First Report of Fusarium subglutinans Causing Leaf Spot Disease on Cymbidium Orchids in Korea

Abstract

In 2006~2010, leaf spot symptoms, that is, small, yellow spots that turned into dark brown-to-black lesions surrounded by a yellow halo, were observed on Cymbidium spp. in Gongju, Taean, and Gapyeong in Korea. A Fusarium species was continuously isolated from symptomatic leaves; in pathogenicity testing, isolates caused leaf spot symptoms consisting of sunken, dark brown lesions similar to the original ones. The causal pathogen was identified as Fusarium subglutinans based on morphological and translation elongation factor 1-alpha sequence analyses. This is the first report of F. subglutinans as the cause of leaf spot disease in Cymbidium spp. in Korea.

Members of the genus Cymbidium, belonging to the family Orchidaceae, are popularly grown as potted plants and used as cut flowers throughout Asia due to their esthetic value. Many cultivars of Cymbidium developed by breeding systems have been released in the commercial market [1]. Cymbidium currently occupies the largest cultivation area of any orchid in Korea, accounting for 47% (ca. 101 ha) of the total cultivated land [2]. During 2006~2010, some leaf spots were observed on Cymbidium species cultivated in greenhouses located in Gongju, Taean, and Gapyeong in Korea. Tiny yellow spots were initially observed on the upper side of the leaves, which later turned dark brown to black with a surrounding yellow halo (Fig. 1A and 1B). The lesions became larger and sunken with dark brown, raised edges. In the advanced stage, the centers of leaf spots that were more than 50mm in size fell out, leaving holes in these older lesions (Fig. 1C and 1D). The disease finally resulted in leaf deformation, thereby reducing the market value of the affected plants.

Fig. 1. Leaf spot symptoms occurring on Cymbidium sp. and morphological characteristics of Fusarium subglutinans. A, B, Early symptoms on leaves appearing as tiny, dark brown lesions surrounded by a yellow halo; C, D, Advanced foliar lesions producing holes; E, F, Leaf spot symptoms on inoculated plants; G, Colony on potato dextrose agar after 1 wk of incubation; H, Conidiophores with polyphialides; I, Macroconidia and microconidia.

Small sections cut from the diseased tissues were surface-disinfected in 70% ethanol and 1% sodium hypochlorite, washed with sterile distilled water, and subsequently placed on potato dextrose agar (PDA) plates supplemented with streptomycin sulfate (200 ppm). The hyphal tips of emerging colonies were transferred onto new PDA plates and subcultured to obtain pure isolates. A total of 5 fungal isolates were derived from different collections and deposited at the Korean Agricultural Culture Collection (Table 1). Fungal structures from fresh materials were examined under a light microscope and their dimensions were measured with the aid of a micrometer eyepiece. Microscopic images were obtained using a Zeiss AXIO microscope equipped with AxioCam ICc3 (Carl Zeiss, Jena, Germany). Colonies on the PDA plates were white to pale pinkish, with sparse, fluffy, aerial mycelium in the outer region; were submerged in the middle region; and had an average diameter of 55 mm after 4 days (Fig. 1G). Morphological characteristics of the present isolates were observed after 7 days of incubation at 25℃. Conidiophores were unbranched or branched, bearing monophialides and polyphialides (Fig. 1H). Microconidia were aseptate, ellipsoid to allantoid, and 8~18 × 2.5~3 µm (Fig. 1I). Macroconidia were straight to slightly curved, 3-5 septate, and 32.5~75 × 2.5~4 µm (Fig. 1I). The morphological and cultural characteristics of the causal fungus were consistent with the description of Fusarium subglutinans (Wollenw. & Reinking) PE Nelson, Toussoun & Marasas [3].

Table 1. Information on Fusarium subglutinans isolates obtained from Cymbidium orchids.

Total genomic DNA was isolated using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Mycelial mats and conidia were harvested by scraping the surface of colonies on PDA plates after for 1 mon. The genomic DNA was used as a template for PCR. A portion of the translation elongation factor 1-alpha gene was amplified with the primers EF1 (5'-ATG-GGT-AAG-GAR-GAC-AAG-AC-3') and EF2 (5'-GGA-RGT-ACC-AGT-SAT-CAT-GTT-3') [4]. The PCR cycling conditions were as follows: initial denaturation for 2min at 96℃; 35 cycles of denaturation for 30 sec at 94℃, annealing for 30 sec at 56℃, and extension for 30 sec at 72℃; and a final extension for 7 min at 72℃. The PCR products were purified using a PCR purification kit (Inclonebiotech Co., Seoul, Korea), and both strands were directly sequenced using the ABI Prism 3730xl automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA). NCBI BLAST analysis showed high similarity (97~98%) to the sequences of F. subglutinans. The resulting sequences of the present isolates were submitted to GenBank (accession Nos. KM213990, KM213991, KM213993, and KM213994). To infer the phylogenetic relationships, 19 reference sequences of F. subglutinans were retrieved from the GenBank database. F. solani (AB674290) was used as an outgroup. A neighbor-joining tree was generated in MEGA5 [5]. In the phylogenetic tree (Fig. 2), the present isolates were grouped into a clade accommodating F. subglutinans isolates with a bootstrap value of 99%. The molecular data verified the identity of the present isolates.

Fig. 2. Phylogenetic tree inferred from translation elongation factor 1-alpha sequences of Fusarium spp. using neighbor-joining analysis. Bootstrap values ≥ 50% obtained from 10,000 replicates are indicated above the branches. The scale bar represents 0.02 nucleotide substitutions per site.

A pathogenicity test was carried out using 6-mon-old Cymbidium plants in a glasshouse. Two isolates (06-115 and 10-173) were used as inocula that consisted of conidial masses and mycelium harvested from the 30-day-old cultures. Each conidial suspension, adjusted to 10⁶ conidia per mL, was sprayed on 3 young plants whose leaves had been injured with needles. Three control plants were sprayed with sterilized water. All plants were covered with polyethylene bags and kept in a growth chamber at 28℃ for 48 hr. Leaf necrosis symptoms appeared on the inoculated leaves within 7 days (Fig. 1E and 1F). The fungal pathogen was successfully reisolated from the leaves of all inoculated plants, confirming Koch's postulate. In contrast, no symptoms were observed in the control plants.

Until date, 3 Fusarium species, viz., F. oxysporum, F. proliferatum, and F. solani, which cause dry rot, have been recorded on Cymbidium spp. in Korea [6]. There have been no previous records of F. subglutinans associated with Cymbidium orchids in Korea, whereas the Fusarium species has been known to occur on these plants in New Zealand [7] and Japan [8]. As leaf spot disease may pose a serious threat to commercial Cymbidium growers, adequate disease control is required to reduce economic losses.