Sporothrix stylites : A New Record from Field Soil in Korea

The genus Sporothrix was established by Hektoen and Perkins [1] who isolated the fungus from infected boy’s finger, for which the binomial Sporothrix schenckii was introduced. Fungal species that related to the genus Sporothrix have a long history of phylogeny and recently summarized by de Beer et al. [2]. As a result, based on phylogenetic analyses of four gene regions they recognized 51 taxa under the genus Sporothrix, with six species complexes. Members of the Sporothrix originate from a wide variety of environments, including wood, plant debris, peat moss, human clinical materials, but mostly also from soil [2, 3]. Amongst Sporothrix taxa there are clinically relevant species such as S. brasiliensis, S. globosa and S. mexicana [4], insect pathogens such as S. insectorum [5] as well as saprophytic species such as S. stylites and S. lignivora [3]. A member of S. pallida species complex, S. stylites, was isolated from pine pole at soil level and was characterized as producing micro- to semimacronematous, solitary, straight conidiophores and nonseptate, hyaline, smooth, thin-walled conidia [3]. Dark secondary conidia are absent in S. stylites therefore its cultures do not darken with age.

During our studies of microbial communities in field soil in Buyeo-gun, Chung-cheongnam-do, Korea, several fungal strains were isolated. With distinctive morphology, one isolate, KNU16-008, was selected for further morphological study and molecular phylogenetic analysis. Based on its morphological characteristics and phylogenetic analysis, this isolate was identified as Sporothrix stylites and named S. stylites KNU16-008. To the best of our knowledge this fungus has not been previously reported in Korea.

Collected soil sample (1 g) was suspended in 10 mL of sterile distilled water, and prepared suspension was vortexed, serially diluted, and then spread on potato dextrose agar (PDA; Difco, Detroit, MI, USA) plates. The plates were incubated at 25°C for 3 days. Single colonies on these plates were purified by transferring them onto new plates and subjecting them to an incubation on PDA at 25°C.

During the past decade, species of Sporothrix were distinguished mainly by the molecular phylogenetic anlaysis, primarily using the internal transcribed spacer (ITS) sequences of rDNA [3, 6]. Other gene regions such as β-tubulin (BT) [3, 7], calmodulin (CAL) [4] or ribosomal large subunit (LSU) [8] were also used as markers for phylogenetic analysis in Sporothrix. In the present phylogenetic analysis, ITS regions and BT gene sequences of related Sporothrix taxa were obtained from the GenBank (Table 1). The recovered sequences were aligned with the ITS and BT sequences of isolate KNU16-008 using the program Clustal X. Gaps and 5′ and 3′ ends of the alignments were edited manually using the BioEdit program. Evolutionary distance matrices for the neighbor- joining algorithm were calculated using Kimura’s two-parameter model [9]. Tree topologies were inferred by the neighbour-joining, maximum-likelihood, and maximum- parsimony methods in the program MEGA7 [10], with bootstrap values based on 1,000 replications.

A BLAST search in the NCBI database revealed that ITS regions and BT gene sequences of KNU16-008 matched with those of Sporothrix stylites CMW 14544 (EF127884, EF139097) with 99.6 and 100% similarities, respectively. Phylogenetic tree based on the combined ITS rDNA and BT gene sequences confirmed the affiliation of the isolate to Sporothrix stylites ; KNU16-008 was clustered together with S. stylites CMW 14544 and CMW 14541 in a monophyletic clade with the maximum bootstrap value. This phylogenetic relationship was supported by three tree-inferring methods employed in this study (Fig. 1).

Morphology of the isolate was examined under an Olympus CX31 light microscope (Olympus, Tokyo, Japan). The isolate KNU16-008 was cultured at 25°C, and colony characteristics such as color, shape and size were recorded. After 14 days of incubation on PDA agar, colony was 3.4~3.5 cm in diameter, pale yellowish in color, smooth, compact and flat (Fig. 2A, 2B). The colony did not darken with age. Conidia were 2.4~3.2× 1.3~1.7 µm, nonseptate, hyaline, smooth, oval to fusiform with a pointed base (Fig. 2C, 2D). These morphological characteristics of isolate KNU16-008 are in good agreement with those of Sporothrix stylites reported by de Meyer et al. [3] (Table 2), which is in line with the phylogenetic results of KNU16-008.

This is the first report on Sporothrix stylites in Korea. The type species of genus Sporothrix, S. schenckii is known as pathogen of human sporotrichosis and other species such as S. brasilensis, S. globosa and S. mexicana also show human infectivity [4, 11]. There were no reported cases of human infection by S. stylites, but that could not eliminate the possibility of infections that may have been erroneously ascribed to other Sporothrix species. Further studies are needed to confirm a low infectious potential of S. stylites.