Introduction
Ascomycota, the largest phylum of fungi, is morpholo- gically diverse group covering from unicellular yeasts to complex cup fungi. During a survey on diversity of soil- inhibiting fungi we encountered sixteen ascomycete’s fungi, which have not been officially reported in Korea; Acremonium cellulolyticus, Acremonium zonatum, Chaeto- mium madrasense, Cladosporium silenes, Humicolopsis ce- phalosporioides, Leptosphaerulina chartarum, Paecilomyces marquandii, Paecilomyces tenuis, Paraphaeosphaeria sporu- losa, Penicillium rubidurum, Pochonia suchlasporia, Sporo- thrix inflata, Thielavia hyrcaniae, Thielavia terricola, Xylo- gone sphaerospora and Zopfiella longicaudata.
The genus Acremonium consists of most simply struc-tured among all filamentous anamorphic fungi with the morphological characteristics of septate hyphae, tapered, and mostly lateral phialides produced singly or in small groups [1]. Acremonium zonatum and Acremonium cellu- lolyticus belong to the family Hypocreceae. The latter spe- cies produces cellulose which is mainly used for manage- ment of silage and has been shown to improve silage qua- lity [2]. Paecilomyces is genus of the order Eurotiales. The genus Paecilomyces was first introduced by Bainier and he described that the genus is differentiated from the closely related genus Penicillium having long divergent phialides with colonies that are never typically green [3]. Paecilo- myces consists of more than 30 recognized species [2]. P. marquandii is known for its ability to degrade herbicides like alachlor [4-5]. The synonymy of P. ma rquandi i is Metarhizium marquandii [6]. Paecilomyces species are abundant on soils, indoor and outdoor air, food and water and have cosmopolitan distributions [4, 5, 7, 8]. Paecilo- myces tenuis is characterized by the very slender phiali- des and fusiform to ellipsoidal conidia [9]. This species are thermophilic or thermotolerent [10].
Since Paecilomyces species are ubiquitous in nature, they are frequently cited in ecological studies. However, there may be many indigenous fungal species of Paecilomyces that have not been investigated yet. Thus, it is necessary to execute investigations regarding unreported indigenous fungal species.
Penicillium is the member of the order Eurotiales and Trichocomaceae family recorded from soil and have a worldwide distribution. Penicillium is a genus of ascomy- cetous fungi which have greater importance in food and drug production as well as in natural environment [1]. Penicillium rubidurum is an anamorph, monoverticillate species of the genus Penicillium [8]. Chaetomium madra- sense, Thielavia terricola and Thielavia hyrcaniae belongs to the family Chaetomiaceae. Members of the family Chaetomiaceae are ubiquitous ascosporulating fungi com- monly found in soil enriched manure or cellulosic mater- ials [11]. C. madrasense is considered to be a rather com- mon species and is known from several types of sub- strate in various regions of the world [12].
Paraphaeosphaeria sporulosa belongs to the family Phae- sphaeriaceae. Conidiogenous cells of P. sporulosa are glo- bose to ampulliform, hyaline, discrete or positioned on aggregated clumps of cells that protrude into the cavity [13]. Zopfiella longicaudata and Sporothrix inflata belong to the family Lasiophaeriaceae. Zopfiella species consists of non-ostiolate ascomata, clavate to cylindrical usually evanescent asci lacking an apical ring [14]. The anamor- phs of most Zopfiella species were unknown. Zopfiella latipes forms a Humicola-like anamorph in culture [15]. The anamorphic fungus Sporothrix inflata, known as a soil-borne fungus, can colonize living, dead as well as partly deteriorated roots [16].
Species of Cladosporium are cosmopolitan in distribu- tion and many of them are agents of decay, deterioration, or a cause of allergy or even plant or animal disease [17]. The Cladosporium spp. are often of high environmental impact, the genus is of interest to researchers in a wide variety of disciplines [17]. Cladosporium silenes was named after the host plant, Silene maritime, on which it was col- lected [18].
Soil fungi play the crucial roles in terrestrial and micro- bial communities and in decomposition of organic matter and nutrient cycling [19]. For sixteen soil fungi newly isolated Ascomycetes from Korea, this work performed the morphological investigation and molecular phylog- enetic analysis of the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) regions.
Materials and Methods
Soil sample collection and fungal isolation
Soil samples were collected in 2014 and 2015 from vari- ous places of Korea (Table 1). Soil samples were taken from 0~15 cm depth after scrapping and removing the leaf litters and other plant debris on soil surface. Appro- ximately 200~250 g soils were collected for each sample. The collected soil samples were prepared by drying at room temperature, grinding and sieving with an auto- clave-sterilized brass sieve of 2 mm aperture size. The fungi were isolated using the dilution technique [20]. Briefly, 1 gram of soil sample was suspended in 9 mL of sterile distilled water and vigorously shaken for 2~3 min. Then, soil suspensions were diluted serially in sterile distilled water. One milliliter of each 10-3, 10-4, 10-5 and 10-6 dilution was pipetted out and poured into petri plates containing potato dextrose agar (PDA; Difco, Detroit, MI, USA); and plates were incubated for 5~7 days at 25oC until growth of fungal colony was observed. The morpho- logically distinct colonies were selected and further puri- fied by sub-culturing on the plates containing PDA. The pure cultures were preserved on 20% glycerol stock at 4oC for further studies.
Morphological characterization
Morphological characteristics of all the isolates were studied by growing on potato dextrose agar (PDA), oat- meal agar (OA), malt extract agar (MEA) and potato car- rot agar (PCA). KNU14-8, KNU14-18-2 and KNU14-2 were additionally grown on czapek yeast extract agar (CYA) and yeast extract sucrose agar (YES). The strains were inoculated at three points on 9-cm petri dishes and incubated for 10 days at 26 oC in dark place. All these media were prepared as described by Samson [21]. After incubation, the colony diameter on the various agar media was measured. Colony color (obverse and reverse sides) and the degree of sporulation were observed. Colony col- ors were described using Kornerup and Wanscher [22]. Microscopic pictures were taken with an HK 3.1 CMOS digital camera (KOPTIC, Seoul, Korea) connect to an Olympus BX50F-3 microscope (Olympus, Tokyo, Japan) and a scanning electron microscope (LEO Model 1450VP Variable Pressure Scanning Electron Microscope; Carl Zeiss, Oberkochen, Germany).
Genomic DNA extraction, sequencing and data analysis
Total genomic DNA of the isolates were extracted using he DNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions. The int- rnal transcribed spacer region (ITS) gene was amplified sing primers ITS1 (5'-TCCGTAGGTGAACCTGCG-3') nd ITS4 (5'-TCCTCCGCTTATTGATATGC-3') [19]. The amplified PCR products were sequenced with the ABI Prism 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA). ITS sequences was implied to compare the sequences of GenBank at National Center for Biotech- nology Information (NCBI) applying the basic local align- ment search tool [23]. The nucleotide sequences were deposited at culture collection of National Institute of Biological Resources (NIBR). The culture numbers assigned by NIBR and accession numbers provided by GenBank were listed in Table 1. Phylogenetic relationships were analyzed using molecular evolutionary genetic analysis (MEGA 6) software [24]. Neighbor-joining phylogenetic tree was constructed to infer the evolutionary relationship between the Korean isolates and the previously reported species with the type strains.
Results
Morphology of all the studied fungal isolates
Acremonium cellulolyticus (KNU14-25): Talaromyces cellulolyticus T. Fujii, FEMS Microbiology Letters 351: 39 (2013) [MB#805758]
Colony on PDA attains 55~60 mm in diameter. Front side of the colony was white at center and light green at the margins and brown at back side (Fig. 1). Mycelium was white centrally and light green peripherally (Fig. 1). Exudate was absent. Irregular form and surface was rough. Sporulation was moderate to dense. Texture was woolly. Conidia was subglobose with the diameter of 0.4~2.1 μm.
Acremonium zonatum (KNU14-15): Acremonium zon- atum (Sawada) W. Gams, Cephalosporium-artige Schi- mmelpilze: 119 (1971) [MB#308213]
Colonies attains 15~20 mm diameter of growth at 25 C on PDA with white to slightly pinkish in color. Conidio- phores are very variable in length. Growth of colony was slow. Texture was wooly. Irregular form and surface was rough. The fungus has herbicidal properties and is used to control water hyacinth (Fig. 2). Exudate was absent. Sporulation was moderate to dense.
Chaetomium madrasense (KNU14-9): Chaetomium ma- drasense Natarajan, Proc. Indian Acad. Sci. (Pl. Sci.): 255-264 (1971) [MB#310909]
Colonies on PDA with aerial mycelium or sometimes partially with quite sparse white to yellowish aerial myce- lium. Reverse light yellow to dark yellowish (Fig. 3). Col- ored exudates absent. Ascomata superficial, in reflected light olivaceous-green at first, then becoming black due to emerging ascospore masses from ascomatal ostioles, obovate, spherical or nearly so, ostiolate, 198~273 μm high, 149~198 μm diam.; ascomatal wall brown, composed of textura intricata in surface view. Terminal hairs flexuous, undulate or slightly coiled, pale brown, indistinctly septate, verrucose, 2~3.5 μm broad near the base. Asci fasciculate, clavate, stalked, without apical structures, 68~90 × 11~16 μm, 8-spored, evanescent. Ascospores limoniform, slightly biapiculate, bilaterally flattened, usually with a lateral bul- ge, olivaceous-brown or brown when mature, containing several droplets, 9~11.5 (~14) × 7.5 ~ 9 (~10) × 6~7.5 μm, with an apical germ pore.
Cladosporium silenes (KNU14-18-1): Crous, P.W; Tan- aka, K: Summerell, B.A; Groenewald, J.Z. 2011. Addi- tions to the Mycospharella complex, IMA Fungus. 2(1): 49-64. [MB#560083]
Colonies of KNU 14-18-1 isolate cultured on different growth media viz., PDA, MEA, OA and PCA media at 25°C showed aerial mycelium, lobate margins and reached 24~25 mm, 22~25 mm, 21~25 mm and 16~18 mm dia- meter, respectively (Fig. 4A~4H). Colony surface was grey- olivaceous, reverse olivaceous-grey. Sporulation was mod- erate to dense. Exudate was absent.
Fig. 4
Morphological characters of Cladosporium silenes (KNU14-18-1) grown for 7 days on PDA, OA, CYA and YES at 26 oC.A~D, obverse colony from left to right grown on PDA, OA, MEA and PCA; E~H, reverse colony from left to right grown on PDA, OA, MEA and PCA; I~J, conidiophores and conidia produced on PDA and MEA; K, simple microscopic picture of coni- diophore; L~N, SEM of conidiophore; O~P, SEM of conidia. PDA, potato dextrose agar; OA, oatmeal agar; CYA, czapek yeast extract agar; YES, yeast extract sucrose agar; MEA, malt extract agar; PCA, potato carrot agar.
Taxonomy of the isolate KNU 14-18-1: Colony mor- phology of the present isolate was similar to one of Clastoridium silenes (Bensch et al., 2010)
Mycelium of the KNU 14-18-1 isolate were septated, constricted, refractive, 2~4(~4.8) μm wide, branched with rare swellings and consisted of pale to light brown hyphae (Fig. 4I~4K). Conidiophores were arising terminally and laterally from ascending hyphae of diameter 14~50 (~150) × 2.5~4 μm, (Fig. 4I~4K). Ramoconidia was aseptate, smooth, slightly curved, cylindrical of 12~22 × 3~5 (~5.4)μm diameter. Conidia were aseptate, ovoid, with darkened and thickened hilum, 4~5 × 3 (~3.5) μm, abundant, con- nected in branched chains of up to 7 branches (Fig. 4I~4K).
Humicolopsis cephalosporioides (KNU15-3): Humicol- opsis cephalosporioides Cabral & Marchand, Boletín de la Sociedad Argentina de Botánica 17 (1-2): 70 (1976) [MB#315257]
Colonies on PDA was flat, smooth, umbonate and yel- lowish white. Colony growth was fast attaining a diameter of 70~75 mm in 7 days at 25o C. Exudate was absent. Sporulation was moderate to dense. Conidiophores were more or less differentiated and mostly unbranched, up to 90 μm long (Fig. 5).
Leptosphaerulina chartarum (KNU14-16): Leptospha- erulina chartarum Cec. Roux, Transactions of the Brit- ish Mycological Society 86 (2): 320 (1986) [MB#103530]
Colony growth was fast and attained 70~80 mm diam- eter in 7 days at 25oC, the front side of the colony was gray brown (Fig. 6) and the reverse side of it was tan. Mycelium was transparent, branched and septated. Vege- tative and spore hypha was 2.0 to -7.5 μm and 2.0 to 5.0 μm in diameter, respectively. Exudate was absent and sporul- ation was moderate. Conidia was dark brown in color and elliptic and obtuse on the apex. Three transverse septum and 0 to 1 longitudinal septa was usually observed in the conidium and size is 10.0 to 19.0 μm.
Paecilomyces marquandii (KNU14-8): Paecilomyces marquandii (Massee) S. Hughes, Mycological Papers 45: 30 (1951) [MB#302194]
Micrographs of morphological structures of the isolate re shown in Fig. 3. Colony grew moderately on PDA ttaining a diameter of approximately 30~36 mm at 26 oC fter 14 days of inoculation. Front side of the colony olor was whitish yellow and back side was light yellow Fig. 7A, 7F). Exudates were absent, margin was irregular. Texture of colony was floccose. Colony margin was un- dulate. Sporulation was moderate to dense. On OA, col- ony was light brown at the center with white in margin n front side and yellow at back side. Colony was grown lowly reaching a diameter of 15~20 mm within 14 days t 26 oC (Fig. 7B, 7G). Exudates absent, margin was slightly rregular. Texture of colony was floccose, and colony mar- gin was undulate. Sporulation was moderate to dense. On CYEA, colony color was powdery white at front and yel- owish brown at back side. Colony grew slowly attaining diameter of 19~24 mm within 14 days at 26 oC (Fig. 7C,7H ). Sporulation was moderate to dense. Exudates was bsent. Texture of colony was floccose, and margin was undulate. On YES, colony grew moderately reaching a dia- meter of 21~25 mm within 14 days at 26 oC (Fig. 7D, 7I ). Front side of the colony color was white in color and back side was yellow in color. Colony was light brown at the center. Exudates was absent, margin was slightly irre- gular, texture of the colony was floccose, and sporulation was moderate to dense. On MEA, colony grew moderately attaining a diameter of 22~26 mm within 14 days at 26 oC (Fig. 7E, 7J). Front and back side of the colony color was light and yellowish brown respectively. Sporulation was moderate to dense. Exudates was absent, margin was sli- ghtly irregular, and texture of the colony was floccose.
Fig. 7
Morphological characters of Paecilomyces marquandii KNU14-8 grown for 7 days on PDA, OA, CYA and YES at 26 oC. A~D, obverse colony from left to right grown on PDA, OA, CYA and YES; E~H, reverse colony from left to right grown on PDA, OA, CYA and YES; I~J, conidiophores and conidia produced on PDA and MEA; K, simple microscopic picture of coni- diophore; L~N, SEM of conidiophore; O~Q, SEM of conidiophore and conidia. PDA, potato dextrose agar; OA, oatmeal agar; CYA, czapek yeast extract agar; YES, yeast extract sucrose agar; MEA, malt extract agar; PCA, potato carrot agar.
Taxonomy of the isolate KNU14-8: Paecilomyces mar- quandii (Massee) S. Hughes, Mycological Papers 45: 30 (1951) [MB#302194] = Metarhizium marquandii (Mas- sae) Kepler S.A Rehner $ Humber. Comb. nov. Mycob- ank [MB806091]
Conidiophores were hyaline, smooth walled. 48~292 × 2.2~2.7 μm in diameter (Fig. 7K~7M, 7O ). Conidia were in chains, ellipsoidal and smooth walled of 2.5~2.9 × 1.5~2 μm diameter and dry chains or aggregating spore group (Fig. 7N, 7P, 7Q). Phialides were short cylindrical of 7~12 × 1.5~2 μm diameter. The phialides consist of a swollen basal part, tapering into thin and distinct necks. Chlamy- dospores-like structures were thin- walled, globose to ellip- soidal and 2.9 μm in diameter.
Paecilomyces tenuis (KNU14-18-2): Paecilomyces ten- uis Y. F. Han & Z.Q. Liang, Mycotaxon 102: 54 (2007) [MB#510919]
Micrographs of morphological structures of the isolate are shown in Fig. 8. Colony on PDA, attaining a diameter of 34~40 mm within 14 days at 26oC. The front side of mycelium was milky white in color while back side was yellowish brown (Fig. 8A, 8F). Sporulation was dense, conidia were in mass. Irregular form, smooth surface. The color was initially white then became light yellow. Colony on OA, attaining a diameter of 33~38 mm within 14 days at 26 oC. The front side of mycelium was wooly white in color while back side was light brown (Fig. 8B, 8G). Spo- rulation was moderate, conidia were in mass. Irregular form, rough surface. On CYA, attaining a diameter of 33 ~38 mm within 14 days at 26 oC. The front side of myce- lium was white and back side was reverse yellowish in color (Fig. 8C, 8H). Sporulation was moderate to dense. Conidia were in mass. Irregular form, smooth surface. On YES, attaining a diameter of 35~39 mm within 14 days at 26 oC. The front side of mycelium was powdery white and back side was yellow in color (Fig. 8D, 8I). Spo- rulation was moderate to dense. Conidia were in mass. Irregular form, smooth surface. On MEA, attaining a dia- meter of 25~34 mm within 14 days at 26 oC. The front side of mycelium was powdery white and back side was yellow in color (Fig. 8E, 8J).
Fig. 8
Morphological characters of Paecilomyces tenuis KNU14-18-2 grown for 7 days on PDA, OA, CYA and YES at 26 oC. A~D, obverse colony from left to right grown on PDA, OA, CYA and YES; E~H, reverse colony from left to right grown on PDA, OA, CYA and YES; I~J, conidiophores and conidia produced on PDA and MEA; K, simple microscopic picture of conidiophore; L~N, SEM of conidiophore; O~Q, SEM of conidiophore and conidia. PDA, potato dextrose agar; OA, oatmeal agar; CYA, czapek yeast extract agar; YES, yeast extract sucrose agar; MEA, malt extract agar; PCA, potato carrot agar.
Taxonomy of the isolate KNU14-18-2. Paecilomyces tenuis Y. F. Han & Z.Q. Liang, Mycotaxon 102: 54 (2007) [MB#510919]
Vegetative hyphae was hyaline, smooth-walled, 1.0~1.2 μm wide. Conidia was hyaline, smooth-walled, fusiform to long-ellipsoidal. Conidiophores were septate, 12~17.3 μm in diameter, forming a veticillate branches with phialides in whorls of 2~4. Conidia were in chains ellipsoidal and smooth walled of 2.3~2.6 × 1.5~1.7 μm diameter Phiali- des were divergent, very slender and consisting of a cylin- drical or clavate basal portion.
Paraphaeosphaeria sporulosa (KNU15-2): Paraphaeo- sphaeria sporulosa (W. Gams & Domsch) Verkley, G Gkl & Stielow, Persoonia 32: 47 (2014) [MB#800768]
Colonies on PDA: growing very fast and attains 80~90 mm diameter in 7 days at 25 oC. The colony color is creamy white (Fig. 9). Exudate was absent. Irregular form and sur- face was rough. Sporulation was slightly moderate to dense. Conidia were globose to sub-globose and 1.0 to 2.5 μm in diameter. Conidiogenous cells were globose to hyaline of 4~6 × 3.2~4.3 μm in size.
Penicillium rubidurum (KNU14-12): Penicillium rubi- durum Udagawa & Y. Horie. Transactions of the Mycol- ogical Society of Japan 14: 381 (1973) [MB # 319295]
Micrographs of morphological structures of the isolate are shown in Fig. 10. Colony on PDA, attaining a diame- ter of 19~21 mm within 7-days at 26 oC. The front side of mycelium was milky white in color while back side was dark yellow (Fig. 10A, 10E). Sporulation was moderate, conidia were in mass, irregular form, smooth surface. The color of colony was initially white then became light yel- low. Colony on OA, attaining a diameter of 22~25 mm within 14 days at 26 oC. The front side of mycelium was white in color while back side was brown (Fig. 10B, 10F). Sporulation was moderate, conidia were in mass. Irregular form, rough surface. On CYA, attaining a diameter of 16~18 mm within 14 days at 26 oC. The front side of myce- lium was white and back side was light red in color (Fig. 10C, 10G). Sporulation was moderate to dense. Conidia were in mass, irregular form and smooth surface. On YES, attaining a diameter of 20~23 mm within 14 days at 26 oC. The front side of mycelium was powdery white and back side was yellowish red in color (Fig. 10D, 10H). Sporula- tion was moderate to dense. Conidia were in mass, irre- gular form and smooth surface (Fig. 10K, 10O). Conidi- ophores were hyaline, smooth walled, 1.3~2.2 μm in dia- meter (Fig. 10I ). Conidiophore was monoverticillate, four phialides per metula. Phialides with terminal whorls. Phi- alides were in verticls of 3~5. Conidia in chains ellipsoi- dal and rough walled, 1.9~2.3 μm (Fig. 10L). Phialides were short cylindrical to ellipsoidal chlamydospore-like structures, thin-walled and globose.
Fig. 10
Morphological characters of Penicillium rubidurum (KNU14-12) grown for 7 days on PDA, OA, CYA and YES at 26 oC. A~D, obverse colony from left to right grown on PDA, OA, CYA and YES; E~H, reverse colony from left to right grown on PDA, OA, CYA and YES; I~J, conidiophores and conidia produced on PDA and MEA; K, simple microscopic picture of conid- iophore; L~N, SEM of conidiophore; O, SEM of conidia. PDA, potato dextrose agar; OA, oatmeal agar; CYA, czapek yeast ext- ract agar; YES, yeast extract sucrose agar; MEA, malt extract agar; PCA, potato carrot agar.
Pochonia suchlasporia (KNU15-6): Pochonia suchla- sporia var. suchlasporia, Nova Hedwigia 73 (1-2): 67 (2001) [MB#485231]
Colony on PDA grew fast reaching 60~70 mm diameter in 10 days 26 oC. Colony color was wooly, white to yellow- ish; reverse yellow to brownish cream (Fig. 11). Exudate was absent. Sporulation was moderate. Texture was woolly. Irregular form and surface was rough. Conidiophores were long, erect and densely verticillate.
Sporothrix inflata (KNU15-8): Sporothrix inflata de Hoog, Studies in Mycology 7: 34 (1974) [MB#323935]
Colony pictures of the present isolate KNU 15-8 on four different culture media, namely PDA, MEA, OA and PCA at 26°C are indicated in Fig. 12A~12H. Sporulation was moderate. Irregular form and surface was rough. Col- ony was white, floccose and smooth and became floccose with few concentric rings at the edges and tufts at the center over time.
Fig. 12
Morphological characters of Sporothrix inflata (KNU15-20) grown for 7 days on PDA, OA, CYA and YES at 26 oC. A~D,obverse colony from left to right grown on PDA, OA, MEA and PCA; E~H, reverse colony from left to right grown on PDA, OA, MEA and PCA; I~J, conidiophores and conidia produced on PDA and MEA; K, simple microscopic picture of conidiophore; L~N, SEM of conidiophore; O~P, SEM of conidia. PDA, potato dextrose agar; OA, oatmeal agar; CYA, czapek yeast extract agar; YES, yeast extract sucrose agar; MEA, malt extract agar; PCA, potato carrot agar.
Taxonomy of the isolate KNU15-8
Colony size of the isolate on PDA, MEA, OA and PCA media was 21~30 mm, 24~29 mm, 21~25 mm and 25~ 30 mm in diameter, respectively. Vegetative hyphae of the isolate was hyaline, smooth-walled, many droplets, bran- ched and 1~1.5 μm wide with swellings (Fig. 12M~12P). Conidiogenous cells were dispersed, arising laterally and terminally on hyphae, 20~50 μm long and 1~2.2 μm wide at the base, swollen clusters formed from intercalary posi- tion (Fig. 12I, 12J, 12L). Two types conidia viz., hyaline (3~4 × 1.5~3.0 μm) and dark (3~4 (~6.5) × 2~4.0 μm)conidia were observed and they were unicellular, smooth, obovate to ellipsoid, pointed at the base (Fig. 12J~12K).
Thielavia hyrcaniae (KNU15-1): Thielavia hyrcaniae Nicot, C. R. Acad. Sci. Paris: 304 (1961) [MB#340053]
Colonies on PDA: fast growing and attains the size of 70~80 mm diameter in 7~10 days at 25 oC. The front side of the colony is white in color and the reverse side of it is brownish white (Fig. 13). Sporulation was moderate to dense. Exudate was absent. Pycnidia were dark brown or black, globose to sub-globose, spores were elliptical, dark brown, apiculate at both ends, 12~16 × 8~11 μm.
Thielavia terricola (KNU14-23-1): Thielavia terricola (J.C. Gilman & E.V. Abbott) C.W. Emmons, Bulletin of the Torrey Botanical Club 57: 123-126 (1930) [MB#255078]
Colonies on PDA was medium to fast growing and att-ained the size of 50~60 mm diameter in 7~10 days at 26oC. The front side of the colony was white in color and the reverse side of it was brownish white (Fig. 14). Exudate was absent. Irregular form and surface was rough. Pycnidia were dark brown or black, globose to sub-glo- bose, mostly 100~200 μm in diameter, with small ostiolar papillae, spores are elliptical, dark brown, apiculate at both ends of 10~15 × 7~9 μm diameter.
Xylogone sphaerospora (KNU15-7): Xylogone sphaero- spora Arx & T. Nilsson, Svensk Botanisk Tidskrift 63: 345 (1969) [MB#325626]
Colonies fast growing. Initially orange-white, darkening to gray (Fig. 15) with development of abundant ascomata. Ascospores smooth, hyaline, subglobose, 3.9~5.2, 3, 2.3~ 4.1 μm.
Zopfiella longicaudata (KNU15-5): Zopfiella longicau- data (Cain) Arx, Proceedings van de Koninklijke Neder- landse Akademie van Wetenschappen Section C 76 (3): 291 (1973) [MB#325685]
Colonies on PDA was fast growing and attained a dia- meter of 60~70 mm in 7 days at 26oC, the front side of the colony was dark to olivaceous brownish white (Fig. 16), usually superficial cleistothecia with a peridium adorned with varying degree of hair. Sporulation was moderate. Exudate was absent. Asci were highly evansescent and are 8-spored, cylindrical to clavate of 73~110 μm.
Molecular phylogeny of all the studied fungal isolates Acremonium cellulolyticus (KNU14-25)
KNU14-25 was isolated from crop field soil in July 2014. ITS rDNA sequences were employed to determine the phy- logenetic relationship between the isolate KNU14-25 and previously defined Acremonium species. The isolate was most closely related to A. cellulolyticus and formed a mo- nophyletic group with a bootstrap value of 99% (Fig. 17).
Fig. 17
Neighbor-joining phylogenetic analysis of the partial 18S-ITS1-5.8S-ITS2-28S rDNA region of (KNU14-25), (KNU14- 15), (KNU14-8), (KNU14-9), (KNU14-18-1), (KNU14-18-2), (KNU15-2) and (KNU15-3) obtained from crop field soil in Korea. Numerical values (>50) on branches are the bootstrap with 1,000 replicates.
Acremonium zonatum (KNU14-15)
KNU14-15 was isolated from crop field soil in July 2014. ITS rDNA sequence of the isolate KNU14-15 was most closely related to A. zonatum and formed monophyletic group with bootstrap value of 99% (Fig. 17). The phylog- enetic analysis showed that the isolate is A. zonatum. This is a common species found especially in soil though it is the first report from Korea.
Chaetomium madrasense (KNU14-9)
KNU14-9 was isolated from crop field soil in July 2014. The phylogenetic tree was inferred from the ITS rDNA sequence. Our study isolate supports the monophyly of Chaetomium. The strain has high similarity with C. mad- rasense with bootstrap value of 98% (Fig. 17). This sug- gests that our study isolate is C. madrasense which is not previously reported in Korea.
Cladosporium silenes (KNU14-18-1)
KNU14-18-1 was isolated from crop field soil in July 2014. The neighbor joining relationship tree of study iso- late (KNU14-18-1) was carried out by using ITS rDNA sequence. According to the result, KNU14-18-1 showed close relation with the C. silenes (Fig. 17) with the simil- arity of 100% (Fig. 17).
Humicolopsis cephalosporioides (KNU15-3)
KNU15-3 was isolated from crop field soil in July 2014. ITS rDNA sequences were employed to determine the phylogenetic relationship between the isolate KNU14-15- 3 and previously defined Humicolopsis species. The acqui- red ITS sequence showed 100% similarity with a H. cepha- losporioides (accession no. KU516491) available in the Gen- Bank database, suggesting the isolate is H. cephalospori- oides. In addition, neighbor joining tree for the identifi- cation of the isolate for clarity of related taxa. Results rev- ealed that the isolate was most closely related with H. ce- phalosporioides with the bootstrap value of 100% (Fig. 17).
Leptosphaerulina chartarum (KNU14-16)
KNU14-16 was isolated from crop field soil in July 2014. ITS rDNA sequences were used to compare and determ- ine the phylogenetic relationship between the isolate KNU 14-8 and previously described Leptosphaerulina species. All the retrieved sequences were aligned by using the Multalin program. Bootstrap analysis was carried out with 1,000 replications to determine the support for each clade. According to the results, ITS sequence of KNU14-16 mat- ched with L. chartarum (HQ248491) with 99% similarity (Fig. 8).
Fig. 18
Neighbor-joining phylogenetic analysis of the partial 18S-ITS1-5.8S-ITS2-28S rDNA region of (KNU14-23-1), (KNU15- 1), (KNU15-5), (KNU15-6), (KNU15-7), (KNU15-8), (KNU14-16) and (KNU14-23-1) obtained from crop field soil in Korea. Numerical values (>50) on branches are the bootstrap with 1,000 replicates.
Paecilomyces marquandii (Metarahzium marquandii) (KNU14-8)
KNU14-8 was isolated from crop field soil in July 2014. ITS rDNA sequences were used to compare and determ- ine the phylogenetic relationship between the isolate KNU 14-8 and previously described Paecilomyces species. The phylogenetic relationship was constructed by using the neighbor joining search option with nearest neighbor int- erchange. Bootstrap analysis was performed with 1,000 replicates to determine the support for each clade. The ITS rDNA region were identical to that of P . marquandii (GU566261) with the bootstrap value of 100 % (Fig. 17). These results suggested that our study isolate, KNU14-8 matched with P. marquandii.
Paecilomyces tenuis (KNU14-18-2)
KNU14-18-2 was isolated from crop field soil in July 2014. ITS rDNA sequences were compared to determine the phylogenetic relationship between the isolate KNU14- 18-2 and previously described Paecilomyces species. The isolate was most closely related to Paecilomyces tenuis (KU 933674) and formed monophyletic group with bootstrap value of 99% (Fig. 17). The phylogenetic analysis showed that the isolate is Paecilomyces tenuis. This is a common species found especially in soil but this is the first report of its occurrence in Korea.
Paraphaeosphaeria sporulosa (KNU15-2)
KNU15-2 was isolated from crop field soil in July 2014. ITS rDNA sequences were compared to determine the phylogenetic relationship between the isolate KNU15-2 and previously described Paraphaeosphaeria species. The isolate was assigned to Parapaheosphaeria on the basis of ITS sequences. The isolate KNU15-2 formed a monophy- letic clade with the sequences of P . sporulosa and had high sequence similarity with P . sporulosa (JX496114) with bootstrap value of 100% (Fig. 17). This is the first report of its existence in Korea.
Penicillum rubidurum (KNU14-12)
ITS rDNA sequences of the isolate KNU14-12 were compared to determine the phylogenetic relationship bet- ween the isolate KNU14-12 and previously described Peni- cillium species. The isolate was most closely related to Penicillum rubidurum and formed a monophyletic group with bootstrap value of 99% (Fig. 18). The phylogenetic analysis showed that the isolate is Penicillum rubidurum.
Pochonia suchlasporia (KNU15-6)
ITS and rDNA sequences of the isolate KNU15-6 were compared to determine the phylogenetic relationship bet- ween the isolate KNU15-6 and previously described Poc- honia species. The ITS region of the KNU15-6 were 98% identical to the culture collection of P. suchlasporia (HG 008759) (Fig. 18). The results strongly suggests that that our study isolate KNU15-6 is P . suchlasporia which is not reported previously in Korea.
Sporothrix inflata (KNU15-8)
KNU15-8 was isolated from crop field soil in July 2014. ITS rDNA sequences were employed to determine the phylogenetic relationship between the isolate KNU15-8 and previously defined Sporothrix species. The ITS sequ- ence of KNU15-8 was aligned with the available sequ- ences of S. inflata strains and its nearest species through the BLAST sequence using the MultAlin program, and a neighbor joining tree was constructed from the aligned sequences. The phylogenetic analysis suggests that the isolate KNU15-8 was 99% similar with the S. inflata (HQ607879) (Fig. 18). The phylogenetic analysis showed that the isolate is S. inflata.
Thielavia hyrcaniae (KNU15-1)
KNU15-1 was isolated from crop field soil in July 2014. ITS rDNA sequences were employed to determine the phylogenetic relationship between the isolate KNU15-1 and previously defined Thielavia species. The isolate sho- wed strong sequence similarity to T. hyrcaniae and formed monophyletic group with bootstrap value of 98% (Fig. 18). The phylogenetic analysis showed that the isolate is Thielavia hyrcaniae.
Thielavia terricola (KNU14-23-1)
KNU14-23-1 was isolated from crop field soil in July 2014. ITS rDNA sequences were employed to determine the phylogenetic relationship between the isolate KNU14- 23-1 and previously defined Thielavia species. The isolate was most closely related to T. ter r icola and formed mono- phyletic group with bootstrap value of 98% (Fig. 18). The phylogenetic analysis showed that the isolate is Thielavia terricola.
Xylogone sphaerospora (KNU15-7)
KNU15-7 was isolated from crop field soil in July 2014. ITS rDNA sequences were employed to determine the phylogenetic relationship between the isolate KNU15-7 and previously defined Xylogone species. The isolate was most closely related to Xylogone sphaerospora and formed monophyletic group with bootstrap value of 98% (Fig. 18). The phylogenetic analysis showed that the isolate is Xylo- gone sphaerospora which is not reported previously in Korea.
Zopfiella longicaudata (KNU15-5)
KNU15-5 was isolated from crop field soil in July 2014. ITS rDNA sequences were employed to determine the phylogenetic relationship between the isolate KNU15-5 and previously defined Zopfiella species. The isolate was most closely related to Zopfiella longicaudata and formed monophyletic group with bootstrap value of 98% (Fig. 18). The phylogenetic analysis showed that the isolate is Zop- fiella longicaudata.
Discussion
It has been reported that the Paecilomyces is mainly dis- tinguished by the shape of the phialides that tapering into a long distinct neck and the divergent aggregation of whorls [25]. Morphologically these two isolates showed variable colony color in all tested growth media. Isolate KNU14-8 grew slowly as compared to the isolate KNU14- 18-2 in all the tested growth media. On the basis of above mentioned taxonomical properties of the isolate KNU14- 8 was considered to be P. mar quandi i . Taxonomical prop- erties of our study isolate KNU14-8 reasonably fits with the description made by Hughes [26]. In addition, P. mar - quandii can be distinguish from all the other species in the genus by producing phialides that often rise from undifferentiated hyphae in whorls, white colonies on cza- pak agar and subglobose conidia [27]. Moreover, Phylog- enetic analysis revealed that the isolate is P. marquandi i. Thus based on these morphological and molecular cha- racteristics it can be concluded that the isolate KNU14-8 is P. mar qu andi i . The other isolate KNU14-18-2 was pre- sumed to be a Paecilomyces on the basis on its phialides. The study isolate reasonably fits with the description of Paecilomyces tenuis [10]. Our study isolate KNU14-18-2 also consists of similar morphological characteristics as described by Han et al. [10]. Moreover, Phylogenetic ana- lysis revealed that the isolate is P. t e nui s. Thus based on these morphological and molecular characteristics it can be concluded that the isolate KNU14-18-2 is P. t e nui s. In addition, P. t e nui s has an importance in producing Hup- erzine (Hup A) which has a potential application in Alz- heimer disease therapy [28]. However further studies in this aspect would be worthwhile.
Penicillium is a genus of ascomycetous fungi that are saprobic, filamentous and typically monomorphic [29]. Penicillium species have septate hyphae (2~5 um in dia- meter) that give rise to branched or unbranched conidio- phores with secondary branches that give Penicillium abrush-like appearance [29]. We also found the similar brush-like appearance of conidiophore in our isolate (Fig. 10L~10N). Moreover, conidiophore and cleistothe- cium characters of Penicillium are of great taxonomic im- portance [30]. The isolate KNU14-12 was presumed to be a Penicillium on the basis of its conidiophores, conidia and its colony appearance. The isolate reasonably fits with the description of Penicillium rubidurum which was des- cribed in Udagawa and Horie [31]. The study isolate KNU14-12 from crop field soil only differs slightly from the original description by size of conidiophores, and co- nidia. We also found the similar morphological charac- teristics in the present isolate. Moreover, several studies showed that Penicillum has a large impact on human life and main function in nature is the decomposition of or- ganic materials, where species cause devastating rots as pre and post-harvest pathogens on food [32]. Further studies on these aspects by the present isolate are needed. On the basis of above mentioned taxonomical properties of the isolate KNU14-12 was considered to be P. r ub i du - rum. This is a common species found especially in soil but this is the first report of the isolation from crop field soil in Korea.
Morphology features and molecular characteristics of KNU15-8 isolate was similar to the morphology Sporo- hrix inflata that has been previously reported [15, 17, 18, 33]. In addition, the results were also is in agreement with the previous study [10] that dark, thick-walled coni- dia referred to as the ‘Humicola-type’ were observed rarely. Colony morphology of the present isolate was similar to he colony image of Clastoridium silenes [18]. Moreover, he morphological feature of KNU 14-18-1 isolate was very imilar to C. silenes [18] that C. silenes differs from C. ladosporioides in having shorter ramo- and intercalary conidia. ITS rDNA sequences were also employed to det- ermine the phylogenetic relationship between the isolate KNU14-18-1 and previously defined Cladosporium species. Hence, the isolate was found to be more closely related to C. silenes with bootstrap value of 98%. In addition, the morphological features of the isolates KNU15-2, KNU15- 6, KNU15-7, KNU15-5, KNU15-1, KNU14-23-1, KNU14- 16 and KNU15-3, was found similar to that of previously eported Paraphaeosphaeria sporulosa, Pochonia suchlas- poria, Xylogone sphaerospora, Zopfiella longicaudata, Thie- avia hyrcaniae, Thielavia terricola, Leptosphaerulina char- arum, and Humicolopsis cephalosporioides isolates respec- ively. Further studies regarding their biotechnological im- portance are worthwhile in the future.
