Ecological significance of mycorrhizal symbiosis in wild orchids. ( Dr. Iwase) MAPK MEK () ( ) 3 Uromyces () ()

() () () (. ) ITS/5.8S rDNA GPD () cichoric acid ()

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( )

() () amphotericin B ()

( ) () Halophytophthora () () () Pseudozyma () () () ( ) ()

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() ( )

() Sacchachitosan ( () () ( )

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() RAW 264.7 ()

Monascus pilosus Monascus purpureus ()

() () SCAR ()

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mRNA ()

() (Pleurotus ferulae) ( ) () () () IGS-RFLP () Achlya () () - () () ()

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Ecological significance of mycorrhizal symbiosis in wild orchidsKoji IwaseFungus/Mushroom Resource and Research Center, Faculty of Agriculture, Tottori University

Orchid is a group of plants, which has specifically evolved characteristics such as very small seeds containing very small amount of energy source (sometimes called as dust seeds), flowers with fantastic morphology, and symbiosis with fungi in root cells. Orchid seeds swell to germinate when the appropriate moisture is available. If the undifferentiated embryo is colonized by suitable and compatible fungi, it could grow to form a clump of cells called protocorm followed by the differentiation of organs to grow into adult plant in nature. Orchid is known to be mycoheterotrophic in germination and protocorm stage in natural habitat because of non-photosynthetic ability. In most photosynthetic orchids, their mycorrhizal fungi have been known to belong to the form-genus Rhizoctonia. On the other hand, other basidiomycete species have been described in many achlorophyllous orchids as the mycorrhizal fungi. Fungal isolation and inoculation experiments have revealed the association of Armillaria spp. in Galeola septentrionalis and in Gastrodia elata, and the association of Erithromyces crocicreas in Galeola altissima. In this study, we conducted to identify the mycorrhizal fungi in acholophyllous species, Epipogium roseum and chlorophyllous species, Cepharanthera falcata. In Epipogium roseum, the several isolates were obtained from peloton, and the DNA analysis

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indicated some of them were classified to gunus Coprinus, and other to genus Psathyrella, both in Coprinaceae. When compatible mycorrhizal fungi is inoculated, seeds could germinate to grow into protocorm, and grew further to form inflorescence under controlled cultivation condition. This is the first study to achieve the whole life cycle from seed to flowering under controlled condition in wild orchid. In a surrounding of the colony of this orchid, many wood logs or fallen leaves are always found, which indicates that those could be used as an energy source for the orchid through the hyphae of mycorrhizal fungi. On the other hand, in C. falcata, the fungi isolated from peloton were clustered with Tomentella in Thelephoraceae or Russula in Russulaceae. Those fungi in Thelephoraceae or Russulaceae are known to from ectomycorrhiza with woody plants. Therefore, in this association among orchid C. falcata, mycorrhizal fungi, and woody plants, tri-partnership symbiosis is suggested.

MAPK MEK

Molecular cloning and functional analysis of MAPKs and MEKs in Phytophthora parasitica-Hao-Zhi Yan, Chien-Yu Huang, and Ruey-Fen Liou (Department of Plant Pathology and Microbiology, National Taiwan University, Taipei). MAPK MAPK RACE 4 MAPK 5 MEK MAPK MEK 4 MAPK MEK MAPK MEK MAPK MAPK The Mitogen-activated protein kinase (MAPK) pathway, with a prototype of the cascade consisting of MAPK kinase kinase, MAPK kinase (MEK), and MAPK, is known to play essential roles in the signaling of stress response, mating, cell integrity, and pathogenicity in a variety of fungi. Not much is known, however, about the structural characteristics and possible roles of genes

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involved in the MAPK signaling pathway in Oomycetes including Phytophthora. In this study, genes encoding MAPK (ppmk) and MEK (ppmek) in P. parasitica, an important plant pathogen, were cloned by PCR using degenerate primers, followed by 5- and 3-RACE. Analysis of the deduced amino acid sequences indicated that ppmks possess the MAPK signature as well as the eleven conserved catalytic subdomains that are typical of Ser/Thr protein kinases and, with the exception of one gene, all ppmeks have the conserved MEK activation site. Phylogenetic analysis indicated that, with both MAPKs and MEKs, genes of P. parasitica form a clade distinct from those of fungi and plants. To further characterize the ppmk genes, their expression in response to a variety of experimental conditions was analyzed by real-time quantitative reverse transcriptasePCR. Besides, recombinant proteins of ppmks were obtained from E. coli and analyzed by in vitro kinase assay. Results obtained from these experiments will be discussed.

Development of fungi-fermented health food using solid state fermentation--Yu-Ling Lee, Hui-Ching Kuo, Shao-Yu Jian, Tsai-Ping Wu and Jeng-Leun MauDepartment of Food Science and Biotechnology, National Chung-Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan Cordyceps sinensisTermitomyces albuminosus Taiwanofungus camphoratus pH

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Recently, the use of mushroom as a food source has changed from personal preference to its physiological activities due to the concern of health. Currently, mushrooms are obtained in the form of fruit bodies and mycelia. Some species of fruit bodies are hardly cultivated, such as Cordyceps sinensis, Termitomyces albuminosus and Taiwanofungus camphoratus. In addition, an excess production of rice became a major issue for farmers. Then, many reports showed that solidstate fermentation may offer numerous advantages for the production of microbial chemicals and enzymes. Therefore, this report was to evaluate the optimal conditions of Cordyceps-fermented rice, Taiwanofungus-fermented rice and Termitomyces-fermented wheat as evidenced by their ergosterol contents and pH values.

3 Uromyces 1 21

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Morphological and molecular phylogenetic studies of three Uromyces species on legumes in Japan Wen-Hsin Chung1, Makoto Kakishima2 (Department of Plant Pathology, National Chung Hsing University, TaiwanLife and Environmental Sciences, University of Tsukuba, Japan) Uromyces viciae-fabae var. viciae-fabae var. orobi Vicia Lathyrus Pisum U. viciae-fabae 94 U. viciae-fabae 23 D1/D2 ITS U. viciae-

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fabae Uromyces appendiculatus U. vignae Phaseolus Vigna Apios Lablab Dunbaria U. appendiculatus U. vignae 225 45 D1/D2 ITS 2 3 ITS Pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciaefabae and var. orobi, based on difference in urediniospore wall thickness and putative host specificity in Japan. Morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus and Pisum did not show definite host specific morphological groups. While 23 Uromyces specimens from Vicia, Lathyrus and Pisum formed a single genetic clade based on D1/D2 and ITS regions. These results suggest that U. viciae-fabae populations on different host plants are not differentiated to groups that can be recognized as varieties. Uromyces appendiculatus, inclusive of three varieties, is distinguished from U. vignae primarily by the position of urediniospore germ pores and putative host specificity. However, opinions over these morphological and physiological features as a taxonomic character have varied greatly and distinction of these species has often been confused. Morphological features of urediniospores and teliospores of 225 rust fungus specimens on species of Phaseolus, Vigna, Apios, Lablab and Dunbaria were examined. The position of germ pores in urediniospores and the teliospore wall thickness were considered as good characters to separate three morphological groups. While 45

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specimens fell into two and three clades based on the nucleotide sequence at D1/D2 and ITS regions, respectively. Neither morphological groups nor molecular clades were host-limited. It is suggested that three morphological groups that corresponded to three distinct ITS clades constitute distinct species.

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Preliminary survey of Ganoderma in Taiwan--Dong Mei Wang1,2, Sheng Hua Wu1 (1Department of Botany, National Museum of Natural Science, Taichung, Taiwan 404, ROC, 2Systematic Mycology and Lichenology Laboratory, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, China)

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25 25 233 79 ITS 21 mitSSU 19 14 G. flexipes Pat. G. multiplicatum (Mont.) Pat. G. australe (Fr.) Pat. G. lucidum (Curtis) P. Karst. Ganoderma P. Karst., a polypore genus, is well known for its economical and ecological importance. Survey of Taiwanian Ganoderma was initiated in early of the 20th century and 25 names, so far, have been recorded from Taiwan by a literature search. Some names were treated as synonyms, or transferred to other genera. The checklist of Taiwanian Ganoderma is being prepared. In our study, 233 Ganoderma specimens have been subjected to morphological examination, including 25 names except for those unidentified materials. Seventy-nine ITS sequences and 21 mitSSU sequences have been obtained from our study. Currently, 19 Ganoderma taxa including 14 from Taiwan have been tentatively recognized from these specimens based on morphological and molecular data. Among these recognized taxa, some are new to Taiwan, e.g. G. flexipes Pat. and G. multiplicatum (Mont.) Pat.. The taxa generally treated as G. australe (Fr.) Pat. and G. lucidum (Curtis) P. Karst. in Taiwan have been proved to comprise several species.

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Investigation and Monitoring of Macrofungal Diversity in a Cryptomeria japonica plantation--PiHan WangDepartment of Life Science, Tunghai University Pi-Han Wang (Department of Life Science, Tunghai University) 14% 95 40 392 15,846 70 1,228 99% 1%

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87%13% 25% 50% Forest ecosystem is one of the most important ecosystems in Taiwan. Among these area, 14% are plantation forests. The new goal is timber production with consideration of its impacts on climate changes, biodiversity losses, ecosystem functions and public acceptance. We propose to monitor and quantify macrofungal community dynamics after 0%, 25% and 50% thinning practices from 2007. We have established 12 one hectare permanent plots in Cryptomerioid japonica plantation forest in central Taiwan. Within this plot, macrofungi are systematically survey and collect as baseline data in 2006. In this year, we collected 392 mushroom species, and 15,846 samples from the plantation plots. Tricholomataceae and Lepiotaceae are dominant in the plantation plots. We collected 70 mushroom species, and 1,228 samples from natural forest plot. Tricholomataceae is dominant in the natural forest plot. There are 99% saprophytic fungi and 1% paragenesis fungi in plantation plots. There are 87% saprophytic fungi and 13% paragenesis fungi in the natural plot.

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Orchid mycorrhizal fungi were isolated from the roots of seven wild grown orchids--Shu-Fen Cheng1, Hsiao-Yi Chen1, Jin-Liang Chen2 , Yuan-Tay Shyu1, and C.N. Chang1(1Department of Horticulture, National Taiwan University, Dept. of Hospital and Health Care Administration) 104 Trichoderma(Mucor) (Xylaria) 57 3

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Roots of five species of Calanthe and two species of Phaius from north, midland and south of Taiwan were collected for the isolation of orchid mycorrhizal fungi. Total for 104 isolates of fungi were isolated. They are identified respectively by the traditional method or DNA sequence. Results showed that Trichoderma and Mucor were most often isolated fungi. The DNA sequence results showed that only Xylaris was most common fungus. Fifty seven isolate species are not identified yet and three isolates were identified with both traditional and DNA sequence of cultured pellets at the same time, the result are not corresponding and needed further investigation.

1 2 3 4 51,2,3,4,5

The diversity of the rice sheath blight pathogens in Taiwan--Sz-Hau Chen1, Siang-Jhe Yang2, Yi-jen Lin3, C.H. Chang4, Lung-Chung Chen5 (1,2,3,4,5Department of Plant Pathology National Chung Hsing University) (Rhizoctonia solani AG1IA)

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Rhizoctonia solani AG1-IA 28 67.3625mm/day 1.198mm (MCGs) 0 5 2.8 3.3 (Single primer PCR) NTSYS UPGMA 68.8% (Rhizoctonia solani AG1-IA) 86% ( ) ( ) ( )( ) 60% 11 I II IV V VIII IX X dsRNA I II III VII VIII Rice is used to be the major agriculture production in lots of country. There are couple pathogens which cause the reduction of yield, especially the rice sheath blight and rice blight. The rice sheath blight is caused by Rhizoctonia solani AG1-IA, and the existing of sheath blight dominantly happened in the area with high temperature and moisture. There fore, the position of Taiwan is the reason why cultivated rice suffered because of the seasons are closed to the weather aforementioned. After isolation of the pathogens collected from each county of Taiwan, and besides the isolation of Rhizoctonia solani AG1-IA, there still have some others with smaller sclerotia or light colored one. Besides, with the inoculation of different isolates collected in Taiwan on rice TaiKing 9 , there seems to be no such difference of disease index surrounded between 2.8 to 3.3 under the circumstance of disease index between 0 and 5.0. Besides, about physiological characteristic test, isolates were cultured at various temperature and pH value, to botanize mycelial growth rate and dry weight of sclerotia, and numbers of sclerotia. The result suggested that the mycelial growth of all isolates was 28 , and the average colony size was 67.3625 mm/day, and then the average mycelial growth rate was 1.198 mm/hr. About mycelial compatible groups (MCGs) test, there were four groups in Taiwan by mycelial compatible and incompatible ration. On the other hand, using single primer PCR to amplify difference fragments and to analysis

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genetic similarity by UPGMA cluster analysis with computer software Gel Compare. Dendrogram based on the analysis of 54 isolates of Rhizoctonia solani AG1-IA, and the result suggested to differentiate Rhizoctonia solani AG1-IA in Taiwan or Japan and other AGs in similarity 68.8%; and further more, isolates of Rhizoctonia solani AG1-IA from Taiwan could differentiate into five groups in similarity 86%. Group north: isolates from Yilan, Hsinchu, Taoyua. Group midland: isolates from Taichung, Hanghua, Nantou, Chiayi, Yunlin. Group east: isolates from Hualien. Group south: Pingtung, Tainan, Chiayi. Group complex: a large breadth group. In deep, the phylogenetic tree was obviously discriminated in to eleven subgroups of the dormant population of R. solani AG1-IA based on the five groups. The subgroups of phylogenetic tree are as follows: Group I includes Taoyuan County, TaiChung City, Changhua County and Yunlin county, Pintung County, HualienCounty. That seems to be a crop production street across the western field of island Taiwan. The Group II is related only with Kaohsiung County. And Group IV is closed to deposit on Chiayi County. The last are that Group V Tainan County and Group VIII is Taoyuan County, Group IX the Naotou County, Group X Chiayi County and Hualien County. The last group XI is prefer to Yilan County. Beside, the phylogenetic tree of the diversity with dsRNAs are constituted by eight subgroups separated to: Group I Changhua County Yunlin County, Chiayi County, Hualien County Group II Taichung County Group III Pingtung County Group VII Tainan County and the Group VIII Yilan County, Taoyuan County and Hsinchu County. We also have the result which is interconnected both with the phylogenetic tree with dsRNA and RAPD, which means the distribution do have specific geographical specification among the dsRNA and the RPAD. The relationship between MCGs and dendrogram based on SSR-PCR analysis showed that diversity of isolates from Taiwan had high degree with geography. This had a great effect upon the diversity of isolates from Taiwan with geography more than with rice variety (host). Therefore under the circumstance with reducing pesticide supplying, to realize the mechanism with virulence and hypovirulence would be a potential plan to manage the sheath blight of rice.

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The research of heavy metal content in Agaricus blazei Murrill in Taiwan--Hsin-Der Shih1, IHsin Lee1, Wen-Yuh Lin1, Chien-Yih Lin3, Horng-Yuh Guo2, Kuan-Tzer Wu1 and Chien-Liang Chu2 (1Divison of Plant Pathology, 2Divison of Agricultural Chemistry, 3Director-general, Taiwan Agricultural Research Institute, Wufeng 413, Taiwan)

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(Agaricus blazei Murrill ABM) -D- 3 1.2. 3. 2 ppm 3 ppm 3.71 187.38 GAP It has been reported in recent years that Agaricus blazei Murrill (ABM) has -D- glucan, which has anticancer effect and was verified in the clinical trials that it is efficient in improving the immunity system, reducing serum lipid, activating the third component in complement system and interferon induction, etc. However, some papers have reported that it is characteristic of ABM to accumulate heavy metals during growth period which influence its quality greatly. ABM could be manufactured into highly potential products for the market place if the heavy metal content of ABMs produced in Taiwan could be lowered. Focusing on major ABM farms in Taiwan, this study investigated cultivation conditions and methods. The farms studied located mostly in Taichung, Nantou, Changhua County in central Taiwan and Tainan, Kaohsiung County in southern Taiwan. Of the seventeen farms investigated, only four utilized air-conditioned growing houses, which produce ABM all year round. Soil used by most farmers to cover the sawdust bags is taken locally from mountain areas, river beds (which contain mostly sand) or crop fields; very few farmers purchase peat moss for the purpose. There are three main cultivation methods; one method places the sawdust bag on the bed and another method places the sawdust bag directly on the ground, while the last method places the sawdust in the tray. The investigation sampled various materials used during the cultivation process, and the materials included compost (fertilizer and raw material), casing mixtures, water, and fruiting body both fresh and dried. The analysis result showed some of the ABM produced domestically contain Cadmimum over 2 ppm, and few ABM

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sampled contain lead over 3 ppm. Based on the finding of this research, we assumed there is a difference of 3.71-187.38 folds in Cadmimum accumulation ability amongst different types of ABMs. The result also showed that the cap of the ABMs contained more Cadmimum concentration than the leg of the ABMs. Further, after boiling the ABM in two separate experiments, one with the whole fruiting body while the other with the fruiting body grounded up into powder, the results showed the Cadmimum in ABM has low solubility. Our data suggested that high Cadmimum concentration casing mixture will surely produce ABMs with high density of Cadmimum. Future research will explore further into the methods of reducing Cadmimum concentration during ABM cultivation. The project is optimistic that a GAP production system of ABM will be established in Taiwan. The production of ABM in Taiwan will not only meet the FDA standard for quality but will also satisfy the quantity required for domestic and international market.

ITS/5.8S rDNA GPD

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Phylogeny of Cercophora and Podospora (Lasiosphaeriaceae) inferred from nuclear internal transcribed spacers and GPD sequences--Jong-How Chang and Yei-Zeng Wang (National Museum of Natural Science, 1 Kuan-Chien Rd. Taichung 404, Taiwan) (Cercophora)(Podospora) 6 22 (hyaline sigmoid ascospore) (subapical globule) (pedicel) (Schizothecium) DNA (polymerase chain reaction, PCR) ITS/5.8S rDNA GPD ( -3- glyceraldehyde-3-phosphate dehydrogenase) ITS/5.8S rDNA GPD (maximum parsimony)(Bayesian Inference) (polyphyletic) Coprophilous Pyrenomycetes of Cercophora and Podospora (Lasiosphaeriaceae) are usually grown on dung of herbivores. Six species of Cercophora and twenty-two species of Podospora have been previously reported from Taiwan. The genus Podospora is characterized by ascospores with hyaline pedicel and gelatinous appendages while Cercophora Fuckel has hyaline, cylindrical ascospore with a swollen, pigmented head. Based on the morphology of perithecia and spore development, Podospora was divided into Podospora and Schizothecium by some mycologists. This study focuses on the phylogenetic relationships of Cercophora and Podospora. Crude genomic DNA from each pure strain was isolated. The ribosomal DNA internal transcribed spacers (ITS/5.8S rDNA) and the fragment of glyceraldehyde-3-phosphate dehydrogenase (GPD) sequences were amplified by polymerase chain reaction (PCR). Multiple gene sequences were analyzed using maximum parsimony and Bayesian analyses. In all analyses, Cercophora and Podospora were found to be polyphyletic, consisting of a group of morphologically heterogeneous and phylogenetically distant species. Those species characterized by perithecia adorned with swollen agglutinated hairs are grouped in one clade. Currently, there is insufficient evidence to support the transfer of P. conica group into the genus

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Schizothecium. Thus, we consider Schizothecium as a synonym under Podospora.

cichoric acid -

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Extraction of cichoric acid-active ingredient of coneflower by microbial fermentation--Chao Feng Tseng, Chi-Guang Wu Department of Biotechnology and Bioinformatics, Asia University, Taichung, Taiwan HPLC cichoric acid cichoric acid VA CK VA cichoric acid 472.520 g/mLCK 245.638g/mL cichoric acid cichoric acid In this study, fermentation was applied to extract cichoric acid from coneflower, which was monitored by HPLC analysis. Our data indicates that the concentration of cichoric acid show significant difference pre- and post- fermentation. The concentration of cichoric acid increased three times if compared with the initial background value. In the mycorrhizal inoculation test, the total dry weight of inoculated coneflower (VA) was more than control. The concentration of cichoric acid in the leaves of inoculated coneflower, 472.520 g/mL was significantly higher than uninoculated control 245.638 g/mL. In this study, a combination of mycorrhizal inoculation and microbial fermentation strategy to promote the use of coneflower and its commercial value was evaluated. Keyword: Coneflower ( Echinacea sp.), Mycorrhiza, Cichoric acid.

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Identification of orchid mycorrhizal fungi and its inoculum composition effects on the growth of seedlings of Anoectochilus formosanus Hayata--Ming-Chih Lee1, Kuo-Chi Lee2 and Doris C.N. Chang1(1Graduate Institute of Horticulture, Taiwan University, No. 1, Sec. 4, Roosevelt Road, 10617 Taipei, Taiwan and 2Council of Agriculture, 37 Nan-Hai Road, 10014 Taipei, Taiwan) (2004) R02 R04 ITS rDNA R01~R09 9 Rhizoctonia endophytica (R01) Rhizoctonia callae (R02) Fusarium oxysporum ( R03 F01)Fusarium solani ( R08 F02) Rhizoctonia solani (R04R05R06R07 R09 AG-6 group) () R09 (R. solani) R07 (R. solani) 2~3 () R09 ( R. solani, AG-6) Chou(2004) indicated that Rhizoctonia spp. of orchid mycorrhizal fungi (OMF) R02 and R04 could highly-enhance the growth of Anoectochilus formosanus Hayata. Morphology observation, keys identification, hyphal anastomosis reactions and internal transcribed spacer ribosomal DNA(ITS rDNA) were used to identify the OMF isotates R01~R09. Results showed that they were Rhizoctonia endophytica (R01), Rhizoctonia callae (R02), Fusarium oxysporum (R03, now changed to F01) Fusarium solani (R08 now as F02) and Rhizoctonia solani (R04, R05, R06, R07 and R09 all belonged to AG-6 group), respectively. When A. formosanus Hayata (Taitung cv.) under plastic bag cultivation method(PBCM) and cultivated at low temperature condition, the fresh weights of seedlings were significantly enhanced by the inoculation of OMF isolates R09 and R07 (both were Rhizoctonia solani, multi-nucleate). To use a mixture of two or three Rhizoctonia spp. of solates on the growth of A. formosanus Hayata by both PBCM and traditional cultivation methods. Results showed that growth of seedlings were about the same. Therefore, single isolate inoculation of OMF was recommended for the cultivation of A. formosanus Hayata. It is recommended to use OMF isolate R09 (R. solani, AG-6) as inoculum for the cultivation of A. formosanus Hayata in commercial use.

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Study of the mechanisms of orchid mycorrhizal fungi on the survival rate and the growth of Phalaenopsis--Po-Hung Wu1, Doris C.N. Chang1 and D.D Huang2(1Graduate Institute of Horticulture, Taiwan University, No. 1, Sec. 4, Roosevelt Road, 10617 Taipei, Taiwan and 2 Department of Life Science, Cheng-Kung University, No1, Ta-Hsueh Road, 701 Tainan City, Taiwan) (Suppression Subtractive HybridizationSSH) 6 cytochrome P450 monooxygenase GDA2 protein pectinesterase PVPR3 glutaredoxin droughtinduced protein RT-PCR KC1111 cytochrome P450 monooxygenase GDA2 protein pectinesterase PVPR3 drought-induced protein It is known from the preliminary study that inoculation of orchid mycorrhizal fungi on Phalaenopsis can elevate the survival rate, vegetable growth and flowering quality of several Phalaenopsis cultivars. In this study, plantlets of Phalaenopsis from the culturing flask were inoculated with orchid mycorrhizal fungi and grown in growth chamber for two- and four- months individually. Then Suppression Subtractive Hybridization (SSH) were used to compare the differential expression of genes between mycorrhiz Phalaenopsis al and nonmycorrhizal roots. It is found that 6 nucleic acid sequences which showed high homology to the gene sequences of cytochrome P450 monooxygenase, GDA2 protein, pectinesterase , PVPR3 , glutaredoxin , and drought-induced protein from other vascular plants. RT-PCR analysis of the transcriptsexpression pattern of these pathogenesis-related protein and stress-resistant protein confirmed that all of them were up-regulated in Phalaenopsis cultivar KC1111. It is therefore proposed that inoculation of orchid mycorrhizal fungi in Phalaenopsis could increase the expression of these pathogenesisrelated and stress-resistant genes.

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Investigation of Bioactivities from culture study of Pycnoporus cinnabarinus -Wei-Ren-Pan1, Chee-Jen-Chen 1, Min-Hsiung-Pan2 (Sounth Taiwan of technology University . Department of Biotechnology. No.1, Nantai St, Yung-Kang City, Tainan and National Kaohsiung Marine University . Department of Sea Food Science. No.142, Haijhuan Rd., Nanzih District, Kaohsiung City 81143, Taiwan ) (Pycnoporus cinnabarinus) NO 80 g/ml iNOS COX-2 20 g/ml LPS iNOS COX-2 EtOHEA MCF-7 Sub G1 In this study. the culture of Pycnoporus cinnabarinus after the investigation of its growing conditions, the laboratory cultivation and shaking flask were carried out and the biological activities analyzed , with extracts of fruit bodies, Divided into anti-inflammatory activity and cell apoptosis. In anti-inflammatory, the best activity has EtOH extracts, inhibits NO activity increased with increasing concentration, best activity concentration is 80g/ml,in iNOS/COX-2 expression, concentration 20g/ml could inhibit iNOS/COX-2 expression in RAW264.7 cells stimulated by LPS. Furthermore, the cells apoptosis ,MCF-7 cells treated by EtOH/EA extracts of P. cinnabarinus fruit bodies, the result indicated that Sub G1 of value increased with increasing concentration.

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amphotericin B 1 12 1 3

Ethanol extract of solid state cultivated Taiwanofungus camphoratus extract enhance synergistically cell death of amphotericin B in human osteosarcoma cell lineLing -Yi Chen1, Chuh-Kai Liao12, Ling-Yo Liu1and Ching-Hua Su3 (1Graduate Institute of Medical Sciences, Taipei Medical University2 Department of Family Medicine, Cardinal Hospital ,3Graduate Institute of Biomedical Materials and Department of Microbiology and Immunology, Taipei Medical University, 250 Wu-Hsing Street, 110 Taipei, Taiwan) , amphotericin B MG63 100 g/ ml 24 amphotericin B 3ug/ml amphotericin B apoptosis 100 g/ml 24 amphotericin B 3ug/ml G2 p 53p21 cyclin B1 CDC2 caspases : amphotericin B Taiwannofungus camphorates (Antrodia camphorate) is a unique fungus found in Taiwan. T. camphoratus contains many active biological compounds which are ethanol souble. According to previous data, ethanol extract from T. camphoratus had reflected anti-tumor effect in some experiments. Among the numerous type of tumors, the osteosarcoma tumors is difficult to be treated by chemotherapy, and the etiology is still remains unclear. The present study is focus on ethanol extract of solid state cultivated (ESSC) T. camphoratus , and their synergistically effect

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with amphotericin B on osteosarcoma cell line as well as title mechanism of inhibition. MG63 osteosarcoma cell lines was used to test the inhibiory concentration of ESSC T. camphoratus, amphotericin B, and their combined effect. We find that the cell line treated with 100g/ml ESSC T. camphoratus, amphotericin B for 24 hr, washed out with PBS and then added 3g/ml of AmB for 24 hour induced more apoptosis and potentated the cytotoxic effect in MG63 cell line that is compare with treating of ESSC T. camphorates alone, or amphotericin B alone. Their synergistic effect causes cell cycle block at G2, upregulates p53, p21, levels ,decreases cyclin B1 and CDC2 expression ,and activation of caspases. The result provide evidences that the synergistic effect acts the apoptotic pathway. Taken together, our findings suggested that TC will be effective in the treatment of osteosarcoma tumors . Abbreviations: Taiwannofungus camphoratus ,TC ethanol extract of solid state cultivated EESC; AmB, amphotericin B

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Macrofungi diversity in different type and thinning degree forestsYi-Ting Wang, Wan-Cheng Chen, Ming- Hsiu Kao Wan-Rou Lin and Pi-Han Wang(Department of Life Science, Tunghai University) 30 (1,500 /)(1,000 /) (825 /) 11 52 1,168 22 98 6,732 22 55 There are about 420,000 ha plantation forests in Taiwan. Different forest management practices will inevitably affect the forest ecosystem. In this study, we compare the macrofungi diversity of nature and plantation forests, and evaluate the effects of different thinning practices on macrofungi biodiversity. The study site is located in Tai-Siet-San forest park. We established three plots in 30 years old Chamaecyparis formodensis plantation forest, and one plot in the natural forest which are mostly dominated by Fagaceae and Lauraceae. Among the plantation plots, one is located in the non-thinning forest (1,500 tree/ha), another is in the moderate-thinning forest (1,000 tree/ha) and still another in the heavy-thinning forest (825 tree/ha). Fungi were inventoried along 200m transects running across each site. We recorded the morphogens, color, substrates and the number of fruiting body. The sporocarps were identified to species level whenever is possible. In May, 1168 sporocarps were counted, including 11 families, 52 species. In September, 6732 sporocarps were counted, including 22 families, 98 species. Both species diversity and richness in

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the fall were higher than those in spring. Tricholomataceae was dominant in the study site. Among the four sampling plots, natural forest plot always has the highest species diversity and the number of fruiting body, in which 22 species were found in spring and 55 species were found in fall. There are four habitats of fungi, including saprobic on decaying wood, saprobic on soil, parasites on wood and paragenetic on soil. There was no paragenesis fungus occurred in the plantation forest plot. Our results suggested that thinning increased fungi diversity. In non-thinning plantation plot, both population size and number of species are the lower than other plots. In addition, fungal diversity in heavy-thinning plot is higher than those in moderate-thinning plot.

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Ramariopsis kunzei, white coral fungi in the Cryptomeria japonica plantationsChan-Ching Hung, Hsin-Yin Chang, Te-Yu Guu and Pi-Han Wang(Department of Life Science, Tunghai University) 1,275 1,500 1,400 1,470 200 200 345 159 20 5.0~11.0 2~5 1.0~3.0 0.1~0.6 4~11 3.96 6.93 1.89 3.96 Ramariopsis kunzei is a wood-saprobe fungi, which varied from ivory to cream white in color. This study surveyed the distribution and fruiting season of Ramariopsis kunzei within a 40 year Cryptomeria japonica plantations forest at Nantou in Taiwan. We surveyed ten 1-ha plots located between1,275m a.s.l. to 1,500m a.s.l. every month in 2006. R. kunzei mainly distributed in four plots, few fruiting bodies were found in two plots, and none in the other four plots. Plots with the presence of R. kunzei are all located at the North-facing slope between altitudes from 1,400 to 1,470. Fruiting season varied between plots: At plot 6, fruiting bodies were abundant in June to August; at plot 7, which the number of fruiting bodies is most abundant, 200 fruiting body were recorded in June and July and the number reached 345 in August, but decreased to 159 in September; at plot 5, only 20 fruiting bodies were found in June. Fruiting bodies of Ramariopsis

30

kunzei are up to 5.0~11.0cm tall, 2~5 branches from the main stem. Each branch is 1.0~3.0cm in length and 0.1~0.6cm in width from which 4~11 dichotomous branching. Tip pointed branchlet on the branch upright, on the surface. Basidiospores shaped wide and oval verrucose, 3.96~6.93 in length and 1.89~3.98 in width. Now we are working on polymorphism population genetics.

Halophytophthora

Taxonomy and Phylogenetic analysis of Halophytophthora--Fu-Ling Chan, Sung-Yuan Hsieh and Gwo-Fang Yuan (Bioresources Collection and Research Center / Food Industrial Research and Development Institute, Hsinchu, Taiwan.) Halophytophthora Halophytophthora Halophytophthora 15 2 28S DNA D1D2 Halophytophthora Halophytophthora species are common zoosporic oomycetes in subtropical and tropical mangrove forest. They were mainly collected from fallen leaves of mangrove, and were thought to be important decomposers in mangrove ecosystem. There are fifteen species and two varieties in the world. Species delimitation in Halophytophthora is based mainly on the morphology of zoosporangium, the presence of vesicle or/and plug, and the mode of zoospore release. Based on 28S large subunit ribosomal RNA gene D1D2 fragment sequence analysis, and the difference in the morphology and structure of zoosporangia, most species except four should move out from Halophytophthora, and several new genus need to be erect for accommodate these fungi.

31

() 1 2 11. 2.

Myxomycetes of Taiwan XIX. The Order Echinosteliales--Chin-Hui Liu1, Jong-How Chang2 and Ya-Fen Chen1 (1. Institute of Plant Science, National Taiwan University, 2. National Museum of Natural Science) 6 3 (Echinostelium apitectum Whitney) (E. arboreum Keller & Brooks) (E. paucifilum Whitney) (Clastoderma) (Echinostelium) Six taxa in the order Echinosteliales are reported from Taiwan. Of the six taxa, Echinostelium apitectum Whitney, E. arboreum Keller & Brooks, and E. paucifilum Whitney are newly recorded, which are all collected from moist-chamber cultures. Keys to the families of Echinosteliales, and to the species of Clastoderma and Echinostelium recorded in Taiwan are also provided.

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Pseudozyma

The collection and phylogenic group of genus Psuedozyma in TaiwanYu-Hui Wei, Guey-Yuh Liou, Fwu-Ling Lee (Bioresource Collection and Research Center (BCRC), Food Industry Research and Development Institute, Hsinchu 300, Taiwan, R.O.C.) rDNA LSU D1, D2 Pseudozyma Pseudozyma 11 rDNA Pseudozyma hubeiensis P. tsukubaensis P. aphidisP. antarctica D1, D2 rDNA P. fusiformata P. shanxiensis Psuedozyma During our study of yeast-like fungi from plants and insects in Taiwan, all strains in this study were collected and deposited in the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute. Based on the D1/D2 region of LSU rDNA sequences, these strains were clustered in the clade of unusual yeast-like fungi, genus Pseudozyma. The sequences of the D1/D2 region of LSU rDNA of fifteen strains were identical to the type strains of P. antarctica, P. aphidis, P. hubeiensis, and P. tsukubaensis. However, the phylogenetic

33

analysis indicated that five strains were clustered in the clades of P. fusiformata and P. shanxiensis, but the sequences of these domestic strains were quite different from the type strains. Genus Pseudozyma is the anamorph of Ustilaginomycetes and currently contains eleven species. The survey of Pseudozyma species in Taiwan showed the presence of at least six distinct species. The phylogenic analysis shows a great biodiversity of the genus Psuedozyma in Taiwan.

1 2 3 4 51,2,3,4,5

Characterization of diversity in Rhizoctonia solani in Taiwan--Sz-Hau Chen1, Siang-Jhe Yang2, Yi-jen Lin3, C.H. Chang4, Lung-Chung Chen5 (1,2,3,4,5Department of Plant Pathology National Chung Hsing University) 20 (MCGs) 0 5 2.8 3.3 (Single primer PCR) NTSYS UPGMA (Rhizoctonia solani AG1IA) 86%( ) ( ) ( )( ) 60% 11

34

dsRNA The sheath blight is still an important one of rice epidemic in Taiwan. There are fewer reports this disease during the last 20 years in the island. We are interested in the change of the disease and want to figure out the difference between nowadays and the past. Therefore, in order to realize the difference between isolates, we have physiological characteristic test. The isolates were cultured at various temperature and pH value, to botanize mycelial growth rate and dry weight of sclerotia, and numbers of sclerotia and base on the difference to discriminate the isolates. On anther hand, with mycelial compatible groups (MCGs) test, there are four groups in Taiwan with the difference between mycelial compatible and incompatible ration. Besides, with the inoculation of different isolates collected in Taiwan from rice TaiKing 9 , there seems to be no such difference of disease index surrounded between 2.8 to 3.3 under the circumstance of disease index between 0 and 5.0. The conclusion with this is different in comparison to the former research. On the other hand, by using single primer PCR to amplify difference fragments and to analysis genetic similarity by UPGMA cluster analysis with computer software NTSYS to have Gel Compare. Dendrogram based on the analysis of 54 isolates of Rhizoctonia solani AG1-IA, and the results suggest the difference between Rhizoctonia solani AG1-IA in Taiwan or Japan and other AGs. Further more, isolates of Rhizoctonia solani AG1-IA from Taiwan could differentiate into five groups in similarity 86%. Group north: isolates from Yilan, Hsinchu, Taoyua. Group midland: isolates from Taichung, Hanghua, Nantou, Chiayi, Yunlin. Group east: isolates from Hualien. Group south: Pingtung, Tainan, Chiayi. Group complex: a large breadth group. In deep, the phylogenetic tree was obviously discriminated in to eleven subgroups of the dormant population of R. solani AG1IA based on the five groups. The subgroups of phylogenetic tree are as follows and which includes Taoyuan County, TaiChung City, Changhua County and Yunlin county, Pintung County, HualienCounty. That seems to be a crop production street across the western field of island Taiwan. The Group two is related only with Kaohsiung County. And Group four is closed to deposit on Chiayi County. The last are that Group five Tainan County and Group eight is Taoyuan County, Group night the Naotou County, Group X Chiayi County and Hualien County. The last group eleven is prefer to Yilan County. Beside, the phylogenetic tree of the diversity with dsRNAs are constituted by eight subgroups separated to: Group one Changhua County Yunlin County, Chiayi County, Hualien County Group two Taichung County Group three Pingtung County Group seven Tainan County and the Group eight Yilan County, Taoyuan County and Hsinchu County. We also have the result which is interconnected both with the phylogenetic tree cross eachother, which means the distribution do have specific geographical specification among the dsRNA and the RPAD. The relationship between MCGs and dendrogram based on SSR-PCR analysis showed that

35

diversity of isolates from Taiwan had high degree with geography. This had a great effect upon the diversity of isolates from Taiwan with geography more than with rice variety (host). Therefore under the circumstance with reducing pesticide supplying, to realize the mechanism with virulence and hypovirulence would be a potential plan to manage the sheath blight of rice.

1 1 2 31

2 3

Characteristics and Distributions of Ambient Fungal Spores in the Taipei Area--Ying-Chen Fang1, Hsing Jasmine Chao1, Chang-Chuan Chan2 and Chung-Te Lee3 (1Graduate Institute of Public Health, Taipei Medical University, 250 Wu-Hsing Street, 110 Taipei, Taiwan 2Institute of Occupational Medicine and Industrial Hygiene, National Taiwan University 3Graduate Institute of Environmental Engineering, National Central University) 2005 112 Burkard 3 1930.62 spores/m ascospores basidiospores Cladosporium unidentified spores Aspergillus/Penicillium Fusarium

36

Biological particulates are ubiquitous in our daily environments, including pollens, fungal spores, bacteria, viruses, or any fragments derived from microorganism, plants and animals. We used a Burkard seven-day recording volumetric spore trap to collect ambient fungal spores from January to December 2005. We evaluated the compositions and distributions of ambient fungi in the Taipei area and examined the relationships between fungi and other environmental parameters. During the study period, the average concentration of total fungal spores was 1930.62 spores/m3, and the predominant fungal taxa were ascospores, basidiospores, Cladosporium, unidentified spores, Aspergillus/Penicillium and Fusarium. In regression models, we found that temperature was the most consistent factor correlated with fungal concentrations. Other environmental parameters, including relative humidity, wind speed, and ozone, also had statistically significant relationships with fungal levels. Future studies should be implemented to evaluate the health impacts of aeroallergens, and to examine the interactions among fungal spores, meteorological factors and air pollutants.

1 11

2

3 1

2 3

Establishment of molecular diagnosis for detecting Phytophthora capsici and genetic diversity of Phytophthora inter- and intra-species--Yi-Wen Chung 1, Wen-Chen Liu 1, Shih-Mei Pan2, PaoJhen Ann3, Lung-Chung Chen1(1Department of Plant Pathology, National Chung Hsing University 2Department of Life Sciences, National Chung Hsing University 3Agricultural Research Institute, Council of Agriculture, Executive Yuan, R.O.C.) () ( ) 1

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A1 OPB07 800 bp 800 bp P. capsici 3 PCR B07F3/B07R3 P. capsici 381 bp 7 ( Phytophthora spp. )4 ( Pythium spp. ) P. capsici DNA PCR 381 bp ( papillate ) 18 RAPD 13 NTSYS-pc UPGMA 0.73~0.86 8 RAPD 8 P. capsici P. cryptogea 0.18~0.26 P. colocasiae P. citrophthora 0.35~0.39P. parasitica P. drechsleri 0.18~0.35 PCR RAPD Phytophthora capsici is an important fungal pathogen that causes seed rot, seedling damping-off, root and crown rot, leaf spots, stem lesions, foliar blight, and fruit rot in many solanaceous crops (pepper, eggplant, tomato) and cucurbits (cantaloupe, cucumber, summer squash, pumpkin, watermelon). Rotting of seedlings prior to emergence (preemergence damping-off) and blighting of recently emerged seedlings (postemergence damping-off) can occur. The roots and plant base may be discolored and infected seedlings often fall over. Crown rot causes the entire plant to collapse and die in a short period of time. Leaf spots are dark brown, one to several centimeters inches in diameter. Vines can be affected at any part. The lesions are dark brown, water-soaked, and girdle the stem, causing the stem to collapse and die. White fungal growth may cover infected areas of blighted seedlings under moist

38

conditions. Phytophthora capsici, as well as other Phytophthora spp., can also produce a wide variety of symptoms on mature plants that vary by host. Since the species P. capsici has been broadened, there is considerable diversity in host-specific pathogenicity. The isolates of P. capsici in Taiwan belonged to A1 mating type , and no chlamydospore was found. The structure or survival of P. capsici in the field is unkown. The molecular marker used to understand the pathogen ecological approach in the nature should be established and developed. Therefore, we searched the molecular marker for detection of P. capsici using by RAPD. The primer OPB-07 can amplify a 800 bp species-specific fragment through RAPD and Southerns hybridization. The species-specific fragment was cloned and designed these PCR primer pairs from 800bp nucleotide sequences. The primer B07F3/B07R3 could amplify a 381 bp fragment to isolates of P. capsici by polymerase chain reaction, but 7 Phytophthora spp., 4 Pythium spp., and health tomato, red pepper, sweet pepper, and eggplant tissues could not amplify any fragment. DNA extracts from pepper after inoculating and infected pepper in the field could be amplified a 381 bp fragment by primer B07F3/B07R3. And the colony morphology of P. capsici isolates from different hosts could be distinguished into unifom, radiate, chrysanthemum, rosette and stellate. The sporangial shapes include ellipsoid, spherical, broadly ovoid, obturbinate, obovoid, fusiform, and pyriform. Each sporangium has one or two papilla. To approach the genetic diversity among isolates of Phytophthora capsici intra-species and Phytophthora inter-species, 13 primer results from 25 primer RAPD profiles with amplification of 18 P. capsici isolates were selected to analysis genetic similarity by UPGMA cluster analysis with computer software NTSYS-pc, and 8 primer results from 25 primer RAPD profiles with amplification of Phytophthora spp. isolates were selected . We found that isolates of P. capsici from different hosts have different genetic diversity. The P. capsici isolates from pepper and tomato have relatively high degree in genetic similarity from 0.73 ~ 0.86. Dendrogram based on the analysis of 27 isolates of P capsici, P. citrophthora, P. parasitica, P. palmivora, P. colocasiae, P. cryptogea, and P. dreschsleri using RAPD showed the relatively higher similarity coefficients for P. capsici and P. cryptogea, ranging from 0.18 ~ 0.26. The high level of similarity between isolates of P. colocasiae and P. citrophthora ranged from 0.35 ~ 0.39, and similarity coefficients of the isolates of P. parasitica and P. dreschsleri ranged from 0.18 ~ 0.35. By the speies-specific molecular marker, we can develope a complete system for detecting P. capsici in the field, and explore the genetic varation of Phytophthora inter-species and intra-species in relation to pathogenicity by RAPD analysis. The establishment of molecular markers and the posses of genetic diversity of Phytophthora capsici intra-species and Phytophthora inter-species are further studied. KeywordsPhytophthora capsiciMolecular diagnosisPhylogenetic analysis

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1 2 11

2

Identification of Ophiostoma species in imported woodH.S. Li1, H.W. Kao2 and C.Y. Chen1 (1Department of Plant Pathology, National Chung-Hsing University 2Department of Life Sciences, National Chung-Hsing University) MSC 88 Ophiostoma piceae complexOphiostoma quercus DNA O. quercus floccosum pluriannulatum abiocarpum O. O. O.

40

The fungi of Ophiostoma species commonly occur on woody plants, and usually adversely affect the commercial wood quality. Eighty eight Ophiostoma isolates have been isolated from imported wood by using MSC selective medium. Three groups of species can be morphologically distinguished according to the anamorphic state, among which Ophiostoma quercus in the group of Ophiostoma piceae complex is dominant. The identification is carried out morphologically, and then is further ascertained in combination of biology, mating type studies and molecular data. Consequently four species have been identified. They are O. quercus O. floccosum O. pluriannulatum, and O. abiocarpum. Lots of isolates are still pending to be identified. Further studies are required.

1 1 21

2

Studies of Exobasidiaceae in TaiwanHsin-Hui Shih1, Huann-Ju Hsieh1 and Chuen-Hsu Fu2(1 Department of Plant Pathology and Microbiology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan2 Division of Forest Protection, Taiwan Forest Research Institute, 6 Floor, Room 610, #67 San-Yuan Street, Zhongzheng District, Taipei, 10079, Taiwan) 14 Exobasidium spp.1 Muribasidiospora sp.Exobasidium 2 E. cylindrosporum pieridis E. yoshinagai E.

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Muribasidiospora 30 Exobasidium ITS1-5.8S rDNA-ITS2 Recently, more than 100 exobasidiaceous specimens were collected, among which were composed of 14 Exobasidium species and 1 Muribasidiaspora. Two Exobasidium species occurred on Vaccinium japonicum var. lasiostemon and Pieridis taiwanensis were new species and three species, E. cylindrosporum on Rhododendron spp., E. pieridis on Lyonia ovalifolia var. ovalifolia and E. yoshinagai on R. mariesii, were newly recorded. Muribasidiospora occurred on Gordonia axillaries was also a new species in Taiwan. Analyzing the ITS1-5.8S rDNA-ITS2 regions of 30 Exobasidium isolates, the phylogenetic tree indicated that Exobasidium species were correlated to their respective hosts and could be grouped into three clades corresponded with host taxonomic groups, Rhododendron, Vaccinioideae and Theaceae.

1 11

2

1

2

Application of molecular diagnosis for detection and quantification of seedborne and soilborne fungi--Wen-Chen Liu1, Yu-Min Chang1, Shih-Mei Pan2 and Lung-Chung Chen1(1Department of Plant Pathology, National Chung Hsing University 2Department of Life Sciences, National Chung Hsing University)

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PCR (PCR-based methods)PCR (PCR--ELISA) (RAPD) RAPD 830 bp PCR Alternaria brassicicola Ab536r Ab536f A. brassicicola 536 bp 50 pg l-1 PCR-ELISA 50 g ml-1 1 pmol DIG primer DIG dNTP Penicillium spp. Aspergillus spp. Peridiospora sp. Sporotrichum sp. PCR DNA PCR Alternaria brassicaeA. brassiaicolaA. raphani Fusarium oxysporum f. sp. lycopersici Leptosphaeria maculans Plasmodiophora brassicaeSclerotinia sclerotiorumRhizoctonia solaniPhytophthora capsici Phytophthora infestans

The determination of a PFA (pest free area) in a country or an area will benefit export of plant products, reduce the risk of import and improve the competitiveness of plant products in markets. A pest free area is an area in which a specific pest does not occur as demonstrated by scientific evidence and in which, where appropriate, this condition is being officially maintained. Three main stages are considered in the establishment and subsequent maintenance of a PEA are systems to establish freedom, phytosanitary measures to maintain freedom and checks to verify freedom has been maintained. The requirement for phytosanitary is the quite important part of quarantine in the world thus we have developed and established the more accurate techniques, including PCR-based methods, PCR-ELISA for quantification and RAPD, to resolve the problems of phytosanitary. In this study, the 830 bp specific DNA fragment derived from RAPD analysis was used to design A. brassicicola specific primer pairs Ab536R (5 -ATATAAAGGCGGGTAACG-3) and Ab536F (5-AGCGCCTTATACTCCTTCT-3). The

43

primer pairs could amplify a specific 536-bp fragment from genomic DNA of A. brassicicola by PCR. The sensitivity of the primer pairs could detect 50 pg of genomic DNA of A. brassicicola. Furthermore, the establishment of PCR-ELISA could quantify PCR products directly. Our results showed that when utilizing DIG-labeled primer, 1 pmol probe and 50 g ml -1 streptavidin, will near the results of utilizing DIG-labeled dNTP, 0.5 pmol probe and 10g ml -1 streptavidin, but is more economic. For isolation of fungi from media and extraction of DNA, the spores inoculated to media were harvested. The template obtained by heat and specific primers were employed to PCR and a specific DNA fragment was amplified. For seed test, extraction of tissue DNA and specific primers were used to PCR and a specific DNA fragment was detected. Besides, according to the reference from the world, what the molecular marker of seaborne fungi which is including Alternaria brassicae A. brassiaicola A. raphani Fusarium oxysporum f. sp. lycopersici Leptosphaeria maculans Plasmodiophora brassicae Sclerotinia sclerotiorum Rhizoctonia solani Phytophthora capsici and Phytophthora infestans. For the above studies, we can set up the accurate methods for seeds and seedlings quarantine systems. KeywordsMolecular diagnosis, Seed inspection, Phytosanitary technique

1 1

Two new species of Syncephalis from Taiwan:Syncephalis obliqua Syncephalis macrospora Hsiao-Man Ho1, Shu-Cheng Chuang1, Dept. of Science education, National Taipei University of Education, 134. Sect. 2, Heping E. Rd. Taipei.

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Syncephalis obliqua Syncephalis macrospora. S. obliqua S. macrospora Two new species of Syncephalis isolated from Taiwan are described and illustrated Syncephalis obliqua and Syncephalis macrospora . S. obliqua is distinguished from other species by the asymmetrical placement of the merosporangia on the vesicle and larger cylindrical spores. S. macrospora is distinghished from the other species by longer sporophores and larger spores. A discussion comparing these new species with other taxa is included.

Sacchachitosan 1. 21 2

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Mode of action on SACCHACHITOSAN for Alkali-burned Cornea of RabbitsShiao-Chuan Liu1, and Ching-Hua Su2(1Graduate Institute of Biomaterials, Taipei Medical University,2Graduate Institute of Biomedical Materials and Department of Microbiology and Immunology,Taipei Medical University, 250 Wu-Hsing Street, 110 Taipei, Taiwan)

: SACCHACHITIN() , SACCHACHITOSAN (deacetylated SACCHACHITIN) SACCHACHITOSAN SACCHACHITOSAN (SIRC) SACCHACHITOSAN 200-300 g/ml SACCHACHITOSAN SACCHACHITOSAN In response to corneal injury, cytokines and growth factors play a crucial role during the healing and reparative processes. These processes included inflammation, cell proliferation, migration and differentiation, synthesis of the extracellular matrix, and remodeling of connective tissue. The aim of the present study was to develop a new type wound healing drops derived from cell wall of fungi and other sources to meet the demands of alkali burn rabbits cornea. SACCHACHITIN extracted from cell wall of Ganoderma tsugae is reported as an accelerator of wound healing, and previous reports indicate that SACCHACHITIN is able to promote wound healing by inducing cell proliferation , increasing the secretion of cell cytokines and growth factors and decreasing matrix metalloproteinase by these cells provided an excellent environment for wound healing. SACCHACHITOSAN, a deacetylated SACCHACHITIN, is water-soluble, thus it is selected for the eye drops of this purpose. Before the animal experiments, SACCHACHITOSAN was estimated for its effects on the proliferation and dose dependent response on rabbit corneal epithelial cell line (Statens Seruminstitut Rabbit Cornea; SIRC). Preliminary results indicated that SACCHACHITOSAN performed as a good mediator for the growth of the cell line in the concentration of 200-300 g/ml. It was suggested that the existence of SACCHACHITOSAN may help in development of better ocular drug.

46

Further experiments on animals are no under investigation.

.

1 2 1 1*1

2

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Study on Chinese herbal medicine for bio-diversion in Antrodia cinnamomeaCh'i -Hung Hsieh1, Chia-Hsiang Chang2, Wei-Chen Ho1, Ch'ang-Wei Hsieh1 (1Department of Bioresources, DA-YEH University2Department and Graduate Program of Bioindustry Technology, DA-YEH University, 112 Shan-Chiao Street, 510 Chen Lin, Taiwan) 30 3.41 2.72 2.2 The objectives of this research were to study different kind of Chinese herbal medicine in solid culture of Antrodia cinnamomea that observed the tinctures and acervate conditions in 30 days. After freezing and desiccation, we aimed at index composition of mycelium such as triterpenoids and polysaccharides analyzed the contents in different times. Through mushroom, used the principle of bio diversion that the substance have none curative effect or bio-activity can transform leavening having bio-activuty. In triterpenoids, Ganoderma Lucidum and Magnolia officinalis have much more bio-diversion that achieved 3.41 and 2.72 time respectively. In polasaccharide, perilla have more bio-diversion that rechieved 2.2 time. Therefore, how to make Antrodia cinnamomea can account high-activity products in artificial culture efficiently are our destination.

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-

A preliminary study on the hyphal growth of Antrodia camphorata-- Chao-Feng Tseng, ChaoEi Wang and Chi-Guang Wu (Department of Biotechnology and Bioinformatics, Asia University, Taichung, Taiwan). Antrodia camphorata 2% agar HPLC In this study, different composition of culture media was designed to cultivate Antrodia camphorata. The results indicated that wood extract of A. camphorata could enhance the hyphal growth, better than other nutrient amendments. Yeast extract amended medium did not stimulate hyphal growth, and the mycelial color was pale whiteand. In contrast, wood extract showed the best hyphal growth if compared with other treatments. Through the HPLC analysis, the composition of hyphal extract was closely similar to that from fruiting body. Keyword: Antrodia camphorata, growth factor, HPLC

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Preparation of the alkali-polysaccharide extracted from -irradiated Ganoderma tsugae residue and its antioxidant properties--Wan-Lih Huang,Pei-Ying Lian and Jeng-Leun Mau (Department of Food Science and Biotechnology, National Chung-Hsing University,Taichung) Ganoderma tsugae Murrill 1 kGy 5 mg/ml 61.54% 1,1--2- 0.5mg/ml43.48%39.96% 5-10mg/ml16.41-39.75%0-2.26% 0.5-20mg/ml68.50-100% 55.62-98.94% The residue of Ganoderma tsugae Murrill, which has been extracted twice by hot water, has no economical value and exploitation. Besides, some research indicated gamma irradiation might degrade the residue and yield polysaccharide with reduced size. Therefore, the research was designed to apply 1 kGy gamma ray irradiation on the residues of G. tsugae in order to explore the utilization of this residue. The alkali- polysaccharide with 1 kGy -irradiation was compared with the control for its antioxidant properties. The alkali- polysaccharides with 1 kGy irradiation dose showed the best antioxidant activity (61.54%) at 5 mg/ml. The alkali-polysaccharides with irradiation increased its reducing power with concentration. The alkali-polysaccharide with 1 kGy -irradiation (43.48%) had higher scavenging ability of alkali-polysaccharides on DPPH radical at low concentration (0.5 mg/ml) than with the control (39.96%). The alkali-polysaccharide with 1 kGy -irradiation (16.41-39.75%) had higher scavenging ability of alkali-polysaccharides on OH radicals (5-10 mg/ml) than with the control (0-2.26%). The alkali-polysaccharide with 1 kGy irradiation (68.50-100%) had higher chelating ability on ferrous ions (0.5-20 mg/ml) than with the control (55.62-98.94%) and increased with concentration.

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Non-volatile taste components of solid state fermented termite fungal wheat--Jia-Li Syu, Shao-Yu Jia, Jeng-Leun Mau (Department of Food Science and Biotechnology, National Chung-Hsing University, 250 Kuokung Road, Taichung 40227, Taiwan, ROC) 14 73.67 320.73 mg/g 28.49 mg/g MSG-like 3.41 6.06 1.63 3.51 mg/g 9.71 mg/g 243.21 g/100g172.29 g/100g 0.93 g/100g3.30 g/100g The research was to inoculate the mycelia of Termitomyces albuminosus into autoclaved polished wheat and incubate for 14 days to produce a product called termite fungal wheat. The product was freeze dried and its non-volatile components were analysed and compared to mycelia, filtrate and wheat. With regards to fatty acid component, linoleic acid was the major fatty acid, and the mycelia were the highest (73.67%). The content of soluble sugars of filtrate was the highest (320.73 mg/g). Furthermore, the major free amino acids of mycelia were arginine and glutamate. The content of total free amino acids for mycelia was the highest (28.49 mg/g). With regards to monosodium glutamate-like components, the contents of mycelia, filtrate, termite fungal wheat and wheat were 3.41, 6.06, 1.63 and 3.51 mg/g, respectively. The content of 5-nucleotide was the highest in mycelia (9.71 mg/g). Besides, the equivalent umami concentration of filtrate (243.21 g/100 g) was higher than those of mycelia (172.29 g/100 g), wheat (3.30 g/100 g) and termite fungal wheat (0.93 g/100 g). Overall, the taste components of termite fungal wheat obtained from solid state fermentation was much less than those in wheat. This might be due to the inadequate growth of termite fungal in wheat. Therefore, the optimal condition for T. albuminosus to grow in wheat could be studied further.

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Antioxidant properties of solid-state fermentated Chang-chin rice--Shin-Yi Lin, Tsai-Ping Wu, Jeng-Leun Mau (Department of Food Science and Biotechnology, National Chung-Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, ROC) Taiwanofungus camphorates Wu et al. 24 1,1--2- 20 mg/ml 88.42 20 mg/ml 100 20 mg/ml 1,1--2- 1 mg/ml 50 Taiwanofungus camphorates Wu et al. (Chang-chih or Niu-chang-ku) is a precious edible and medicinal mushroom due to the fact that it contains many physiologically active substances. The objectives of this study were to prepare Chang-chih rice using embryo rice as a substate, Changchih was initially grown on the rice for 24 days, and then freeze-dry and analyze its antioxidant properties and compare with mycelia, embryo rice and some commonly used antioxidants. With regard to reducing power of hot water extracts, the mycelia better than Chang-chih rice. At 20 mg/ml, scavenging ability of Chang-chih rice on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals were 88.42, better than ascorbic acid that of at the same concentration. Chang-chih rice chelated ferrous ions by 100% at 20 mg/ml. All of ethanolic extracts had the highest antioxidant activity at 20 mg/ml. For reducing power, Chang-chih rice was higher than others. At 1 mg/ml, scavenging ability of Chang-chih rice on DPPH radicals were higher than 50%. Bsae on the results obtained, the Chang-chih rice prepared from the solid state fermentation of embryo rice possessed better antioxidant properties.

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RAW 264.7

The anti-tumor effect of polysaccharides and exopolysaccharides of Sparassis crispa under submerged fermentation from activated RAW 264.7 macrophages Chun-Sheng Chang, KaiYuer Tang, Chee-Jen Chen, Jian-Chyi-Chen (Southern Taiwan University of Technology. Department of Biotechnology. 1, Nan-Tai St, Yung-Kang City, Tainan ) (Sparassis crispa) cyclophosphamide DBA/1 DBA/2 IgM RAW264.7 (Corn Starch) 11.98 g/L (Glucose)117.71 mg/g(Saccharose)40.17 mg/g F-F- F- (Raw 264.7) (Fructose) F-1(MCF-7) (50.16% 1.87%) F- FIII 39.99% 2.62 %58.49 % 2.25% (HEP G2) F-F- F- RAW 264.7 MCF-7 The experiments were to investigate the anti-tumor effect of polysaccharides and exopolysaccharides of Sparassis crispa under submerged fermentation from activated RAW 264.7 macrophages. Screening the six different carbon sources, the corn starch as the carbon source can harvest the highest concentration of biomass, 11.984 g/L. When glucose used as the carbon source,

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the highest production of polysaccharide per gram of mycelium was obtained at 117.71 mg/g. The highest production of exopolysaccharide per gram mycelium was 40.17 mg/g which was used saccharose as the carbon source. Polysaccharide fraction (F-I) was extracted the air-dried and powdered S. crispa with hot water. The resulting residue was then extracted with cold alkali to obtain the second polysaccharide fraction (F-II), and then extracted with hot alkali to obtain the third polysaccharide fraction (F-III). Those polysaccharide fractions were collected after extensive dialysis. To test the anti-tumor activity of mediator released from the macrophage Raw264.7 were stimulated by the polysaccharide fractions, respectively. The F-I cultured from the fructose as carbon source that showed the lowest survival ratio of MCF-7 cell line was 50.16% 1.87%. Both of the F-II and F-III were cultured from the corn starch as the carbon source. Their best anti-tumor activity for MCF-7 was showed at 39.99% 2.62 % and 58.49 % 2.25%, respectively. Those results showed the polysaccharide of S. crispa can apparently inhibit the cell growth of MCF-7 from activated Raw 264.7.

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Monascus pilosus Monascus purpureus

Comparison of Genes Expression in Monascus pilosus and Monascus purpureus using Microarray--Ching-Yu Tu, Ing-Er Hwang, Li-Ling Liaw (Food Industry Research and Development Institute, Bioresource Collection and Research Center, P.O. Box246, Hsinchu 300, Taiwan, R.O.C.) 596 Monascus pilosus (BCRC38093) M. purpureus (BCRC38113) pilosus monacolin K M. purpureus GABA M. (1) monacolin K M. pilosus monacolin K mkA~mkI M. purpureus 2-20 (2) M. purpureus M. pilosus M. purpureus (3) M. purpureus alpha-amylase A nonhemolytic phospholipase C alpha-glucosidase galactonate dehydratase alphaxylosidase lipase 11,3-beta-glucan synthase component bgs2 oligo-1,6glucosidase lysophospholipase 3-phytase A uricase chitin synthase Gendochitinase 1bifunctional endo-1,4-betaxylanase xylAglucan 1,3-beta-glucosidase acetylxylan esterase In Eastern, Monasus is a fungus used extensively in the food industry. Many researches reported that Monascus could produce many secondary metabolites and hydrolysis enzymes. According to these results, Monascus was applied wildly in the development of healthy food, medicine and industrial enzymes. A Monascus microarray chip, included of 596 genes, was used to study different genes expression in M. pilosus (BCRC38093) and M. purpureus (BCRC31499), which were cultured at liquid medium. M. pilosus is a highly monacolin K producing strain and M. purpureus is a GABA-rich strain. The results from microarry analysis were summarized as follow(1) Monacolin K genes cluster (mkA~mkI) were expressed 2-20 fold higher in M. pilosus than in M. purpureus. (2) In particular, transcription levels of most catabolic genes of the glycolytic pathway were higher in M. purpureus than in M. pilosus. It implies that M. purpureus expresses more glycolytic activity than M. pilosus. (3) We further studied the expression of genes encoding hydrolytic enzymes. M. purpureus gave the higher gene expression profile of alpha-

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amylase Anon-hemolytic phospholipase Calpha-glucosidasegalactonate dehydratasealphaxylosidaselipase 1 1,3-beta-glucan synthase component bgs2 and oligo-1,6-glucosidase genes and lower expression levels of lysophospholipase 3-phytase A uricase chitin synthase G endochitinase 1bifunctional endo-1,4-beta-xylanase xylAglucan 1,3-beta-glucosidase and acetylxylan esterasegenes.

Antioxidant properties of methanolic and ethanolic extracts from Monascus fermented soybeans--Yu-Ling Lee and Jeng-Leun MauDepartment of Food Science and Biotechnology, National Chung-Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan Monascus pilosus BCRC 31527 Monascus purpureus BCRC 31499 5 mg/ml 78.1-87.4% 31499 5 mg/ml DPPH 3149931527 95.2% DPPH The objective of this study was to evaluate the antioxidant properties of methanolic and ethanolic extracts from Monascus purpureus BCRC 31499 fermented soybeans (MFS-31499) and Monascis pilosus BCRC 31527 fermented soybeans (MFS-31527) as compared to uninoculated soybean product. Using the conjugated diene method, antioxidant activities of ethanolic and methanolic extracts were similar and in the range of 78.1-87.4% at 5 mg/ml. Concerning to three samples, methanolic extracts showed higher reducing powers than ethanolic extracts and MFS31499 exhibited better reducing power. Regarding to three samples, methanolic extracts showed better scavenging DPPH abilities than ethanolic extracts and in the descending order: MFS-31499 > MFS-31527 > soybeans. In chelating abilities in ferrous ions, the results showed similar pattern as those of scavenging DPPH abilities. The contents of total antioxidant components assayed for

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methanolic and ethanolic extracts were in the descending order: MFS-31499 > MFS-31527 > soybeans. Overall, the result of total phenols increased by incubation with Monascus, MFS-31499 and MFS-31527 showed higher phenolic components and better antioxidant activities.

Development of functional Monascus Fermented Black Soybean Products.--Hu-Lun Lee, YuLing Lee and Jeng-Leun Mau (Departmemt of Food Science and Biotechnology, National ChungHsing University, 250 Kuokuang Road, Taichung, Taiwan) Monascus purpureus Went 31499 monacolin K (daidzin daidzein genistin genistein) 50% 3% 13mg/g 25 10 monacolin K 216 ppm 10% monacolin K 140 ppm0.22ppm monacolin K daidzin genistin daidzein genistein 25 50% 3% 10% 10 monacolin K The study used black soybean as the substrate and inoculated Monascus purpureus Went 31499 to cultivate the fermented black soybean product, and analyzed its content of ergosterol, monacolin K, isoflavones (daidzin, daidzein, genistin, genistein) and citrinin to find the suitable conditions. The results showed that under different moisture content and inoculation rates, the 50% moisture and 3% inoculation of Monascus purpureus Went 31499 yields better results, and the content of ergosterol is 13mg/g; The cultivation at 25 for 10 days under different temperature and days could get the highest amount of monacolin K (216 ppm), and did not detect out any citrinin. In carbon source, addition of 10% glucose could get the highest amount of monacolin K

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(140 ppm) and its citrinin content is 0.22 ppm; We found that addition of nitrogens could not increase the content of monacolin K, but reduce it. Besides, following the cultivation of days, the content of daidzin and genistin decreased, but daidzein and genistein increased. Above all, the optimal conditions were 50% moisture content, 3% inoculation rate, 10% glucose, then cultivated at 25 for 10 days, so that we could obtain the best yield of monacolin K.

SCAR

Identification of Taiwan cultivated strains of Lentinula edodes by SCAR markers Chiu-Chien Su and Lung-Chung Chen (Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan 402, R. O. C.) 48 10 21 10 10 RAPD 0.18 kb-2.52 kb DNA 7-19 10 NTSYS-pc UPGMA 0.68 DNA 0.91 DNA RAPD 850 bp 884 bp primer 5.0 LE7 Forward LE7 Reverse SCAR-PCR RAPD SCAR 623 bp DNA ITS1 ITS4 SCAR 58 921 SCAR : Lentinula edodes (Berk.)Pegler, the shiitake mushroom, is one of the most widely cultivated mushrooms worldwide. The purpose of the project is to investigate the characteristics of 48 isolates or cultivated strains and 10 foreign countries strains. 21 isolated from Taiwan and 10 foreign countries strains were selected. Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the shiitake mushroom, L. edodes. 10 primers

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produced polymorphisms in all 31 strains tested, producing 7-19 bands ranging from 0.18 to 2.52 kb.10 primers results from 27 primers RAPD profiles with amplification of 31 L. edodes isolates were select to analysis genetic similarity by UPGMA cluster analysis with computer software NTSYS-pc. We found that isolates of L. edodes had different genetic diversity. It indicated that 31 isolates of L.edodes could be divided into three groups under DNA similar coefficient of 0.68 mainly in Taiwan, some isolates were similar to foreigen countries. It indicated that 31 isolates of L. edodes could be divided into severn groups under DNA similar coefficient of 0.91. It indicated that Taiwan cultivate L. edodes have rich genetic diversity.We used results of RAPD to select special bands, cloned and sequenced, then used as bases for designing SCAR marks.We used primer 5.0 to design primer LE7 forward and primer LE7 Reverse.Then the RAPD marks were converted into SCAR marks,which were much more specific and stable and the band size was 623bp. In order to prove the DNA sequence completely and specifical characteristics we obtained.We also took primers ITS1 and ITS4 to prove the possible of false positive.When the SCAR mark was tested using 58 strains collected from Taiwan, its feasibility and reliability was validated. The special could amplify 921 strains and completed with foreign countries not the specifical band. The strain-specific SCAR marks were suitable for the rapid identification of L. edodes Key : Lentinula edodes, random amplified polymorphic DNA, sequence characterized amplified region.

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mRNA

Cloning and characterization of genes differentially expressed during fruit body development of Lentinula edodes and Pleurotus species by differential display of RAPD-Chiu-Chien Su and LungChung Chen (Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan 402, R. O. C. ) LE16 P5 P6 RNA RT-PCR cDNA 28 cDNA cDNA gal F1 (200 bp) P2 (300 bp) P1 F2 (250 bp) F1 173 bp NCBI EST Neurospora crassa P6 P13 A (200 bp) S23 E (500 bp) F (690 bp)P9 S24 G (300 bp) K (700 bp) AEF NCBI EST A E 484 bp Ustilago maydis hyposis protein F 684 bp Nurospora crassa filment H protein

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: mRNA Knowledge of the seat of growth with drives morphogensis and appreciation of the balance between physical, biological phenomena and gene expressed intisisted by fungist. In this study, we studied on Lentinula edodes (LE16) and Pleurotus species (P6 and P9). To analysis genes involved in fruit body development of L. edoeds and Pleurotus species, mRNA from three different developmental stages: vegetative mycelium, and fruit body. Total mRNA were isolated and reverse-transcribed to cDNAs. Twenty eight random PCR amplifications were perform with cDNAs, which F1, F2, P1, P2 cDNA fragments from different stage in L. edoeds. By using primer gal, four cDNA clones specifically expressed in primordium (P1, P2)or mature fruit body (F1, F2) were detected. Sequence analysis and databases searchs revealed significant similarity with Neurospora crassa hypothetical protein partial mRNA (F1) and others are still studying. In Pleurotus species respect, Twenty eight random PCR amplifications were performed with the cDNAs, which A, E, F, cDNA fragments from different developmental stages. Sequence analysis and database searches reveraled significant similarity with Ustilago maydis hyposis protein (E), Nurospora crassa filment H protein ( F). In the future, we will use Northern analysis to confirme and protein extracted and analized by different fruit body development stages. Key world: Lentinula edodes, Pleurotus species, differential display of RAPD

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1 21

2

Study on the fermentation broth of Tremella strains for the potential use in cosmetic.--YungChuan Hsieh1Chee-Jen Chen2 (Institute of Biotechnology, Southern Taiwan University of Technology) (Tremella) Tremella encephala T897Tremella nivalis CCJ911Tremella flava CCJ928Tremella sp. CCJ933 Tremella fucifomis CCJ960(B16F10) CCJ897 tyrosin hydroxylase 11.44% dopa oxidase 27.43%UVA NO(Nitric oxide) NO CCJ960 nitrite nitrite 27.36% (H2O2)(Hela cell) CCJ911 H2O2 H2O2 Hela cell 17.15% The fermentation broth of five Tremella strains was compared and its character was explored in this study. The five Tremella strains: Tremella encephala CCJ897 Tremella nivalis CCJ911 Tremella flava CCJ928 Tremella sp. CCJ933 and Tremella fucifomis CCJ960. The

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inhibitory effect on the melanin synthetic enzyme via cell culture of mouse melanoma (B16F10) was studied. The culture T897 showed higher inhibitory activities, 11.44% to tyrosin hydroxylase and 27.43% to dopa oxidase, than the other strains respectively. UVA radiation may induce the generation of NO which will stimulate melanocytes to generate and accumulate melanin. NO may induce inflammatory, suggesting the anti-inflammatory response prevent pigmenting of the skin. The result indicated that the culture CCJ960 has inhibited LPS-induced nitrite production up to 27.36% better than the other strains. Meanwhile, the antioxidant activity showed that the culture CCJ911 increased 17.15% of viability of Hela cells prevented the damage from hydrogen peroxide. This study provides some clues towards the understanding the role and mechanisms of action of five Tremella strains by using liquid fermentation for the application in cosmetic.

(Pleurotus ferulae) 1 21

2

The effect of low temperature and mechanical damages on inducement of the primordia formation in Pleurotus ferulaeChin-Hsiung HungShin-Hua Lee (1Department of Horticultural Science, National Chia-Yi University2Graduate Institute of Horticulture, National Chia-Yi University) (Pleurotus ferulae) 10 33.4 73.3% 43.3 32.2 36.7% 42.9% 10 The stimulation of low temperature and mechanical damages were used to the mycelium and induce the primordia formation of Pleurotus ferulae, the average days of primordial formation by 10 treatments were 33.4 days the percentage of primordia appeared in the Petri dishes were 73.3%. and primordia were only located at peripheral regions of Petri dish, it would be regarded that primordial only appeared at the young mycelia. and mechanical treatment can induce

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regenerate of mycelia, but didn`t affect the location of primordial., it could be suggested that the formation of primodia need the certain mature stages of mycelia. The stimulation by mechanical damages to mycelia could shorten the average days of primordial formation from 43.3 days to 32.2 days. The percentage of primordia appeared in the Petri dishes were increased from 36.7 % to 42.9%, it showed that the mechanical stimulation could promote the formation of primordial, 10 and mechanical stimulation obtained the best induction results.

1 21

2

Polysaccharides composition of Coprinus comatus and its cytotoxic activities on cancer cells Shu-Yao Tsai1 and Jeng-Leun Mau 2 (1 Department of Applied Life Science, Asia University; 2 Department of Food Science and Biotechnology, National Chung-Hsing University) Coprinus comatus (Mull.: Fr.) S. F. Gray NC H IC50 A549 0.24 2.58 mg/mL HCT116 1.14 1.58 mg/mL MCF-7 1.11 3.22 mg/mL SKHep-1 2.56 8.46 mg/mL Coprinus comatus (Muller: Fries) S. F. Gray (Coprinaceae), the shaggy mane or chicken drumstick mushroom and also known as the lawyers wig mushroom, is a newly cultivated edible and medicinal mushroom. With regard to the hot water or alkali extraction from C. comatus, the yield was the higher for alkali extraction polysaccharides. In elemental analysis, nitrogen, carbon

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and hydrogen contents of alkali extracts polysaccharides from C. comatus were higher than that of their hot water extracts polysaccharides. Neutral sugars were mannose and glucose for all polysaccharides isolated. Using gel filtration, the molecular weights of hot water extracts from C. comatus were higher than that of their alkali extracts. Besides, the studies of the cancer cell viability of hot water and alkali extracts polysaccharides of C. comatus. IC50 values in A549 cell line were 0.24 and 2.58 mg/mL; in HCT116 cell line were 1.14 and 1.58 mg/mL; in MCF-7 cell line were 1.11 and 3.22 mg/mL; in SK-Hep-1 cell line were 2.56 and 8.46 mg/mL for fruit body in alkali extracts polysaccharide, and mycelia in alkali extracts polysaccharide from C. comatus, respectively. Overall, C. comatus contained abundant bioactive polysaccharides, tumor cytotoxicity. However, how to inhibition cancer cell grown of C. comatus polysaccharide need to further research.

1 1 2

1 2

Study on the Effects of Nutrients on Growth Characteristics of Tricholoma matsutake Sheng-Jye Yang1, Shih-Hao Lee1, Yue Ken Liao2 (1Department and Graduate Institute of Forest Products Science, 2Department and Graduate Institute of Forestry and Natural Resources, National Chiayi University, 300 University Road, 600 Chiayi, Taiwan) Tricholoma matsutakeIto et ImaiSing. Agaricales Tricholomataceae Tricholoma MNC MNC MNC MNC Tricholoma matsutake is attributed to Tricholomataceae. It is famous for edible fungi all over the

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world. However, due to tremendous decrease in nature culture land and difficulty to cultivate by human, it is highly desired to develop technology to cultivate this valuable fungi. Diminishing aging speed of mycelium is investigated by adding various nutrients MNC, Ebios, extract from peanut hullin the culture medium. It is expected to discover the optimal medium in this work. The results show: The growth speed of mycelium in MNC and Ebios culture medium is the best The MNC and extract from peanut hull culture medium is the next; the MNC culture medium is the worst. The aging of mycelium in the Ebios culture medium is observed in the fourth week. There is no such phenomenon in the MNC culture medium.

1. 21 2

The Inhibitory Effect of Chitosans from Fungal Materials on the Activity of Lipase of Propionibacterium acnes --Chun-Chung Fang1 and Ching-Hua Su2 (1Graduate Institute of Cell and Molecular Biology, Taipei Medical University 2Graduate Institute of Biomedical Materials and Department of Microbiology and Immunology, Taipei Medical University, 250 Wu-Hsing Street, 110 Taipei, Taiwan) (Propionibacterium acnes) ( Acne vulgaris ) - (Lipase)(Chitosan) (Chitosan) -(Lipase)

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Gene cloning lipase gene(GehA) contract pDNR ampicillin agarose gel 1197bps DNA GehA gene T7 promoter p7GFP2-MCS-Rluc IPTG E.coli lipase protein Rhizopus stolonifer Cordyceps bifusispora Ganoderma tsugae Lipase Propionibacterium acnes ( P. acnes ) played an important role in the pathogenesis of acne vulgaris . Extracellular lipases produced by P. acnes in vivo act as key enzyme in the hydrolyzation pathway of native sebum triacylglycerols to glycerols and free fatty acids ( FFA ). We contract lipase gene(GehA) into pDNR vector and then subclone this GehA gene into another p7-GFP2MCS-Rluc vector which has T7 promoter to overexpress lipase protein by IPTG induced in E.coli . Finally, we expect to investigate whether or not chitosan from different materials such as cell wall component from Rhizopus stolonifer Cordyceps bifusispora and Ganoderma tsugaeand possessed the inhibitory potency on the growth of P. acnes and/or the activity of lipase. Putative lipase activity was detected by lipase zymography and the observation of the interactive precipitate between FFA and Victoria Blue B ( VBB ).

IGS-RFLP

IGS-RFLP analysis of pathogenic and nonpathogenic Fusarium oxysporum f. sp. gladioli. -- TingLin Chi and Ruey-Shyang Chen (Graduate Institute of Biotechnology, National Chiayi University, Chiayi 600, Taiwan) Fusarium oxysporum 68 F. oxysporum f. sp. gladioli DNA intergenic spacer (IGS) BstEII ScaI XhoI F. oxysporum f. sp. gladioli

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Pathogenic isolates of Fusarium oxysporum often display a high degree of host specificity and may be subdivided into formae speciales based on the host species attacked. In the present study, sixty-eight isolates of F. oxysporum f. sp. gladioli were analyzed for genetic diversity. The intergenic spacer (IGS) regions of the rDNA of F. oxysporum f. sp. gladioli were amplified by PCR and an IGS-RFLP analysis was performed. Restriction digestion with BstEII, ScaI and XhoI allowed differentiation between pathogenic and nonpathogenic isolates of F. oxysporum f. sp. gladioli. Pathogenic and nonpathogenic isolates were separated into different groups based on RFLP patterns, although some nonpathogenic isolates grouped with pathogenic isolates. The population of pathogenic isolates was less diverse than that of the nonpathogenic isolates, suggesting that the pathogenic isolates were possibly of monophyletic origin.

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1 2 31

2 3

Antimutagenic properties of fungal chitosans from shiitake stipes--Ming-Tsung Yen1 Yu-Hsiu Tseng2 and Jeng-Leun Mau3(1Department of Applied Life Science and Health, Chia Nan University of Pharmacy and Science, 60 Erchjen Road, Section 1, Jente, Tainan, Taiwan 71710, R.O.C. 2Department of Baking Technology and Management, National Kaohsiung Hospitality College, 1 Sung-Ho Road, Kaohsiung 812, Taiwan, R.O.C. 3Department of Food Science and Biotechnology, National Chung-Hsing University, 250 Kuokuang Road, Taichung, Taiwan 40227, R.O.C. B90B120 C90C120 0.055 mg/plate S9 B90 B120 C90 C120 S. typhimurium TA98 TA100 100% 0.05 5 mg/plate S9 B90 B120 C90 C120 S. typhimurium TA98 TA100 0.55 1.11 0.571.07

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The cancer to come about the maximun to be bound up with of diet, this food to impersonate is being of attention. Also have been reported of discover that has natural to send turn of events, carcinogenic composition and antimutagenic material from food. Chitin and derivatives have antimutagenic and to restrain a sudden change of metabolism activity effect. Furthermore, the fungal chitosan of immediate in an opposite to send change matter of antimutagenic from shiitake stipes were studied. Fungal chitosan showed is no toxicity was found in Salmonella typhimurium TA98 and TA100 was observed at 0.05-5 mg/plate of fungal chitosan B90, B120, C90 and C120 with a survival of 100%, respectively. The result showed is no mutagenicity found in Salmonella typhimurium TA98 and TA100 was observed at 0.05-5 mg/plate of fungal chitosan B90, B120, C90 and C120 with the mutagenicity of rate range 0.551.11and 0.571.07, respectively.

-*

Study of production of poly(-glutamic acid) by Bacillius subtilis (natto) -- Shun-Lai Li*, Ming-Fwu Jeng and Meng-Bor Hsu ( Graduate Institute of Biotechnology, Southern Taiwan University of Technology, No.1, Nantai St, Yung-Kang City, Tainan, Taiwan) [poly(-glutamic acid)-PGA] -PGA N1 -PGA 37 150 rpm : . N1 LB ( Yeast extract 15g/L Tryptone 5g/L Sodium chloride 4g/L Glucose 30g/L ). LB : 60 . N1 M2 ( Monosodium glutamate monohydrate 171.7g/L Glucose 80g/L Peptone 15g/L Yeast extract 5g/L Urea 3g/L 2HPO4 2g/L ) -PGA . M2 K

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63.1( -PGA ) 13.5 : LB N1 M2 PGA Poly(-glutamic acid) (-PGA) is a water-soluble biopolymer that can be found in Japanese traditional fermented food, natto, which has potential applications in the environmental protection, health food, medicine and cosmetics. In general, the increase in broth viscosity implies the increase of- PGA content. This study has focused on the screening of cultural parameters for high -PGA production by B. subtilis (natto) N1. In this study, B. subtilis (natto) was incubated at 37 and 150 rpm. The following results were obtained: 1, N1(natto) generated higher biomass as cultured in LB medium ( Yeast extract 15g/L, Tryptone 5g/L, Sodium chloride 4g/L and Glucose 30g/L ). 2, The biomass increased up to 60% by using the inoculum that was activated twice in LB medium. 3, Higher -PGA production was obtained when N1(natto) was fermented in modified M2 medium ( Monosodium glutamate monohydrate 171.7g/L, Glucose 80g/L, Peptone 15g/L, Yeast extract 5g/L, Urea 3g/L and K2HPO4 2g/L ). 4, Feeding modified M2 medium during fermentation could not only raise 63.1% of the broth viscosity, corresponding with higher- PGA production, but also reduce 13.5% of the fermentation time. In conclusionThe two-step activation procedure of N1 in the LB medium and then cultivated in the modified M2 medium with step-increase