Yeo Hong Yun

Fungal and Plant Phenylalanine Ammonia-lyase

Abstract L-Phenylalanine is one of the essential amino acids that cannot be synthesized in mammals in adequate amounts to meet the requirements for protein synthesis. Fungi and plants are able to synthesize phenylalanine via the shikimic acid pathway. L-Phenylalanine, derived from the shikimic acid pathway, is used directly for protein synthesis in plants or metabolized through the phenylpropanoid pathway. This phenylpropanoid metabolism leads to the biosynthesis of a wide array of phenylpropanoid secondary products. The first step in this metabolic sequence involves the action of phenylalanine ammonia-lyase (PAL). The discovery of PAL enzyme in fungi and the detection of 14CO2 production from 14C-ring-labeled phenylalanine and cinnamic acid demonstrated that certain fungi can degrade phenylalanine by a pathway involving an initial deamination to cinnamic acid, as happens in plants. In this review, we provide background information on PAL and a recent update on the presence of PAL genes in fungi.

Identification and Characterization of Eurotium rubrum Isolated from Meju in Korea.

We isolated and identified a strain of Eurotium rubrum from Meju that has not been reported in Korea. This fungus is yellowish brown; reverse dark brown on CYA and PDA while yellow on 2% MEA at 25°C. Cleistothecia are first bright yellow and gradually turned brown. Mycerial growth on CYA attained a diameter of 30 mm at 20°C, 37 mm at 25°C and 32 mm at 30°C after 15 days. The isolate grew slower on 2% MEA (< 20 mm 15 days at 25°C) compared to CYA and PDA (< 40 mm 15 days at 25°C). Cleistothecia are superficial, yellow to light brown, globose to subglobose, 40~75 μm in diameter. Asci are 8-spored and globose to subglobose 8~11 μm. Ascospores are disciform, 4.0~5.0 μm in length and 4.2~4.5 μm in width. Conidia are ovate or bacillar, finely roughened to densely spinulose, 4.6~6.0 μm in length and 3.0~4.3 μm in width. Compared to known Eurotium rubrum, the Korean isolate showed 99% sequence similarity in ITS rDNA (554 bp) and calmodulin (750 bp) gene and 100% in β-tubulin (1016 bp) gene. The E. rubrum isolate also had weak β-glucosidase and protease activities.

Yeast Associated with the Ambrosia Beetle, Platypus koryoensis, the Pest of Oak Trees in Korea

Abstract Oak tree death caused by symbiosis of an ambrosia beetle, Platypus koryoensis, and an ophiostomatoid filamentous fungus, Raffaelea quercus-mongolicae, has been a nationwide problem in Korea since 2004. In this study, we surveyed the yeast species associated with P. koryoensis to better understand the diversity of fungal associates of the beetle pest. In 2009, a total of 195 yeast isolates were sampled from larvae and adult beetles (female and male) of P. koryoensis in Cheonan, Goyang, and Paju; 8 species were identified by based on their morphological, biochemical and molecular analyses. Meyerozyma guilliermondii and Candida kashinagacola were found to be the two dominant species. Among the 8 species, Candida homilentoma was a newly recorded yeast species in Korea, and thus, its mycological characteristics were described. The P. koryoensis symbiont R. quercus-mongolicae did not show extracelluar CM-cellulase, xylanase and avicelase activity that are responsible for degradation of wood structure; however, C. kashinagacola and M. guilliermondii did show the three extracellular enzymatic activities. Extracelluar CM-cellulase activity was also found in Ambrosiozyma sp., C. homilentoma, C. kashinagacola, and Candida sp. Extracelluar pectinase activity was detected in Ambrosiozyma sp., C. homilentoma, Candida sp., and M. guilliermondii. All the 8 yeast species displayed compatible relationships with R. quercus-mongolicae when they were co-cultivated on yeast extract-malt extract plates. Overall, our results demonstrated that P. koryoensis carries the yeast species as a symbiotic fungal associate. This is first report of yeast diversity associated with P. koryoensis.

New Record of Mariannaea elegans var. elegans in Korea

A Mariannaea fungus was isolated during investigation of an elm tree infested with unidentified beetles. Based on morphological characteristics and molecular analysis of the internal transcribed spacer rDNA sequence, the fungus was identified as Mariannaea elegans var. elegans. Fungal growth was better on malt extract agar than on potato dextrose agar and oatmeal agar. Optimal temperature and pH for growth of the fungus were 30°C and pH 7.0, respectively. The fungus was found to have the ability to produce extracellular enzymes such as amylase, β-glucosidase, cellulase, and protease. This is first report on M. elegans var. elegans in Korea.

Identification and Characterization of Gliocladium viride Isolated from Mushroom Fly Infested Oak Log Beds Used for Shiitake Cultivation

Abstract A green mold species that has not previously been reported in Korea was isolated from oak log beds used for shiitake (Lentinula edodes) cultivation that were infested by mushroom flies. In this study, we identify the mold species as Gliocladium viride (an anamorph of Hypocrea lutea) and describe its myeologieal properties. The fungus was cottony on both potato dextrose agar (PDA) and Czapek yeast extract agar (CYA), but was colored white on PDA and became yellowish green and brown on CYA. Mycelial growth on PDA attained a diameter of 73 mm at 30°C after 5 days. The fungus grew faster on malt extract agar (> 80 mm, 5 days at 25°C) compared to CYA and PDA (< 68 mm, 5 days at 25°C). Penieillate eonidiophores of the fungus are hyaline, smooth walled, branching above ty pically in four stages, and 120–240 pm in length. Club-shaped or slender phialides are formed on the metulae. Conidia of the fungus were ovate and elliptic, yellowish brown and green, and 2.5~3.0 μm × 1.8–2.3 μm in size. Typically, slimy conidia are formed in a mass and colored brown to dark green to almost black. The internal transcribed spacer rDNA and translation elongation factor 1 alpha gene sequences of the fungus isolated here show 99% identity with previously identified G viride strains.

Mold Occurring on the Air Cleaner High- Efficiency Particulate Air Filters Used in the Houses of Child Patients with Atopic Dermatitis

Abstract Fungi are the known sources of irritation associated with atopic diseases (e.g., asthma, allergic rhinoconjunctivitis, and atopic eczema). To quantitatively estimate their presence in the indoor environment of atopic dermatitis-inflicted child patient’s houses (ADCPHs), the high-efficiency particulate air (HEPA) filters installed inside the air cleaners of three different ADCPHs were investigated for the presence of mold. The air cleaner HEPA filters obtained from the three different ADCPHs were coded as HEPA-A, -B, and -C, respectively, and tested for the presence of mold. The colony forming units (CFUs) corresponding to the HEPA-A, -B, and -C filters were estimated to be 6.51 × 102 ± 1.50 × 102 CFU/cm2, 8.72 × 102 ± 1.69 × 102 CFU/cm2, and 9.71 × 102 ± 1.35 × 102 CFU/cm2, respectively. Aspergillus, Penicillium, Alternaria, Cladosporium, Trichoderma, and other fungal groups were detected in the 2,494 isolates. The distribution of these fungal groups differed among the three filters. Cladosporium was the major fungal group in filters HEPA-A and -C, whereas Penicillium was the major fungal group in the filter HEPA-B. Nine fungal species, including some of the known allergenic species, were identified in these isolates. Cladosporium cladosporioides was the most common mold among all the three filters. This is the first report on the presence of fungi in the air cleaner HEPA filters from ADCPHs in Korea.

Identification and Characterization of Trichoderma Species Damaging Shiitake Mushroom Bed-Logs Infested by Camptomyia Pest.

The shiitake mushroom industry has suffered from Camptomyia (gall midges) pest, which feeds on the mycelium of shiitake mushroom during its cultivation. It has been postulated that fungal damage of shiitake bed-logs is associated with infestation by the insect pest, but this is not well understood. To understand the fungal damage associated with Camptomyia pest, various Trichoderma species were isolated, identified, and characterized. In addition to two previously known Trichoderma species, T. citrinoviride and T. deliquescens, two other Trichoderma species, T. harzianum and T. atroviride, were newly identified from the pestinfested bed-log samples obtained at three mushroom farms in Cheonan, Korea. Among these four species, T. harzianum was the most evident. The results of a chromogenic media-based assay for extracellular enzymes showed that these four species have the ability to produce amylase, carboxyl-methyl cellulase, avicelase, pectinase, and β-glucosidase, thus indicating that they can degrade wood components. A dual culture assay on PDA indicated that T. harzianum, T. atroviride, and T. citrinoviride were antagonistic against the mycelial growth of a shiitake strain (Lentinula edodes). Inoculation tests on shiitake bed-logs revealed that all four species were able to damage the wood of bed-logs. Our results provide evidence that the four green mold species are the causal agents involved in fungal damage of shiitake bed-logs infested by Camptomyia pest.

Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes.

Abstract Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi.

Mariannaea samuelsii Isolated from a Bark Beetle-Infested Elm Tree in Korea.

During an investigation of fungi from an elm tree infested with bark beetles in Korea, one isolate, DUCC401, was isolated from elm wood. Based on morphological characteristics and phylogenetic analysis of the internal transcribed spacer and 28S rDNA (large subunit) sequences, the isolate, DUCC401, was identified as Mariannaea samuelsii. Mycelia of the fungus grew faster on malt extract agar than on potato dextrose agar and oatmeal agar media. Temperature and pH for optimal growth of fungal mycelia were 25°C and pH 7.0, respectively. The fungus demonstrated the capacity to degrade cellobiose, starch, and xylan. This is the first report on isolation of Mariannaea samuelsii in Korea.

UmTco1, a Hybrid Histidine Kinase Gene, Is Essential for the Sexual Development and Virulence of Ustilago maydis

Hybrid histidine kinase is part of a two-component system that is required for various stress responses and pathogenesis of pathogenic fungi. The Tco1 gene in human pathogen Cryptococcus neoformans encodes a hybrid histidine kinase and is important for pathogenesis. In this study, we identified a Tco1 homolog, UmTco1, in the maize pathogen Ustilago maydis by bioinformatics analysis. To explore the role of UmTco1 in the survival of U. maydis under environmental stresses and its pathogenesis, Δumtco1 mutants were constructed by allelic exchange. The growth of Δumtco1 mutants was significantly impaired when they were cultured under hyperosmotic stress. The Δumtco1 mutants exhibited increased resistance to antifungal agent fludioxonil. In particular, the Δumtco1 mutants were unable to produce cytokinesis or conjugation tubes, and to develop fuzzy filaments, resulting in impaired mating between compatible strains. The expression levels of Prf1, Pra1, and Mfa1, which are involved in the pheromone pathway, were significantly decreased in the Δumtco1 mutants. In inoculation tests to the host plant, the Δumtco1 mutants showed significantly reduced ability in the production of anthocyanin pigments and tumor development on maize leaves. Overall, the combined results indicated that UmTco1 plays important roles in the survival under hyperosmotic stress, and contributes to cytokinesis, sexual development, and virulence of U. maydis by regulating the expression of the genes involved in the pheromone pathway.