎍/㎖ of streptomycin to prevent bacterial contamination. Water agar blocks containing newly growing fungal hyphae were transferred on potato dextrose agar(PDA) media and the PDA was incubated at 25℃ for 10 days.
  2. Microscopic observations
  The fungal colonies containing hyphae, conidiophores and conidia were transferred to a drop of sterilized distilled water on a slide glass and observed under a light microscopy(Axioplan 2, Carl Zeiss, Jena, Germany). For scanning electron microscopic(SEM) observation, we followed the protocol as described in a previous report(Kwon et al., 2017b). Prepared specimens were observed under a scanning electron microscopy(LEO 1420VP, LEO Electron Microscopy Ltd., Cambridge, UK).
  3. Pathogenicity tests
  The pathogenicity of the isolate was then explored on 2-month-old potted blueberry plants(Vaccinium corymbosum cultivar Northland). There presentative fungal isolate F130 was grown on PDA plates for 10 days. Culture plates were flooded with sterile distilled water and rubbed with an artist's paintbrush to obtain conidial suspensions, which were then filtered through sterile cheesecloth and suspended in sterile distilled water. Spore suspension was adjusted to 3×106 conidia/ml using a hemocytometer. Fifty leaves from one blueberry plant were inoculated by spraying with the conidial suspension until run-off. Ten negative control leaves of another blueberry plant were treated with sterile distilled water. The inoculated and noninoculated plants were incubated in a humid growth chamber with >90% relative humidity at 25℃ for 24 h and then maintained in a greenhouse.
  4. Molecular identification
  To confirm the identity of the fungus(F130), the 18S rDNA as well as actin and β-tubulin gene sequences were polymerase chain reaction(PCR)- amplified and sequenced using primers V9G(5'-TGC GTTGATTACGTCCCTGC) / LS266(5'-GCATTCCCA AACAACTCGACTC-3') (Zalar et al., 2007), ACT- 512F(5'-ATGTG CAAGGCCGGTTTCGC-3') / ACT- 783R(5'-TACGAGTCCTTCTGGCCCAT-3') (Carbone & Kohn, 1999), and T1(5'-AACATGCGTGAGATTGTA AGT-3') / T22(5'-TCTGGATGTTGTTGGGAATCC-3') (O'Donnell & Cigelnik, 1997), respectively. The DNA extraction and PCR were followed by the protocol as described in a previous report(Kwon et al., 2017a). The resulting gene sequences of the 18S rDNA(557 bp), actin(243 bp) and β-tubulin(1354 bp) of F130 isolate were compared with those in the NCBI / GenBank database(http://www.ncbi.nlm.gov/blast/) and were deposited in GenBank.
  1. Typical symptoms
  Typical symptoms included a black or dark brown, superficial fungal growth on the leaves, stems, and fruit of blueberry plants(Fig. 1). The fungal growth could sometimes be washed away, leaving a healthylooking leaf surface beneath.
  2. Fungal isolation and microscopic observation
  Cladosporium fungus was consistently isolated from the symptomatic tissues, while other fungi were not isolated. The fungal colonies on PDA were flat and black(Fig. 2A). Fifty individual conidia and conidiophores were measured under a microscope. The morphological characteristics of the fungus were summarized in Table 1. The conidia were dark green in color, globose to sub-globose in shape and 3-5×3-4 μm in size. The ramoconidia were cylindrical and 18-42×3 μm in size. There were 0 to 4 septa. The conidiophores were dark brown, cylindricaloblong in shape and 42-138×3-4 μm in size(Fig. 2B-C). The species was placed in the Cladosporium spp. group. All morphological features of the fungus were consistent with those of C. sphaerospermum (Bensch et al., 2012).
  3 Pathogenicity tests
  To fulfill Koch's postulates, the representative isolate F130 was used for pathogenicity tests. Eighteen days post-inoculation, the blueberry leaves that had been inoculated with the fungus showed sooty mold symptoms(Fig. 2D), whereas the control seedlings were symptomless. Fungus re-isolated from the artificially inoculated plants had the same morphological features as the original isolates. PCR amplification and DNA sequence analysis of re-isolated fungus confirmed the consistency.
  4 Molecular identification
  The 18S rDNA(GenBank accession no. KY952177) and actin(KY952175) gene sequences of the isolate of F130 exhibited 100% identity with those of C. sphaerospermum strains DTO 161-D9(KP701961) and CPC 13368(EU570272), respectively. The β-tubulin (KY952176) gene sequence was ≥99% homologous with the sequence of C. sphaerospermum EXF-458 (EF101409). Because of high nucleotide sequence identity of the genes, further phylogenetic analysis was not considered in the present study.
  Based on the mycological characteristics, molecular identification, and pathogenicity on host plants, this fungus was identified as C. sphaerospermum. This is the first report on sooty mold of blueberry caused by C. sphaerospermum in Korea. We believe that our works will provide necessary information to develop effective disease control strategy for blueberry cultivation and production.
  This research was carried out with the support of Cooperative Research Program for Agriculture Science and Technology Development(Project No. PJ012826 092018) Rural Development Administration, Republic of Korea.
  Symptom of sooty mold of blueberry(Vaccinium spp.) caused by Cladosporium sphaerospermum. A and B: Typical symptoms of sooty mold on leaves, branch and fruits on blueberry of disease outbreak in the field.
  Morphological characteristics of Cladosporium sphaerospermum isolated from blueberry(Vaccinium spp.). A: Mycelial colony grown on PDA for 20 days; B: image under the light microscopy(bar=10μm); C: scanning electron microscope of conidia, ramoconidia and conidiophore(bar=2μm); D: symptom induced by artificial inoculation after 20 days of incubation.
  Comparison of morphological characteristics pathogenic fungus isolated from sooty mold of blueberry with the previous descriptions of Cladosporium sphaerospermum
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