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.

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.

Characterization of chikusaku-eki and mokusaku-eki and its inhibitory effect on sapstaining fungal growth in laboratory scale

Chikusaku-eki and Mokusaku-eki are natural resources and acidic liquid by-products of bamboo and broad leaved trees charcoal burner. These products contain more than 200 ingredients, including phenols, poly phenols and acetic acids. These by-products were tested for their fungicidal activity against sapstaining fungi. There are no studies about the antifungal activity of Chikusaku-eki and Mokusaku-eki against wood staining fungi in the literature. According to the recent findings, this is the first report about the antifungal activity of Chikusaku-eki and Mokusaku-eki against wood staining fungi. These extracts were more effective against sapstaining fungi at minimum concentrations [0.10-1.0%] used in 2% [malt extract agar] medium. Three Chikusaku-eki [Chikusaku-eki-I, Chikusaku-eki-II and Chikusaku-eki-III] and 2 Mokusaku-eki [Mokusaku-eki-I, Mokusaku-eki-II] extracts were tested against 4 sapstaining fungal samples to evaluate the inhibition range on sapstaining fungal growth. Ophiostoma flexuosum, Ophiostoma tetropii, Ophiostoma polonicum and Ophiostoma ips were the sapstaining fungi used in this study against bamboo and wood extracts. The initial calibration of extracts was done by calculating the specific gravity, tar calculation and total organic content. The chloroform fractions of these extracts were analyzed by gas chromatography-mass spectrometry and both the extracts contained 2,6 dimethoxy phenol, dehydroacetic acid and 2,3,5 trimethoxytoluene. Results revealed that compounds of Chikusaku-eki and Mokusaku-eki markedly inhibited fungal growth at lower concentration. The Chikusaku-eki and Mokusaku-eki have both antifungal, antioxidant properties and a potential to be used as natural preservative in wood industries. Wood slice tests displayed the effective role of both extracts in laboratory level

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.

Detection of Cellulolytic Activity in Ophiostoma and Leptographium species by Chromogenic Reaction

To understand the ability of producing cellulolytic enzyme activity in the sapstaining fungi, four species of Ophiostoma and two species of Leptographium were investigated in the culture media containing each of cellulose substrates such as CM-cellulose, Avicel and D-cellobiose and each of chromogenic dyes such as Congo-Red, Phenol Red, Remazol Brilliant Blue and Tryphan Blue. When the fungi were grown for 5~7 days at 25degrees C, the formation of clear zone by chromogenic reaction around the margin of the fungal colony was demonstrated in all the culture media Congo-Red containing CM-cellulose. There was difference in the formation of clear zone among the dyes. Only Ophiostoma setosum and Leptographium spp. showed cellulolytic activity to the three substrates. Overall, the results of this study show that ophiostomatoid sapstaining fungi can produce cellulolytic enzymes.

Analysis of Random Amplicied Polyorphic DNA (RAPD) for Sporothrix schenckii and Related Fungi / 대한의진균학회지

BACKGROUND: Sporotrichosis is a common deep cutaneous fungal disease caused by Sporothtix (S.) schenckii. The recent development of polymerase chain reaction (PCR) technology, in particular, arbitrarily primed PCR (AP-PCR) or random amplified polymorphic DNA (RAPD), has greatly enhanced the molecular detection and identification of various pathogenic agents, including fungi. OBJECTIVE: This study was aimed to differentiate Sporothrix schenckii, and related fungi such as S. schenckii var. luriei, S. flocculosa, S. nivea, Ophiostoma stenoceras, and clinical isolates on the basis of distinct DNA band patterns in the RAPD. METHODS: S. schendcii, S. schenckii var. luriei, S. flocculosa, S. nivea, Ophiostoma stenoceras from ATCC and KCCM, and clinical 10 isolates from Chonnam University Hospital were used for RAPD analysis. For RAPD, 3 random primers were used. Genomic DNA was extracted by Liu method. Amplification reactions were performed in volumes of 50 microL containing 10 mM Tris-HCI (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 200microM dNTP mixture, 40 pM primer, 1 U of Taq polymerase, DNA 20 ng. RESULTS: 3 decamers (5'-TGCCGAGCTG-3', 5'-AGTCAGCCAC-3', 5'-AATCGGGCTG-3') are generated in the RAPD, distinct DNA products from S. schenckii forming characteristic band patterns upon gel electrophoresis. Each random primer amplified characteristic same band patterns in DNA from clinical 8 isolates among 10 isolates, 2 isolates have different DNA band patterns. These results suggest of being a Sporothrix anamorph different from S. schenckii in Korea. CONCLUSION: With 2 random primers (5'-TGCCGAGCTG-3', 5'-AGTCAGCCAC-3') S. schenckii and related fungi investigated produced distinct DNA band patterns on gel electrophoresis. The RAPD was a very valuable laboratory method for identification of S. schenckii isolates.