Neosartorya is a widespread genus of the Trichocomoceae family. It is teloemorph of Aspergillus section Fumigati include many species which is important because of its pathogenic or allergenic to human [1]. Most members of the genus Neosartorya Malloch & Cain [2] in the Eurotiales are of worldwide distribution and are very abundant, occurring nearly everywhere in soil, air, dust, food etc. [3-7]. A few Neosartorya species are mycotoxigenic and causal agents of human diseases like aspergillosis, osteomyelitis, endocarditis and mycotic keratitis. [8-16]. Certain species of the genus are used in the production of bioactive metabolites [17]. All of the Neosartyora species produce heat resistant ascospores that are frequently encountered in different food products and cause the spoilage of processed food products [18, 19]. Thus, there has been considerable recent interest of mycologists in working with Neosartorya aureola.
During studies on the diversity of fungal communities in field soil of Gangwon-do, Korea, a species of Neosartorya was encountered. Based on its morphological and molecular characteristics, this species was identified as N. aureola and named N. aureola KNU14-7. To the best of our knowledge this fungus has not been officially reported in Korea.
Collection of soil samples and fungal isolation
Soil samples were collected from different locations in Samcheok city, Korea in 2014. Soil samples were taken from (0~15 cm) depth, air dried and stored in plastic bags at 4oC until used. The fungi were isolated by conventional dilution method and supplemented with 100 μg chloramphenicol per mL potato dextrose agar (PDA; Difco, Detroit, USA) and grown for 7 days at 28oC until the growth of colonies was observed.
ITS, β-tubulin and calmodulin gene sequencing analysis
Genomic DNA of the strain was extracted using DNeasy Plant Mini Kit (QIAGEN, Germantown, MD, USA) following the manufacturer’s instructions. The internal transcribed spacer (ITS) regions, including the 5.8S were amplified with the primers ITS1 and ITS4 [20]. For the sequencing of partial β-tubulin and calmodulin genes, the segments of β-tubulin and calmodulin were amplified with the primers BT2a and BT2b and cmd5 and cmd6 [21, 22]. The amplified polymerase chain reaction (PCR) product was purified using QIAquick PCR purification Kit (QIAGEN) following the manufacturer’s instructions. The PCR product was sequenced using ABI Prism 3730 DNA analyzer (Applied Biosystems, Foster city, CA, USA). The sequence was compared with reference ITS1-ITS4 rDNA and β-tubulin and calmodulin rDNA sequences from GenBank using BLAST analysis (http://www.ncbi.nlm. nihgob/blast). The nucleotide sequence was deposited in GenBank and assigned accession number KP966615 for isolate KNU14-7. The sequences of closely related strains were aligned using the MultAlin program. The DNA sequences were analyzed for phylogenetic relationship using Molecular Evolutionary Genetics Analysis (MEGA 6) software [23] sequence of present isolate, Neosartorya aureola KNU14-7 was compared with the sequences in the Gen- Bank by using Basic Local Alignment Search Tool (BLAST). Neighbor-joining tree was constructed by using Kimmura 2-parameter substitution model [24]. The phylogenetic tree was inferred using the maximum-likelihood heuristic search option with the nearest-neighbor interchange. Bootstrap analysis was performed with 1,000 replications to determine the support for each clade. The isolate was most closely related to N. aureola (EF669954) and formed a monophyletic group with bootstrap value of 98% (Fig. 1), N. aureola (CBS105.55) with bootstrap value of 98% and 99%, respectively (Fig. 2 and 3). The phylogenetic analysis showed that the isolate is N. aureola.
Table 1. Morphological characteristics of Neosartorya aureola isolated in this study
a Source of description [25].
MEA, malt extract agar; CYA, czapek yeast extract agar.
Fig. 1. Neighbor-joining phylogenetic analysis of Neosartorya aureola KNU14-7 partial 18S-ITS1-5.8S-ITS2-28S rDNA region sequence obtained from crop field soil in Korea. Sequence obtained in the study is shown in boldface. Numerical values (> 50) on branches are the bootstrap values as percentage of bootstrap replication from 1,000 replicate analysis. Neosartorya stramania (AB299411) was used as out group.
Morphological characteristics and identification
Morphological features were observed on malt extract agar (MEA; Fig. 4A and 4B) and czapek yeast extract agar (CYA; Fig. 4C and 4D) by doing three point inoculations in 9 cm petri plates which were incubated in the dark at 28oC for 7 days. The morphological characteristics were examined with the aid of differential interference contrast microscopy. Photomicrographs were taken with a Kodak 14n digital camera (Rochester, NY, USA) attached to the microscope. Slide material was mounted in water and sometimes with aniline blue staining. Colonies on MEA were slow growing, white, attaining a diameter of 25~30 mm after 7 days at 28oC (Fig. 4A and 4B). Colonies on CYA were moderate growing attaining a diameter 40~45 mm (Fig. 4C and 4D).
Fig. 4. Morphological characterization of Neosartorya aureola KNU14-7 observed using a compound microscope and scanning electron microscope (SEM). Malt extract agar media: A, Colony in reverse; B, Colony in front. Czapek yeast extract agar media: C, Colony in reverse D, Colony in front; E~G, Conidiophores; H~I, Simple microscopic picture of ascospores; J~K, SEM of conidiophores; L, SEM of ascospores.
Cleistothecia superficial, scattered, white to yellowish white, globose to ovoid, 350~500 μm in diameter (Fig. 4E and 4F), surrounded by a loose covering of yellow to orange, 2~4 μm wide aerial hyphae. Cleistothecial peridium hyaline to pale yellowish brown, thin, consisting of irregular, 4~10 μm diam cells. Asci globose to ovoid, 11~ 12 × 9.5~11 μm evanescent at maturity (Fig. 4G). Ascospores hyaline to pale yellowish brown, broadly lenticular, spore body 4~4.5 × 3.5~4 μm (Fig. 4H~4L), provided with two equatorial crests which are rather appressed up to 1 μm wide and rarely dissected and with the convex surfaces aculeate with spines up to 0.5 μm long.
In conclusion, we identified and reported Neosartorya aureola KNU14-7 as a new record for Korea. The species of Neosartorya are used in the production of bioactive metabolites. Neosartyora species have been described as causal agents of human diseases including invasive aspergillosis, osteomyelitis, endocarditis and mycotic keratitis [12]. Thus, in the future, further investigation in this respect would be worthwhile.
