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

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. quercusmongolicae 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.

Ophiostoma ips from Pinewood Nematode Vector, Japanese Pine Sawyer Beetle (Monochamus alternatus), in Korea

Japanese pine sawyer beetle (Monochamus alternatus) is an economically important pest in coniferous trees. Ophiostoma ips was isolated from the beetle and identified based on analysis of morphological properties and the beta-tubulin gene sequence. The fungus easily produced perithecia with a long neck on malt extract agar and its ascospores were rectangular shaped. This is first report of Ophiostoma species associated with the pinewood nematode vector beetle in Korea.

A New Record of Candida kashinagacola (Synonym Ambrosiozyma kashinagacola) from Galleries of Platypus koryoensis, the Oak Wilt Disease Vector, in Korea

The ambrosia beetle, Platypus koryoensis, is an economically important pest affecting oak trees in Korea. Candida kashinagacola was isolated from galleries of the beetle in oak wood and identified by analyses of morphology, physiological properties, and nucleotide sequence of the large subunit ribosomal DNA. This is the first report on Candida species associated with oak wilt disease vectored by the ambrosia beetle, Platypus koryoensis, in Korea.

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 25degrees 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.

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 30degrees C and pH 7.0, respectively. The fungus was found to have the ability to produce extracellular enzymes such as amylase, beta-glucosidase, cellulase, and protease. This is first report on M. elegans var. elegans in Korea.

Filamentous Fungi Isolated from Platypus koryoensis, the Insect Vector of Oak Wilt Disease in Korea

The ambrosia beetle, Platypus koryoensis, is a serious pest of oak trees in Korea. In this study we investigated filamentous fungi present in the body of the beetle. Fourteen genera of filamentous fungi belonging to Ascomycota and Basidiomycota were isolated. Among the isolated fungi, some were able to produce wood degrading enzymes. This is first report of fungi associated with P. koryoensis.

Characterization of a Sapstaining Fungus, Ophiostoma floccosum, Isolated from the Sapwood of Pinus thunbergii in Korea

An Ophiostoma fungus was isolated from a stump of Pinus thunbergii in a forest on the West coast of Korea. Microscopic analysis using a light microscope, a stereo microscope, and a scanning electron microscope revealed that it had morphological features of Pesotum and Sporothix synanarmorphs. Based on the beta-tubulin gene sequence analysis, the fungus was identified as the anamorph of Ophiostoma floccosum. Mycological properties of the species including its growth properties on different culture media were described.

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 25degrees C. Cleistothecia are first bright yellow and gradually turned brown. Mycerial growth on CYA attained a diameter of 30 mm at 20degrees C, 37 mm at 25degrees C and 32 mm at 30degrees C after 15 days. The isolate grew slower on 2% MEA (< 20 mm 15 days at 25degrees C) compared to CYA and PDA (< 40 mm 15 days at 25degrees C). Cleistothecia are superficial, yellow to light brown, globose to subglobose, 40~75 microm in diameter. Asci are 8-spored and globose to subglobose 8~11 microm. Ascospores are disciform, 4.0~5.0 microm in length and 4.2~4.5 microm in width. Conidia are ovate or bacillar, finely roughened to densely spinulose, 4.6~6.0 microm in length and 3.0~4.3 microm 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 beta-tubulin (1016 bp) gene. The E. rubrum isolate also had weak beta-glucosidase and protease activities.

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.

Fungi Isolated from Pine Wood Nematode, Its Vector Japanese Pine Sawyer, and the Nematode-Infected Japanese Black Pine Wood in Korea

Pine tree death caused by pine wood nematode (PWN) involves phoretic relationships between PWN and its vector Japanese pine sawyer beetle (JPS). In an effort to understand the diversity of fungi involved in PWN life cycle, a total of 176 fungal isolates were collected from PWNs, adults and larvae of JPS, PWN-diseased Japanese black pine that was cut down in 2005 at Jinju, Korea. Based on microscopic observation and colony morphology, and sequence analysis of the ITS rDNA, the fungal isolates were identified at the level of genus. Three genera including Mucor, Ophiostoma, and Penicillium were identified from PWN. Two genera of Ophiostoma and Penicillium were discovered from JPS larvae. From JPS adult beetles, nine genera of Aspergillus, Gibberella, Hypocrea, Irpex, Leptosphaeria, Ophiostoma, Penicillium, and Plectosphaerella and unknown basidiomycetes were found. Ten genera from PWN-infected wood were confirmed as Bionectria, Botrytis, Camarops, Fusarium, Hypocrea, Nectrtia, Mucor, Ophiostoma, Penicillium, and Trichoderma. Penicillium and Ophiostoma were commonly distributed on PWN and its vector and host. This is first report of the fungi associated with PWN and its vector and host in Korea.