Abstract

Morphological characteristics of Cercospora malayensis.

To examine morphological characteristics, fungal structures from fresh samples were mounted on a glass slide with a drop of water and examined using bright-field and differential interference contrast light microscopy with an Olympus BX51 microscope (Tokyo, Japan) for measurements and Zeiss AX10 microscope equipped with an AxioCam MRc5 (Carl Zeiss, Göttingen, Germany) for imaging. Thirty measurements were taken at 100× and 1,000× magnification for each sample.

A voucher specimen was submitted to the Korea University Herbarium (KUS) under accession number KUS-F27687. Pathogenic agents were collected from the leaves naturally infected with Cercospora. To obtain a pure isolate, conidia were collected from infected leaf tissues using sterile forceps under a dissecting microscope and placed on a drop of sterile water on a glass slide. The conidial suspension was streaked onto 2% water agar plates supplemented with 100 mg/L of streptomycin sulfate and incubated at 25 for 4 days. Suitable germinated conidia were transferred to potato dextrose agar plates using a sterile needle under a dissecting microscope. These single-spore isolates were used to evaluate colony characteristics and colors. An isolate from KUS-F27687 was deposited with the Korean Agricultural Culture Collection, Rural Development Administration, Wanju, Korea (accession No. KACC47655).

After 7 days incubation at 25 on potato dextrose agar in the dark, raised fungal colonies had formed and showed moderate aerial mycelium with smooth and erose or dentate margins. Fruitings were amphigenous. Stromata were not present or weakly developed, subimmersed, globular, dark brown, small to moderate, and composed of swollen hyphal cells (Fig. 1C and 1D). Conidiophores emerged through the cuticle, were fasciculate (n = 3–12), olivaceous to brown, straight to mildly curved, and geniculate, 100–230 µm long, 3.0–5.0 µm wide, had 1- to 5-septate, and showed conspicuous conidial scars (Fig. 1E). Conidia were hyaline, acicular, subacute to obtuse at the apex, and truncate to obconically truncate at the base, had 3- to 20-septate, were 50–240 × 4.0–5.5 µm, guttulate or aguttulate, and showed thickened, darkened hila at the base (Fig. 1F and 1G). These morphological characteristics were consistent with those of Cercospora malayensis F. Stevens & Solheim [3].

Phylogenetic analysis of Cercospora malayensis.

Genomic DNA was extracted from 200–400 mg of fungal mycelia (KACC47655) harvested from 7-day potato dextrose agar cultures grown at 25 using a DNeasy Plant Mini Kit (Qiagen Inc., Hilden, Germany). A sterile blade was used to scrape the mycelia from the surface of the plate. Five nuclear gene regions were targeted for PCR amplification and subsequent sequencing. The primers ITS4 and ITS5 [10] were used to amplify the internal transcribed spacer areas and 5.8S rRNA gene (ITS). Part of the EF-1α gene using the primers EF728F [11] and EF2Rd [6] and part of the ACT gene was amplified using ACT512F [11] and ACT2Rd primers [6]. The CAL228F and CAL737R primers [11] were used to amplify part of the CAL gene, the primers CylH3F and CylH3R [12] to amplify part of the HIS gene, and 28S nrDNA using primers described previously [6]. All PCR mixtures and conditions were as described by Hunter et al. [13]. The PCR products were separated by electrophoresis at 100 V for 40 min on a 0.8% (wt/vol) agarose gel containing ethidium bromide at 0.1 µg/mL in 1× Tris-acetate-EDTA buffer (0.4 M Tris, 0.05M NaAc, and 0.01 M EDTA, pH 7.85) and visualized under UV light, followed by purification with a PCR purification kit (Core-one; Core-Bio, Seoul, Korea). The amplicons were sequenced in both directions using the same PCR primers used for the initial amplification according to the manufacturer's recommendations. The reactions were monitored using BigDye Terminator Cycle Sequencing Kits (Applied Biosystems, Foster City, CA, USA) as indicated by the manufacturer and analyzed on an ABI 3130 automated DNA sequencer (Applied Biosystems). The possible identity of the isolates was established by comparing their ITS, EF-1α, ACT, CAL, and HIS sequences with those in the GenBank database (National Center for Biotechnology Information [NCBI] US National Institute of Health, Bethesda, MD, USA; http://www.ncbi.nlm.nih.gov/BLAST). Selected Cercospora spp. sequences, including ITS, EF-1α, ACT, CAL, and HIS, were retrieved from GenBank for phylogenetic analysis. These retrieved sequences were included in the Cercospora phylogenetic tree constructed by Crous et al. [14]. The obtained sequences were edited and assembled using SeqMan software (Lasergene; DNASTAR, Madison, WI, USA). A neighbor-joining phylogenetic tree was constructed using the maximum composite likelihood method by MEGA6 [15].

The representative resulting 497-bp ITS, 525-bp EF-1α, 603-bp ACT, 304-bp CAL, and 391-bp HIS sequences obtained from KACC47655 were deposited in GenBank (accession Nos. KR400012, KY082663, KY082664, KY082665, and KY082666, respectively). A BLAST search in GenBank using the ITS sequences revealed that the sequences showed over 99–100% identity with several sequences of Cercospora species, including C. cyperina, C. zebrina, C. kikuchii, C. capsici, and C. malayensis. Importantly, ITS sequences shared 99% identity with an isolate from leaf spot of okra (Abelmoschus esculentus (L.) Moench), a plant belongs to the family Malvaceae. Furthermore, CAL and HIS sequences showed 99–100% identity with C. cf. sigesbeckiae from Paulownia coreana and C. cf. richardiicola from Ajuga multiflora, respectively. Although the ITS, CAL, and HIS sequences of the fungus were the same as several Cercospora sequences, the ACT and EF-1α sequences showed that the isolate was a distinct species. The phylogenetic tree, created using a five combined sequence ITS, EF-1α, ACT, CAL, and HIS dataset, showed that C. malayensis from H. cannabinus formed a well-supported clade that was sister to a clade consisting of Cercospora spp., as well as revealed a separate clade distinct from other genera such as Septoria, with bootstrap values greater than 98% for each clade. Furthermore, the C. malayensis pathogen isolated from leaf spot on H. cannabinus was closely related to C. cf. sigesbeckiae on Malva verticillata, Cercospora sp. on Hibiscus sabdariffa, and C. fagopyri on H. syriacus (Fig. 2).

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Fig. 2

Neighbor-joining tree of Cercospora malayensis based on the multigene dataset (internal transcribed spacer [ITS], translation elongation factor 1-alpha [EF-1α], actin [ACT], calmodulin [CAL], and histone 3 [HIS]). The numbers above the nodes are the bootstrap values obtained from 1,000 replicates. The GenBank numbers are represented in order of ITS, EF-1α, ACT, CAL, and HIS, respectively. The isolate obtained in this study is shown in boldface.

Pathogenicity test.

Koch's postulates were evaluated to establish the pathogenicity of the isolated fungus. An isolate of KACC47655 was used in the pathogenicity tests. To prepare hyphal suspensions, 3-week-old colonies grown on potato dextrose agar plates at 25 were homogenized in distilled water. Ten leaves (2 per plant) were inoculated with hyphal suspensions and ten leaves were sprayed with sterile distilled water on the young leaves of 1-year-old healthy kenaf plants ‘Jangdae’ until the water began to run off. The leaves were individually covered with plastic bags to maintain a relative humidity of 100% for 24 hr and then maintained in a greenhouse at 28 ± 2 with a 12-hr photoperiod. Typical symptoms of necrotic spots appeared on the inoculated leaves 10 days after inoculation and were identical to those observed in the field. Cercospora malayensis re-isolated from symptomatic leaf tissues was morphologically identical to the original isolate. The pathogenicity test was conducted twice and showed similar results, fulfilling Koch's postulates. No symptoms were observed on control plants.

Identification and discussion.

The classification of this Cercospora was mainly based on a combination of characteristics such as morphological characteristics, host specificity, and molecular analyses. Although morphological characteristics are frequently used to identify newly isolated fungi, it is not possible to distinguish Cercospora spp. based solely on morphology. Molecular techniques are commonly used to overcome taxonomic problems posed by the limitations of morphological characteristics or in cases where morphological characteristics are in conflict, ambiguous, or missing [16]. Extensive studies of Cercospora and related genera in Korea have generated records of numerous species. However, C. malayensis has not been detected in Korea [7]. Leaf spot caused by C. malayensis was previously been reported on Abelmoschus, Hibiscus, Lavatera (Rosaceae), and Hyptis (Lamiaceae) plants. Leaf spot caused by C. malayensis is common among Hibiscus species, including H. manihot, H. cannabinus, H. esculentus, H. meeusei, H. mutabilis, H. rosa-sinensis, H. sabdariffa, H. syriacus, and H. tiliaceus [7]. However, these reports were mainly reliant on a combination of morphological characteristics and host specificity without an analysis of molecular characteristics of the pathogen. As these sequences of C. malayensis were not previously reported in phylogenetic studies, they will provide a platform for future studies of Cercospora taxonomy and further analyses of Cercospora-Hibiscus associations.

Kenaf leaf spots associated with C. malayensis have been reported in Africa (South Africa, Tanzania, Zambia, and Zimbabwe) and South Asia (Cambodia and China) [7]. However, this is the first report of C. malayensis infection of kenaf in Korea. Our observations in the fields of several farmers showed that the presence of C. malayensis on kenaf in Korea is a potentially serious threat to this plant.

ACKNOWLEDGEMENTS

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