In this study, we examined arbuscular mycorrhizal fungal (AMF) community structure colonizing field-cultivated ginseng roots according of different ages, such as 1- to 5-year-old plant, collected from Geumsan-gun, Korea. A total of seven AMF species namely, Funnelliformis caledonium, F. moseae, Gigaspora margarita, Paraglomus laccatum, P. occultum, Rhizophagus irregularis, and Scutellospora heterogama were identified from the roots using cloning, PCR-restriction fragment length polymorphism and sequence analysis of the large subunit region in rDNA. AMF species diversity in the ginseng roots decreased with the increase in root age because of the decreased species evenness. In addition, the community structures of AMF in the roots became more uniform. These results suggest that the age of ginseng affects mycorrhizal colonization and its community structure.
AMF; Korean ginseng; LSU; Species diversity
Lichen studies, including biodiversity, phylogenetic relationships, and conservation concerns require definitive species identification, however many lichens can be challenging to identify at the species level. Molecular techniques have shown efficacy in discriminating among lichen taxa, however, obtaining genomic DNA from herbarium and fresh lichen thalli by conventional methods has been difficult, because lichens contain high proteins, polysaccharides, and other complex compounds in their cell walls. Here we report a rapid, easy, and inexpensive protocol for extracting PCR-quality DNA from various lichen species. This method involves the following two steps: first, cell breakage using a beadbeater; and second, extraction, isolation, and precipitation of genomic DNA. The procedure requires approximately 10 mg of lichen thalli and can be completed within 20 min. The obtained DNAs were of sufficient quality and quantity to amplify the internal transcribed spacer region from the fungal and algal lichen components, as well as to sequence the amplified products. In addition, 26 different lichen taxa were tested, resulting in successful PCR products. The results of this study validated the experimental protocols, and clearly demonstrated the efficacy and value of our KCl extraction method applied in the fungal and algal samples.
Lichens; Fungi; Algae; Genomic DNA; rRNA; Sequencing
Fruiting bodies similar to those of the ascomycete fungi Podostroma cornu-damae and Cordyceps militaris were collected from Mt. Seunghak in Busan, Korea on August 21, 2012. The fruiting bodies were cylindrical, with tapered ends and golden red in color. The fruiting bodies contained abundant conidiophores bearing single-celled conidia, but no perithecia or asci. Pure culture of the fungal isolates was obtained through single-spore isolation. Analyses of morphological characteristics, including conidia shape, and phylogenetic traits, using internal transcribed spacer sequences, showed that these isolates belonged to the species Simplicillium lanosoniveum. Although this fungal species is known to be mycoparasitic, the isolates obtained in this study were unable to infect fungi. However, silkworms (Bombyx mori) inoculated with the fungal isolates died during the larval or pupal stages, as has been shown for the strongly entomopathogenic fungus Beauveria bassiana. This study is the first report of the entomopathogenicity of S. lanosoniveum and indicates its potential for use in biological control of insects.
Ascomata; Entomoparasite; Fruiting body; Mycoparasite; Simplicillium lanosoniveum
The aim of this study was to identify and characterize new Flammulina velutipes laccases from its whole-genome sequence. Of the 15 putative laccase genes detected in the F. velutipes genome, four new laccase genes (fvLac-1, fvLac-2, fvLac3, and fvLac-4) were found to contain four complete copper-binding regions (ten histidine residues and one cysteine residue) and four cysteine residues involved in forming disulfide bridges, fvLac-1, fvLac-2, fvLac3, and fvLac-4, encoding proteins consisting of 516, 518, 515, and 533 amino acid residues, respectively. Potential N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified in the cDNA sequence of fvLac-1 (Asn-454), fvLac-2 (Asn-437 and Asn-455), fvLac-3 (Asn-111 and Asn-237), and fvLac4 (Asn-402 and Asn-457). In addition, the first 19~20 amino acid residues of these proteins were predicted to comprise signal peptides. Laccase activity assays and reverse transcription polymerase chain reaction analyses clearly reveal that CuSO4 affects the induction and the transcription level of these laccase genes.
Copper sulfate; Flammulina velutipes; Genome; Laccase
To examine the effects of water stress and Cenangium ferruginosum (CF) on the fungal endophytic community of needles of Pinus koraiensis (PK), fungal endophytes isolated from the needles of 5-year-old PK seedlings were compared before and after exposure to water stress conditions and artificial inoculation with CF ascospores. Artificial CF inoculation was successfully confirmed using PCR with CF-specific primers (CfF and CfR). For comparison of the degree of water deficit in water-stressed and control groups of PK seedlings infected with CF, the water saturation deficit and water potential were measured. Lower water potential estimates were found in the water-stressed seedlings than in the control group. The fungal endophytes isolated from the second-year needles of non-water-stressed seedlings before and after CF inoculation revealed that primary saprobes were approximately 30% and 71.7%, respectively, and the remaining endophytes were rot fungi or pathogens. Sixty days after CF inoculation, diverse fungal endophytes in the first-year needles were isolated from the water-stressed seedlings. However, some fungal endophytes isolated from the non-water-stressed seedlings were also identified. Fungal endophytes in the second-year needles of the water-stressed and non-water-stressed seedlings were approximately 8% and 71.7% of saprobes, respectively, and the remaining endophytes were rot fungi or pathogens. On the basis of the results, we conclude that water deficit and CF can have an effect on fungal endophytic communities in the needles of PK seedlings.
Cenangium ferruginosum; Fungal endophytic community; Pinus koriensis; Water stress
During 2011~2013, a total of 729 samples for 19 types of medicinal plant were collected from Seoulyekryungsi in Seoul, Korea, and investigated for the presence of aflatoxins. The samples were analyzed using immunoaffinity column cleanup and high-performance liquid chromatography coupled to a fluorescence detector after post-column derivatization. Aflatoxins were found in 124 out of the 729 analyzed samples: 65 containing aflatoxin B1 (AFB1), 24 with aflatoxin B2 (AFB2), 15 with aflatoxin G1 (AFG1), and 20 samples with aflatoxin G2 (AFG2). The ranges for positive samples were 0.1~404.7 µg/kg for AFB1, 0.1~10.0 µg/kg for AFB2, 0.1~635.3 µg/kg for AFG1, 0.1~182.5 µg/kg for AFG2, and 0.1~1,043.9 µg/kg for total aflatoxins. Most of the medicinal plant samples (721, 98.9%) were below legal limits, but 8 samples exceeded the legal limits of 10 and 15 µg/kg established by the Korean standard for AFB1 and total aflatoxins (the sum of AFB1, AFB2, AFG1 and AFG2), respectively.
Aflatoxins; High-performance liquid chromatography; Total aflatoxins
Recently, the Q biotype of tobacco whitefly has been recognized as the most hazardous strain of Bemisia tabaci worldwide, because of its increased resistance to some insecticide groups. As an alternative control agent, we selected an Isaria javanica isolate as a candidate for the development of a mycopesticide against the Q biotype of sweet potato whitefly. To select optimal mass production media for solid-state fermentation, we compared the production yield and virulence of conidia between 2 substrates (barley and brown rice), and we also compared the effects of various additives on conidia production and virulence. Barley was a better substrate for conidia production, producing 3.43 × 1010 conidia/g, compared with 3.05 × 1010 conidia/g for brown rice. The addition of 2% CaCO3 + 2% CaSO4 to barley significantly increased conidia production. Addition of yeast extract, casein, or gluten also improved conidia production on barley. Gluten addition (3% and 1.32%) to brown rice improved conidia production by 14 and 6 times, respectively, relative to brown rice without additives. Conidia cultivated on barley produced a mortality rate of 62% in the sweet potato whitefly after 4-day treatment, compared with 53% for conidia cultivated on brown rice. The amendment of solid substrate cultivation with additives changed the virulence of the conidia produced; the median lethal time (LT50) was shorter for conidia produced on barley and brown rice with added yeast extract (1.32% and 3%, respectively), KNO3 (0.6% and 1%), or gluten (1.32% and 3%) compared with conidia produced on substrates without additives.
Bemisia tabaci; Biological control; Entomopathogenic fungi; Isaria javanica; Mass production; Solid-state fermentation
Makgeolli is a traditional Korean alcoholic beverage. The flavor of makgeolli is primarily determined by metabolic products such as free sugars, amino acids, organic acids, and aromatic compounds, which are produced during the fermentation of raw materials by molds and yeasts present in nuruk, a Korean fermentation starter. In this study, makgeolli was brewed using the wild yeast strain Saccharomyces cerevisiae Y98-5, and temporal changes in the metabolites during fermentation were analyzed by ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. The resultant data were analyzed by partial least squares-discriminant analysis (PLS-DA). Various metabolites, including amino acids, organic acids, sugar alcohols, small peptides, and nucleosides, were obviously altered by increasing the fermentation period. Changes in these metabolites allowed us to distinguish among makgeolli samples with different fermentation periods (1, 2, 3, 6, 7, and 8 days) on a PLS-DA score plot. In the makgeolli brewed in this study, the amounts of tyrosine (463.13 µg/mL) and leucine (362.77 µg/mL) were high. Therefore, our results indicate that monitoring the changes in metabolites during makgeolli fermentation might be important for brewing makgeolli with good nutritional quality.
Fermentation; Makgeolli; Metabolite; Saccharomyces cerevisiae
Makgeolli, also known as Takju, is a non-filtered traditional Korean alcoholic beverage that contains various floating matter, including yeast cells, which contributes to its high physiological functionality. In the present study, we assessed the levels of β-glucan and glutathione in various yeast strains isolated from traditional Korean Nuruk and selected a β-glucan- and glutathione-rich yeast strain to add value to Makgeolli by enhancing its physiological functionality through increased levels of these compounds. Yeast β-glucan levels ranged from 6.26% to 32.69% (dry basis) and were strongly species-dependent. Dried Saccharomyces cerevisiae isolated from Nuruk contained 25.53 µg/mg glutathione, 0.70 µg/mg oxidized glutathione, and 11.69 µg/g and 47.85 µg/g spermidine and L-ornithine monohydrochloride, respectively. To produce functional Makgeolli, a β-glucan- and glutathione-rich yeast strain was selected in a screening analysis. Makgeolli fermented with the selected yeast strain contained higher β-glucan and glutathione levels than commercial Makgeolli. Using the selected yeast strain to produce Makgeolli with high β-glucan and glutathione content may enable the production of functional Makgeolli.
β-Glucan; Glutathione; Makgeolli; Yeast
Red ginseng (Panax ginseng), a Korean traditional medicinal plant, contains a variety of ginsenosides as major functional components. It is necessary to remove sugar moieties from the major ginsenosides, which have a lower absorption rate into the intestine, to obtain the aglycone form. To screen for microorganisms showing bioconversion activity for ginsenosides from red ginseng, 50 yeast strains were isolated from Korean traditional meju (a starter culture made with soybean and wheat flour for the fermentation of soybean paste). Twenty strains in which a black zone formed around the colony on esculin-yeast malt agar plates were screened first, and among them 5 strains having high β-glucosidase activity on p-nitrophenyl-β-D-glucopyranoside as a substrate were then selected. Strain JNO301 was finally chosen as a bioconverting strain in this study on the basis of its high bioconversion activity for red ginseng extract as determined by thin-layer chromatography (TLC) analysis. The selected bioconversion strain was identified as Candida allociferrii JNO301 based on the nucleotide sequence analysis of the 18S rRNA gene. The optimum temperature and pH for the cell growth were 20~30℃ and pH 5~8, respectively. TLC analysis confirmed that C. allociferrii JNO301 converted ginsenoside Rb1 into Rd and then into F2, Rb2 into compound O, Rc into compound Mc1, and Rf into Rh1. Quantitative analysis using high-performance liquid chromatography showed that bioconversion of red ginseng extract resulted in an increase of 2.73, 3.32, 33.87, 16, and 5.48 fold in the concentration of Rd, F2, compound O, compound Mc1, and Rh1, respectively.
β-Glucosidase; Bioconversion; Ginsenosides; Red ginseng extract; Yeast
Roselle (Hibiscus sabdariffa L.) is one of the most important medicinal crops in many parts of the world. In this study, the effects of microelements, antioxidants, and bioagents on Fusarium oxysporum, F. solani, and Macrophomina phaseolina, the causal pathogens of root rot and wilt diseases in roselle, were examined under field conditions. Preliminary studies were carried out in vitro in order to select the most effective members to be used in field control trials. Our results showed that microelements (copper and manganese), antioxidants (salicylic acid, ascorbic acid, and EDTA), a fungicide (Dithane M45) and biological control agents (Trichoderma harzianum and Bacillus subtilis) were significantly reduced the linear growth of the causal pathogens. Additionally, application of the previous microelements, antioxidants, a fungicide and biological control agents significantly reduced disease incidence of root rot and wilt diseases under field conditions. Copper, salicylic acid, and T. harzianum showed the best results in this respect. In conclusion, microelements, antioxidants, and biocontrol agents could be used as alternative strategies to fungicides for controlling root rot and wilt diseases in roselle.
Biological control; Hibiscus; Root rot disease; Wilt disease
The beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) is difficult to control using chemical insecticides because of the development of insecticide resistance. Several pest control agents are used to control the beet armyworm. Entomopathogenic fungi are one of the candidates for eco-friendly pest control instead of chemical control agents. In this study, among various entomopathogenic fungal strains isolated from soil two isolates were selected as high virulence pathogens against larva of beet armyworm. Control efficacy of fungal conidia was influenced by conidia concentration, temperature, and relative humidity (RH). The isolates Metarhizium anisopliae FT83 showed 100% cumulative mortality against second instar larvae of S. exigua 3 days after treatment at 1 × 107 conidia/mL and Paecilomyces fumosoroseus FG340 caused 100% mortality 6 days after treatment at 1 × 104 conidia/mL. Both M. anisopliae FT83 and P. fumosoroseus FG340 effectively controlled the moth at 20~30℃. M. anisopliae FT83 was significantly affected mortality by RH: mortality was 86.7% at 85% RH and 13.4% at 45% RH. P. fumosoroseus FG340 showed high mortality as 90% at 45% RH and 100% at 75% RH 6 days after conidia treatments. These results suggest that P. fumosoroseus FG340 and M. anisopliae FT83 have high potential to develop as a biocontrol agent against the beet armyworm.
Beet armyworm; Entomopathogenic fungi; Metarhizium anisopliae; Paecilomyces fumosoroseus; Spodoptera exigua
In the past two decades, European ash trees (Fraxinus spp.) have been severely damaged due to ash dieback disease, which is caused by the fungal species Hymenoscyphus fraxineus (Chalara fraxinea in the anamorphic stage). Recent molecular phylogenetic and population genetic studies have suggested that this fungus has been introduced from Asia to Europe. During a fungal survey in Korea, H. fraxineus-like apothecia were collected from fallen leaves, rachises, and petioles of Korean ash and Manchurian ash trees. The morphological and ecological traits of these materials are described with the internal transcribed spacer rDNA sequence comparison of H. fraxineus strains collected from Korea, China and Japan.
Ash dieback; Chalara fraxinea; Hymenoscyphus fraxineus; Korean ash; Manchurian ash
Pseudallescheria boydii KNU13-2 was isolated from crop field soil and identified by analysis of internal transcribed spacer regions of rDNA and morphological characteristics. In the literature, P. boydii has been mentioned as a human pathogen. This is the first record of P. boydii isolated from crop field soil in Korea.
Fungi; Molecular identification; Morphology; Pathogenic fungi; Pseudallescheria
A new recorded species of Mortierella was recovered during the investigation of fungal communities in soil samples collected from different locations of Gangwon-do, Korea. The species was identified and described as Mortierella alpina on the basis of phylogenetic analysis of internal transcribed spacer sequences and morphological characteristics. This species has not been officially reported from Korea thus far.
Morphology; Mortierella alpina; Soil fungal community
Leaf spot disease was observed in sesame (Sesamum indicum L.) during 2009 and 2010 in Korea. The pathogen was identified as Alternaria simsimi based on morphological and cultural characteristics. The morphological identification was well supported by phylogenetic analysis of the ribosomal DNA-internal transcribed spacer region. A. simsimi isolates caused spot symptoms on leaves and stems of sesame plants 2 wk after artificial inoculation, which were similar to those observed in the field. This is the first record of leaf spot disease in Korea caused by A. simsimi.
Alternaria simsimi; Identification; Leaf spot; Pathogenicity; Sesame
In March 2014, a kohlrabi stem rot sample was collected from the cold storage room of Daegwallyong Horticultural Cooperative, Korea. White and fuzzy mycelial growth was observed on the stem, symptomatic of stem rot disease. The pathogen was isolated from the infected stem and cultured on potato dextrose agar for further fungal morphological observation and to confirm its pathogenicity, according to Koch's postulates. Morphological data, pathogenicity test results, and rDNA sequences of internal transcribed spacer regions (ITS 1 and 4) showed that the postharvest stem rot of kohlrabi was caused by Sclerotinia sclerotiorum. This is the first report of postharvest stem rot of kohlrabi in Korea.
Kohlrabi; Pathogenicity; Postharvest disease; Sclerotinia sclerotiorum; Sclerotinia stem rot
Alternaria from different Allium plants was characterized by multilocus sequence analysis. Based on sequences of the β-tubulin (BT2b), the Alternaria allergen a1 (Alt a1), and the RNA polymerase II second largest subunit (RPB2) genes and phylogenetic data analysis, isolates were divided into two groups. The two groups were identical to representative isolates of A. porri (EGS48-147) and A. vanuatuensis (EGS45-018). The conidial characteristics and pathogenicity of A. vanuatuensis also well supported the molecular characteristics. This is the first record of A. vanuatuensis E. G. Simmons & C. F. Hill from Korea and China.
Allium plants; Alternaria vanuatuensis; China; Morphology; Phylogeny; Korea
Depending on the acquisition of developmental competence, the expression of genes for β-1,3-glucan synthase and chitin synthase was affected in different ways by Aspergillus nidulans LAMMER kinase. LAMMER kinase deletion, ΔlkhA, led to decrease in β-1,3-glucan, but increase in chitin content. The ΔlkhA strain was also resistant to nikkomycin Z.
Aspergillus nidulans; Cell wall polysaccharides; Cell wall-related genes; LAMMER kinase; qRT-PCR
Mitochondrial protein Nfu1 plays an important role in the assembly of mitochondrial Fe-S clusters and intracellular iron homeostasis in the model yeast Saccharomyces cerevisiae. In this study, we identified the Nfu1 ortholog in the human fungal pathogen Cryptococcus neoformans. Our data showed that C. neoformans Nfu1 localized in the mitochondria and influenced homeostasis of essential metals such as iron, copper and manganese. Marked growth defects were observed in the mutant lacking NFU1, which suggests a critical role of Nfu1 in Fe-S cluster biosynthesis and intracellular metal homeostasis in C. neoformans.
Cryptococcus neoformans; Fe-S cluster; Metals; Mitochondria; Nfu1
Mushrooms are a recognized component of the human diet, with versatile medicinal properties. Some mushrooms are popular worldwide for their nutritional and therapeutic properties. However, some species are dangerous because they cause toxicity. There are many reports explaining the medicinal and/or toxic effects of these fungal species. Cases of serious human poisoning generally caused by the improper identification of toxic mushroom species are reported every year. Different substances responsible for the fatal signs and symptoms of mushroom toxicity have been identified from various poisonous mushrooms. Toxicity studies of mushroom species have demonstrated that mushroom poisoning can cause adverse effects such as liver failure, bradycardia, chest pain, seizures, gastroenteritis, intestinal fibrosis, renal failure, erythromelalgia, and rhabdomyolysis. Correct categorization and better understanding are essential for the safe and healthy consumption of mushrooms as functional foods as well as for their medicinal use.
Mushroom toxicity; Poisonous mushroom; Toxic compounds
The fungal genus Suillus Micheli ex S. F. Gray plays important roles in the survival and growth of plant seedlings. Humans have utilized these ectomycorrhizal fungi to enhance the nutrient uptake and defense systems of plants, particularly in the reforestation of coniferous forests. The genus Suillus is easily distinguishable by its distinctive morphological features, although the morphology of the fruiting body does not facilitate reliable interspecies discrimination. On the basis of micro-morphological features and internal transcribed spacer sequence analysis, we found that 51 of 117 Korean Suillus specimens had initially been misidentified. The list of the 12 Suillus species previously recorded in Korea was re-evaluated and revised to only eight distinct species: S. americanus, S. bovinus, S. granulatus, S. grevillei, S. luteus, S. pictus, S. placidus, and S. viscidus. We provide taxonomical descriptions for six of these species from the sample specimens.
Ectomycorrhizal fungi; Internal transcribed spacer; Seoul National University Fungal Collection; Suillus; S. luteus
On the basis of extensive field investigation and a series of herbarium specimen identifications, we present and discuss the descriptions and distribution of 22 species of Ramalina found in the Hengduan Mountains of southwestern China. In this revisionary study, representatives of the Ramalina genus, including R. americana, R. confirmata, R. dendriscoides, R. obtusata, R. pacifica, R. pentecostii, R. peruviana, R. shinanoana, and R. subcomplanata are found for the first time in this area. In addition, R. holstii is reported for the first time China. Finally, a newly described species identified as Ramalina hengduanshanensis S. O. Oh & L. S. Wang is reported. It is characterized as growing from a narrow holdfast, solid, sparsely or richly and irregularly dichotomously branched, palmate and flattened lobes with distinctly dorsiventral appearance, surface rugose to reticulate, surface rugosely cracked, dense chondroid tissue, helmet shaped soralia at the tip. The species grows on rock and tree at the highest elevations in this area. Although very few lichen species belonging to the genus Ramalina have been collected above 4,000 m, this new species is found at this elevation. We present detailed morphological, anatomical, and chemical descriptions of this species along with molecular phylogenetic analysis of the internal transcribed spacer rDNA sequences.
Hengduanshan; Key; Molecular phylogeny; New species
NADPH oxidases (Noxes), transmembrane proteins found in most eukaryotic species, generate reactive oxygen species and are thereby involved in essential biological processes. However, the fact that genes encoding ferric reductases and ferric-chelate reductases share high sequence similarities and domains with Nox genes represents a challenge for bioinformatic approaches used to identify Nox-encoding genes. Further, most studies on fungal Nox genes have focused mainly on functionality, rather than sequence properties, and consequently clear differentiation among the various Nox isoforms has not been achieved. We conducted an extensive sequence analysis to identify putative Nox genes among 34 eukaryotes, including 28 fungal genomes and one Oomycota genome. Analyses were performed with respect to phylogeny, transmembrane helices, di-histidine distance and glycosylation. Our analyses indicate that the sequence properties of fungal Nox genes are different from those of human and plant Nox genes, thus providing novel insight that will enable more accurate identification and characterization of fungal Nox genes.
Fungal genome; NADPH oxidase; Nox; Phylogenetics; ROS
We evaluated a more practical and cost-effective immobilization carriers for ethanol production using the yeast Saccharomyces cerevisiae. Three candidate materials-rice hull, rice straw, and sawdust-were tested for their cell-adsorption capacity and operational durability. Derivatizations of rice hull, rice straw, and sawdust with the optimal concentration of 0.5 M of 2-(diethylamino)ethyl chloride hydrochloride (DEAE · HCl) resulted in > 95% adsorption of the initial yeast cells at 2 hr for DEAE-rice hull and DEAE-sawdust and in only approximately 80% adsorption for DEAE-rice straw. In addition, DEAE-sawdust was found to be a more practical carrier for immobilizing yeast cells in terms of operational durability in shaking flask cultures with two different speeds of 60 and 150 rpm. Furthermore, the biosorption isotherms of DEAE-rice hull, -rice straw, and -sawdust for yeast cells revealed that the Qmax of DEAE-sawdust (82.6 mg/g) was greater than that of DEAE-rice hull and DEAE-rice straw. During the 404-hr of continuous column reactor operation using yeast cells immobilized on DEAE-sawdust, no serious detachment of the yeast cells from the DEAE-sawdust was recorded. Ethanol yield of approximately 3.04 g/L was produced steadily, and glucose was completely converted to ethanol at a yield of 0.375 g-ethanol/g-glucose (73.4% of the theoretical value). Thus, sawdust is a promising practical immobilization carrier for ethanol production, with significance in the production of bioethanol as a biofuel.
Continuous column reactor; Ethanol production; Immobilization carrier; Saccharomyces cerevisiae; Sawdust