Molecular Markers for Detecting a Wide Range of Trichoderma spp. that Might Potentially Cause Green Mold in Pleurotus eryngii

In Pleurotus sp., green mold, which is considered a major epidemic, is caused by several Trichoderma species. To develop a rapid molecular marker specific for Trichoderma spp. that potentially cause green mold, eleven Trichoderma species were collected from mushroom farms and the Korean Agricultural Culture Collection (KACC). A dominant fungal isolate from a green mold-infected substrate was identified as Trichoderma pleuroticola based on the sequences of its internal transcribed spacer (ITS) and translation elongation factor 1-a (tef1) genes. In artificial inoculation tests, all Trichoderma spp., including T. atroviride, T. cf. virens, T.citrinoviride, T. harzianum, T. koningii, T. longibrachiatum, T. pleurotum, and T. pleuroticola, showed pathogenicity to some extent, and the observed symptoms were soaked mycelia with a red-brown pigment and retarded mycelium regeneration. A molecular marker was developed for the rapid detection of wide range ofTrichoderma spp. based on the DNA sequence alignment of the ITS1 and ITS2 regions ofTrichoderma spp. The developed primer set detected only Trichoderma spp., and no cross reactivity with edible mushrooms was observed. The detection limits for the PCR assay of T. harzianum (KACC40558), T. pleurotum (KACC44537), and T. pleuroticola (CAF-TP3) were found to be 500, 50, and 5 fg, respectively, and the detection limit for the pathogen-to-host ratio was approximately 1:10,000 (wt/wt).

First Record of the Complete Mitochondrial Genome of a Saprotrophic and Opportunistic Human Pathogenic Fungus, Scopulariopsis brevicaulis

Scopulariopsis brevicaulis is a widely distributed soil fungus known as a common saprotroph of biodegradation. It is also an opportunistic human pathogen that can produce various secondary metabolites. Here, we report the first complete mitochondrial genome sequence of S. brevicaulis isolated from air in South Korea. Total length of the mitochondrial genome is 28,829 bp and encoded 42 genes (15 protein-coding genes, 2 rRNAs, and 25 tRNAs). Nucleotide sequence of coding region takes over 26.2%, and overall GC content is 27.6%. Phylogenetic trees present that S. brevicaulis is clustered with Lomentospora prolificans with presenting various mitochondrial genome length.

First Record of the Complete Mitochondrial Genome of a Saprotrophic and Opportunistic Human Pathogenic Fungus, Scopulariopsis brevicaulis

Scopulariopsis brevicaulis is a widely distributed soil fungus known as a common saprotroph of biodegradation. It is also an opportunistic human pathogen that can produce various secondary metabolites. Here, we report the first complete mitochondrial genome sequence of S. brevicaulis isolated from air in South Korea. Total length of the mitochondrial genome is 28,829 bp and encoded 42 genes (15 protein-coding genes, 2 rRNAs, and 25 tRNAs). Nucleotide sequence of coding region takes over 26.2%, and overall GC content is 27.6%. Phylogenetic trees present that S. brevicaulis is clustered with Lomentospora prolificans with presenting various mitochondrial genome length.

Diversity of Aspergillus, Penicillium, and Talaromyces Species Isolated from Freshwater Environments in Korea

In order to elucidate the fungal diversity and community structure in freshwater environments, numerous fungal strains were isolated from freshwater, submerged soils, twigs, dead insects, etc. Among them, the present study has focused specifically on Aspergillus, Penicillium, and Talaromyces species, which produce diverse useful metabolites in general. Twelve strains of Aspergillus isolated were identified as A. japonicus (n = 5), A. tubingensis (3), A. niger (2), and A. flavus (2), 10 strains of which belong to Aspergillus section Nigri, named black Aspergillus. Eight strains of Penicillium were identified as P. brasilianim (n = 3), P. oxalicum (2), P. crustosum (1), P. expansum (1), and P. piscarium (1). Two different strains of Talaromyces were identified as T. pinophilus and T. versatilis. Thus far, Penicillium piscarium and Talaromyces versatilis have been unrecorded in Korea, for which we provide detailed morphological and molecular characteristics.

Analysis of the MAT1-1 and MAT1-2 Gene Ratio in Black Koji Molds Isolated from Meju

Aspergillus luchuensis is known as an industrially important fungal species used for making fermented foods such as awamori and shochu in Japan, makgeolli and Meju in Korea, and Pu-erh tea in China. Nonetheless, this species has not yet been widely studied regarding mating-type genes. In this study, we examined the MAT1-1 and MAT1-2 gene ratio in black koji molds (A. luchuensis, Aspergillus niger, and Aspergillus tubingensis) and in Aspergillus welwitschiae isolated from Meju, a fermented soybean starting material for traditional soy sauce and soybean paste in Korea. The number of strains with the MAT1-1 locus was 2 of 23 (A. luchuensis), 6 of 13 (A. tubingensis), 21 of 28 (A. niger), and 5 of 10 (A. welwitschiae). Fungal species A. tubingensis and A. welwitschiae showed a 1 : 1 ratio of MAT1-1 and MAT1-2 mating-type loci. In contrast, A. luchuensis revealed predominance of MAT1-2 (91.3%) and A. niger of MAT1-1 (75%). We isolated and identified 2 A. luchuensis MAT1-1 strains from Meju, although all strains for making shochu in Japan are of the MAT1-2 type. These strains may be a good resource for breeding of A. luchuensis to be used in the Asian fermented-food industry.

Aspergillus Associated with Meju, a Fermented Soybean Starting Material for Traditional Soy Sauce and Soybean Paste in Korea

Aspergillus is an important fungal genus used for the fermentation of Asian foods; this genus is referred to as koji mold in Japan and China. A. oryzae, A. sojae, and A. tamari are used in the production of miso and shoyu in Japan, but a comprehensive taxonomic study of Aspergillus isolated from Meju, a fermented soybean starting material for traditional soy sauce and soybean paste in Korea, has not been conducted. In this study, various Aspergillus species were isolated during a study of the mycobiota of Meju, and the aspergilli were identified based on phenotypic characteristics and sequencing of the beta-tubulin gene. Most strains of Aspergillus were found to belong to the following sections: Aspergillus (n = 220), Flavi (n = 213), and Nigri (n = 54). The most commonly identified species were A. oryzae (n = 183), A. pseudoglaucus (Eurotium repens) (n = 81), A. chevalieri (E. chevalieri) (n = 62), A. montevidensis (E. amstelodami) (n = 34), A. niger (n = 21), A. tamari (n = 15), A. ruber (E. rubrum) (n = 15), A. proliferans (n = 14), and A. luchuensis (n = 14); 25 species were identified from 533 Aspergillus strains. Aspergillus strains were mainly found during the high temperature fermentation period in the later steps of Meju fermentation.

The Mycobiota of Air Inside and Outside the Meju Fermentation Room and the Origin of Meju Fungi

The fungi on Meju are known to play an important role as degrader of macromolecule of soybeans. In order to elucidate the origin of fungi on traditional Meju, mycobiota of the air both inside and outside traditional Meju fermentation rooms was examined. From 11 samples of air collected from inside and outside of 7 Meju fermentation rooms, 37 genera and 90 species of fungi were identified. In outside air of the fermentation room, Cladosporium sp. and Cladosporium cladosporioides were the dominant species, followed by Cladosporium tenuissimum, Eurotium sp., Phoma sp., Sistotrema brinkmannii, Alternaria sp., Aspergillus fumigatus, Schizophyllum commune, and Penicillium glabrum. In inside air of the fermentation room, Cladosporium sp., Aspergillus oryzae, Penicillium chrysogenum, Asp. nidulans, Aspergillus sp., Cla. cladosporioides, Eurotium sp., Penicillium sp., Cla. tenuissimum, Asp. niger, Eur. herbariorum, Asp. sydowii, and Eur. repens were collected with high frequency. The concentrations of the genera Aspergillus, Eurotium, and Penicillium were significantly higher in inside air than outside air. From this result and those of previous reports, the origin of fungi present on Meju was inferred. Of the dominant fungal species present on Meju, Lichtheimia ramosa, Mucor circinelloides, Mucor racemosus, and Scopulariopsis brevicaulis are thought to be originated from outside air, because these species are not or are rarely isolated from rice straw and soybean; however, they were detected outside air of fermentation room and are species commonly found in indoor environments. However, Asp. oryzae, Pen. polonicum, Eur. repens, Pen. solitum, and Eur. chevalieri, which are frequently found on Meju, are common in rice straw and could be transferred from rice straw to Meju. The fungi grow and produce abundant spores during Meju fermentation, and after the spores accumulate in the air of fermentation room, they could influence mycobiota of Meju fermentation in the following year. This could explain why concentrations of the genera Aspergillus, Eurotium, and Penicillium are much higher inside than outside of the fermentation rooms.

Erratum: Mycoflora of Soybeans Used for Meju Fermentation

There was a spelling error in the Table 2.

Mycoflora of Soybeans Used for Meju Fermentation

Diverse fungi are present in Korean traditional meju and they are known to play an important role in fermented soybean products. To determine the origin of the fungi in meju, we examined the mycoflora of soybeans from 10 traditional meju factories. The samples were untreated or treated with sodium hypochlorite, and placed on malt extract agar (MEA), dichloran 18% glycerol agar (DG18), and dichloran rose bengal chloramphenicol agar (DRBC) medium. A total of 794 fungal strains were isolated and they were identified as 41 genera and 86 species. From sodium hypochlorite untreated soybeans, the genera, Cladosporium (55%), Eurotium (51%), Fusarium (33%), Penicillium (22%), and Aspergillus (exclusion of Eurotium) (20%), were mainly isolated, and Eurotium herbariorum (22%), Eurotium repens (18%), Cladosporium tenuissimum (18%), F. fujikuroi (18%), Aspergillus oryzae/flavus (7%), and Penicillium steckii (6%) were the predominant species. In case of sodium hypochlorite-treated soybeans, Eurotium (31%) and Cladosporium (5%) were frequently isolated, but Aspergillus (excluding Eurotium), Penicillium and Fusarium which were frequently isolated from untreated soybeans, were rarely isolated. Eurotium herbariorum (21%), Eurotium repens (8%), and Cladosporium tenuissimum (3%) were the predominant species. Of the 41 genera and 86 species isolated from soybeans, 13 genera and 33 species were also found in meju. These results suggest that the fungi on soybeans may influence the mycoflora of meju.

Isolation and Identification of Aspergillus Section Fumigati Strains from Arable Soil in Korea

63 strains of Aspergillus section Fumigati were isolated from 17 samples of arable soil in a central province of Korea. Based on the results of genotypic and phenotypic analyses, they were identified as Aspergillus fumigatus, A. lentulus, Neosartorya coreana, N. fennelliae, N. fischeri, N. glabra, N. hiratsukae, N. laciniosa, N. pseudofischeri, N. quadricincta, N. spinosa and N. udagawae. Among these, N. fennelliae, N. hiratsukae, N. quadricincta, and N. udagawae had not been previously recorded in Korea. The diversity of Aspergillus section Fumigati species from arable soil in Korea is also addressed.

Reidentification of Aspergillus spp. Isolated from Clinical Specimens of Patients Suspected as Pulmonary Aspergillosis in Korea / 대한의진균학회지

Korean Institute of Tuberculosis (KIT) collected 129 strains of Aspergillus spp. from clinical specimens of patient suspected as pulmonary Aspergillosis. On the basis of morphological characteristics, these strains were identified as A. fumigatus (62 strains), A. niger (37), A. flavus (26), A. versicolor (1), A. nidulans (1), A. clavatus (1) and Neosartorya fennelliae (1). These strains were re-identified according to recent Aspergillus classification system which is mainly based on molecular characters. The strains were grouped by Randomly Amplified Polymorphic DNA (RAPD). The representative strains from each group were sequenced with partial betatubulin gene and compared with those of reference strains in the Aspergillus and were identified by the sequence. The identification was confirmed by morphological examination. As the results, they are reidentified as A. fumigatus (58), A. niger (11), A. tubingensis (26), A. flavus (27), A. sydowii (3), A. nidulans (1), A. clavatus (1), Neosartorya fennelliae (1), N. fischeri (1). This is also the first report of A. tubuingensis in clinical field in Korea.

Reidentification of Aspergillus spp. Isolated from Clinical Specimens of Patients Suspected as Pulmonary Aspergillosis in Korea / 대한의진균학회지

Korean Institute of Tuberculosis (KIT) collected 129 strains of Aspergillus spp. from clinical specimens of patient suspected as pulmonary Aspergillosis. On the basis of morphological characteristics, these strains were identified as A. fumigatus (62 strains), A. niger (37), A. flavus (26), A. versicolor (1), A. nidulans (1), A. clavatus (1) and Neosartorya fennelliae (1). These strains were re-identified according to recent Aspergillus classification system which is mainly based on molecular characters. The strains were grouped by Randomly Amplified Polymorphic DNA (RAPD). The representative strains from each group were sequenced with partial betatubulin gene and compared with those of reference strains in the Aspergillus and were identified by the sequence. The identification was confirmed by morphological examination. As the results, they are reidentified as A. fumigatus (58), A. niger (11), A. tubingensis (26), A. flavus (27), A. sydowii (3), A. nidulans (1), A. clavatus (1), Neosartorya fennelliae (1), N. fischeri (1). This is also the first report of A. tubuingensis in clinical field in Korea.

An Integrated Database and Web Service for Microbial Resources at KACC

The Korean Agricultural Culture Collection (KACC) has developed a web-based system to provide an integrated database with information updates about microbial resources. This integrated database consists of 5 major functions and contains general information, which includes identification numbers, culture media composition, image information, DNA sequences, patent information, and general forms for ordering and depositing microorganisms. In 2008, KACC started providing characterization information. KACC maintains 9,801 cultures of microorganisms, including 3,296 strains of bacteria, 4,734 fungi, 784 actinomycetes, 64 yeasts, and 923 others.

Detection of Extracellular enzymes Activities in Various Fusarium spp

Thirty seven species of Fusarium were evaluated for their ability of producing extracellular enzymes using chromogenic medium containing substrates such as starch, cellobiose, CM-cellulose, xylan, and pectin. Among the tested species Fusarium mesoamericanum, F. graminearum, F. asiaticum, and F. acuminatum showed high beta-glucosidase acitivity. Xylanase activity was strongly detected in F. proliferatum and F. oxysporum. Strong pectinase activity was also found in F. oxysporum and F. proliferatum. Amylase activity was apparent in F. oxysporum. No clear activity in cellulase was found from all the Fusarium species tested.

Detection of Extracellular Enzyme Activity in Penicillium using Chromogenic Media

A total of 106 Penicillium species were tested to examine their ability of degrading cellobiose, pectin and xylan. The activity of beta-glucosidase was generally strong in all the Penicillium species tested. P. citrinum, P. charlesii, P. manginii and P. aurantiacum showed the higher ability of producing beta-glucosidase than other tested species. Pectinase activity was detected in 24 Penicillium species. P. paracanescens, P. sizovae, P. sartoryi, P. chrysogenum, and P. claviforme showed strong pectinase activity. In xylanase assay, 84 Penicillium species showed activity. Strong xylanase activity was detected from P. megasporum, P. sartoryi, P. chrysogenum, P. glandicola, P. discolor, and P. coprophilum. Overall, most of the Penicillium species tested showed strong beta-glucosidase activity. The degree of pectinase and xylanase activity varied depending on Penicillium species.

Comparison of Dyes for Easy Detection of Extracellular Cellulases in Fungi

To evaluate which dye is effective in a plate assay for detecting extracellular cellulase activity produced by fungi, four chromogenic dyes including remazol brilliant blue, phenol red, congo red, and tryphan blue, were compared using chromagenic media. For the comparison, 19 fungal species belonging to three phyla, ascomycota, basidiomycota, and zygomycota were inoculated onto yeast nitrogen-based media containing different carbon substrates such as cellulose (carboxylmethyl and avicel types) and cellobiose labeled with each of the four dyes. Overall, the formation of clear zone on agar media resulting from the degradation of the substrates by the enzymes secreted from the test fungi was most apparent with media containing congo red. The detection frequency of cellulase activity was also most high on congo red-supplemented media. The results of this study showed that congo red is better dye than other three dyes in a plate assay for fungal enzyme detection.

First Report of Penicillium brasilianum and P. daleae Isolated from Soil in Korea

In this study, a total of 300 isolates of Penicillium and related teleomorphic genera were collected from soils of 17 locations in Korea from April to May, 2004. Ninety four isolates were identified as the species of Penicillium subgenus Furcatum based on cultural and morphological characteristics and beta-tubulin gene sequences. Among the species, Korean isolates of P. brasilianum Bat. and P. daleae K. M. Zalessky were phylogenetically identical to the reference species based on DNA sequence of the beta-tubulin gene. Here we described and illustrated P. brasilianum and P. daleae that are new in Korea.

Soft Rot of Tomato Caused by Mucor racemosus in Korea

A soft rot of fruits caused by Mucor racemosus occurred on cherry tomato collected in Agricultural Products Wholesale Market in Jinju, Korea. The disease infection usually occurred wounded areas after cracking of fruits. At first, the lesions started with water soaked and rapidly softened and diseased lesion gradually expanded. Colonies were white to brownish to gray in color. Sporangia were 32~54 microm in size and globose in shape. Sporangiophores were 8~14 microm in width. Sporangiospores were 5~12 x 4~8 microm in size, ellipsoidal to subglobose in shape. Columella was 27~42 microm in size, obovoid, ellipsoidal, cylindrical-ellipsoidal, slightly pyriform in shape. Chlamydospores were numerous in sporangiophores and barrelshaped when young, subglobose in old cultures. Optimum growth temperature was about 25degrees C. The fungus was identified as M. racemosus Fres. This is the first report of soft rot on cherry tomato caused by M. racemosus in Korea.

Contribution to the Checklist of Soil-inhabiting Fungi in Korea

A total of 82 genera and 271 species of soil-inhabiting fungi including saprobic, nematode-trapping, and arbuscular mycorrhizal but plant pathogenic fungi published hitherto in South Korea are listed with the information on geographic location, habitat, vegetation when available, and relevant literatures.

Molecular and Morphological Characterization of Green Mold, Trichoderma spp. isolated from Oyster Mushrooms

Isolates of Trichoderma spp. collected from Pleurotus ostreatus and P. eryngii beds, which included loosened substrate compactness and development of green colour, were grouped into three species. The occurrence of different species of Trichoderma was as T. cf. virens (70.8%), T. longibrachiatum (16.7%) and T. harzianum (12.5%). The conidia of Trichoderma spp. were ellipsoidal, obovoid and phialides were bowling pins, lageniform and the length of phialides was 3.5~10.0 x 1.3~3.3 micromm. Phialides of T. cf. virens and T. harzianum were tending clustered, but it was solitary disposition in T. longibrachiatum. T. cf. virens was characterized by predominantly effuse conidiation, sparingly branched, and fertile to the apex and it was penicillate type. RAPD analysis could detect variability amongst three different species of Trichoderma using two newly designed URP-primers. However, intra-specific variation could not be detected in all the isolates except for rDNA sequence data classified Trichoderma isolates into three distinct groups representing three species. The profiles of rDNA sequences of isolates representing a species showed high similarity in T. cf. virens and T. harzianum. However, there was a variation in rDNA sequences of isolates representing T. longibrachiatum. The results of present study reveals that molecular techniques of RAPD and rDNA sequencing can greatly aid in classification based on morphology and precise identification of fast evolving species of Trichoderma.