PCR-Based Sensitive Detection of Medicinal Fungi Hericium Species from Ribosomal Internal Transcribed Spacer (ITS) Sequences

Home , Auriscalpium, Auriscalpium vulgare, Hericiaceae, Hericium, Hericium abietis, Hericium americanum

August 2002 Biol. Pharm. Bull. 25(8) 975—980 (2002) 975

Ling LU,* Jieli LI, and Yihua CANG College of Life Science, Nanjing Normal University; Nanjing 210097, P.R. China. Received February 13, 2002; accepted April 16, 2002

Based on phylogenetic analysis of rDNA internal transcribed spacer (ITS) sequences, a pair of specific primers were designed for differentiating the Chinese traditional medicine Hericium species from other mushrooms by PCR. PCR was performed, with total DNAs as a template at an annealing temperature of 52—57 °C. Positive amplification was obtained from H. erinaceus with all DNA templates from different resources, but not from other related species. The result indicated that H. erinaceus could be clearly distinguished from other fungi by detection PCR, and no incorrect discrimination was found under the same reaction conditions. The primers were also successfully employed to identify H. erinaceus with different tissue types. Key words Hericium erinaceus; detection PCR; internal transcribed spacer (ITS); medicinal fungi; specific primer

Hericium species are considered precious edible mush- Phellinus linteus based on the rDNA ITS sequence data. In rooms, among which Hericium erinaceus has long been used this study, a preliminary phylogenetic analysis of ITS se- as a Chinese traditional medicine to treat various diseases of quences was performed and used to assess the phylogenetic the digestive system.1) The cultures of H. erinaceus or their relationship between Hericium and other relevant species. extracts processed in tablets have been put into production on Furthermore, a set of PCR primers specific to Hericium was a large scale, mainly for curing gastric ulcer and chronic gas- designed for establishing a convenient and accurate method tritisin. Recently, Hericium erinaceus has attracted great at- for the identification of medicinal mushrooms. tention owing to its anti-tumor, immunomodulatory and lipid-lowering effects, as well as other new medical values.2) MATERIALS AND METHODS Understandably, the physiological, biochemical and pharma- cological properties of Hericium erinaceus are being increas- Sample Sources Sixteen samples of 13 species from dif- ingly studied in China, Korea and Japan, etc.3—6) Accurate ferent locations and various types of tissues used in this taxonomic identification and phylogenetic classification will study are listed in Table 3. The specimens have been placed help to predict novel genes and novel useful metabolites, and in the collection of Nanjing Normal University (NJNU), provide useful information for genetic engineering or cultiva- China. Hericium was maintained on 1.5% malt-extract agar, tion of commercial strains.7) However, only a few studies and was grown in the liquid media (1% malt extract, 1% have focused on the molecular systematics of medicinal yeast extract , 0.4% glucose) at 26 °C for 6 d before its fungi. In addition, the evolutionary history and relationships mycelium culture was harvested, freeze-dried, and stored at of Hericium with other basidiomycetes remains controversial Ϫ20 °C for analysis. The solid culture was obtained by trans- in spite of its economic importance.8,9) To solve such prob- ferring the fresh colonies into bagasse media and allowing lems, correct identification of medicinal fungi is necessary. them to grow at 22 °C for 30 d. The fruiting body of HTDB Unfortunately, the traditional methods for the identification developed 20 d after the fruit body appeared by ordinary of mushrooms are problematical, because most of them in methods. mycelia phase are very similar to each other in both morpho- DNA Extraction Total genomic DNA was extracted as logical characteristics and organic or inorganic component; described by Graham et al.16) Lyophilized sample was ground in addition, culturing the fruiting body for identification is in liquid nitrogen and transferred into a 1.5 ml microfuge both time-consuming and inefficient. Thus, a more advanced tube. Lysis buffer (200 mM Tris–HCl, pH8.0, 100 mM NaCl, method should be developed for the identification of H. eri- 25 mm EDTA, 0.5% sodium dodecyl sulfate (SDS)) was then naceus and other medicinal fungi. added. After incubation at 37 °C for 1 h, 2ϫCTAB buffer With the current rapid progress in biological techniques, (2% CTAB [w/v], 100 mM Tris–HCl pH 8.0, 1.4 M NaCl) was molecular genetic markers have been employed for sensitive added, and the mixture was placed in a water bath at 65 °C and rapid identification of medicinal materials.10) The devel- for 15 min. The solution was extracted at least twice with opment of DNA-based PCR11) and taxon species primers12) is equal volume of phenol : chloroform : isoamyl alcohol making it increasingly feasible to detect and study fungi. The (25 : 24 : 1, v/v), then centrifuged at 12000 rpm for 10 min, internal transcribed spacer (ITS) region has been generally followed by extraction with chloroform : isoamyl alcohol considered a convenient target for the molecular identifica- (24 : 1) until the top phase was clear. The upper phase was tion of fungi.13) For example, Henry, T. et al.14) identified As- transferred to new tube, and DNA was precipitated with 0.7 pergillus at the species level and differentiated it from other volume isopropanol, washed with 70% ethanol, dried, and re- true pathogenic and opportunistic molds using ITS 1 and 2 suspended in 100—200 ml TE buffer (10 mM Tris–HCl, 1 mM for earlier diagnosis and screening of effective antifungal EDTA, pH 8.0). Genomic DNA was visualized in 1% agents for patients. Park, D. S. et al.15) designed a pair of spe- agarose gel. The concentration and purity of the DNA were cific primers for PCR detection of wood-decaying fungus determined using a UV spectrophotometer (BIO-RAD

SmartspecTM 3000). Seven ITS sequences of ﬁve Hericium species, H. erinaceum rDNA ITS Region PCR Ampliﬁcation and Sequencing 48001, 48002, H. coralloides, H. abietis, H. alpestre, H.

Amplification and sequencing of HTDB involved using a pair americanum from Genbank were aligned with HTDB by of universal primers ITS5 (5Ј-GGAAGTAAAAGTCGTAA- CLUSTALX (1.8).18) The similarity (%) of ITS sequences CAAGG-3Ј) and ITS4 (5Ј-TCCTCCGCTTATTGATATGC- and values of base transition/transversion were estimated 3Ј)17) for the region containing ITS1 and ITS2 and the 5.8S using MEGA 2.0 based on the Kimura2-Parameter Distance rDNA, and they were also employed as a positive control in model.19) subsequent diagnostic PCR. The amplification was carried Phylogenetic Analysis Sequence data from ribosomal out in a PTC-200 thermocycler (MJ Research, Inc.) in a ITS genes of Homobasidiomyetes were used to perform a 50 ml reaction mixture containing 10 mM Tris–HCl pH 8.3, preliminary phylogenetic analysis. Twenty-eight ITS se- 1.5 mM MgCl2, 50 mM KCl, 0.2 mM of each dNTP, 0.4 m M of quences were obtained from Genbank (Table 1). These each primer, 50 ng template DNA, and 1.5 U Taq DNA poly- species designations were based on morphological species merase (Promega). The reaction mixtures were denatured at concept or referred to previous reports.20,21) Phylogenetic 95 °C for 5 min and subjected to 35 cycles of 40 s at 95 °C, analysis of the aligned sequences was performed by Neigh- 1 min at 52—57 °C, 1 min at 72 °C, and a final extension step bor-Joining (NJ) analysis, using the MEGA (2.0) Kimura 2- of 6 min at 72 °C. Amplified products were purified using a Parameter Distance model, in which gaps were treated as DNA purification kit (Shanghai Watson Bioengineering, missing data. The resulting trees were rooted with Tremella. Inc.), according to the manufacturer’s instructions, and then Bootstrapping was performed with NJ analysis using 300 sequenced directly with BigDyeTM on a ABI-PRISM 310 au- replicates. tomated DNA sequencer (PE Applied Biosystems Co.). The Primer Design Based on the results of the phylogenetic ITS sequence of HTDB (561 bp) was deposited in Genbank analyses, we investigated ITS sequences of the nearest rela- (accession number AF397469). tives and of the relevant species (Table 2). A pair of primers, Sequence Analysis of Hericium Based on ITS Region HT-U1 and HT-L1, was designed for PCR detection of au- thentic species of Hericium (Fig. 1). Primer pairs were tested Table 1. Species of Homobasidiomyetes used for Phylogenetic Anaysis of for specificity with total genomic DNA from 16 samples of Hericium

Length Accession Table 2. Species of Hericium and Relevant Fungi Used in Identiﬁcation Classiﬁcation Source (bp) number Primer Design

Auriscalpiaceae Length Accession Number Classiﬁcation Source Clavicorona microspora Ky5352 665 AF336137 (bp) number Clavicorona pyxidata 7263 640 AF336139

Auriscalpium vulgare 4242 639 AF454430 1 Hericium erinaceum HTDB 561 AF397469 Auriscalpium villipes 4396 655 AF454429 2 Hericium erinaceum 48001 543 AF146779 Lentinellus subaustralis 9159 636 AF454427 3 Hericium erinaceum 48002 543 AF126009 Corticiaceae 4 Hericium coralloides 1 ATCC 52480 543 AF287981 Entonocorticium dendroctoni — 547 AF119506 5 Hericium coralloides 2 CBS 447.85 543 AF173216 Peniophora pithya Tppt 2226 546 AF119520 6 Hericium abietis CBS 243.48 543 AF287979 Peniophora aurantiaca NK 98/32 684 AF210819 7 Hericium americanum CBS 493.7 543 AF287980 Dendrophora albobadia Tdeab 1029 551 AF119522 8 Hericium alpestre CBS 539.90 543 AF287978 Echinadontiaceae 9 Auricularia auricula — 581 AF291268 Echinadontium tsugicola B1377 532 AF218398 10 Tremella fuciformis CCJ 1531 507 AF042436 Echinadontium japonicum B1375 534 AF218399 11 Lactarius intermedius — 675 AF140257 Hericiaceae 12 Zelleromyces hispanicus MA-Fungi 37497 638 AF231911

Hericium erinaceum HTDB 561 AF397469 13 Russula postiana MA-Fungi 42068 626 AF230898 48001 543 AF146779 14 Stereum annosum — 549 AF218401 48002 543 AF126009 15 Heterobasidion annosum 99005/1 547 AF289929 Hericium coralloides ATCC 52480 543 AF287981 16 Clavicorona pyxidata 7263 640 AF336139 CBS 447.85 543 AF173216 17 Lentinellus subaustralis 9159 636 AF454427 Hericium abietis CBS 243.48 544 AF287979 18 Auriscalpium vulgare 4242 639 AF454430 Hericium americanum CBS 493.7 543 AF287980 19 Ganoderma lucidum wc724 600 AF079584 Hericium alpestre CBS 539.90 543 AF287978 20 Lentinula edodes wc394 707 AF079572 Russulaceae 21 Agaricus campestris CCRC 36325 566 AJ133389 Lactarius intermedius — 675 AF140257 22 Flammulina velutipes MH 09210 802 AB064957 Lactarius scrobiculatus — 736 AF140262 23 Pleurotus ostreatus — 537 AF423120 Russula postiana MA-Fungi 42068 626 AF230898 24 Vlovariella volvacea — 728 U15973 Zelleromyces hispanicus MA-Fungi 37497 638 AF231911 25 Dictyophora indusiata ATCC 60890 565 AF324160 Tremellaceae Tremella fuciformis CCJ 1531 507 AF042436 Schizophyllaceae Heterobasidion annosum 99005/1 547 AF289929 Heterobasidion insulare 97007/5 564 AF289932 Stereaceae Amylostereum ferreum B1359 518 AF218390 Amylostereum chailletii B1354 518 AF218391 Stereum annosum — 549 AF218401 Fig. 1. Locations of PCR Primers in the Nuclear rDNAs and ITS Regions Stereum hirsutum — 568 AF218400 The arrowheads represent the 3Ј end and approximate position of each primer. The nuclear large rDNA is truncated in this ﬁgure. August 2002 977

Table 3. Species Used in Identiﬁcation Assay

Number Name Locality Tissue type Voucher no.

1 H. erinaceus Jiling Liquid shaken mycelia NJNU L-99HTDB 2 H. erinaceus Chejiang Liquid shaken mycelia NJNU L-99HTZJ 3 H. erinaceus Jiangsu Liquid shaken mycelia NJNU L-00HT12 4 H. erinaceus Heilong Jiang Liquid shaken mycelia NJNU L-00HT43 5 Pleurotus ostreatus Jiangsu Basidiocarp NJNU L-01CR 6 Vlovariella volvacea Jiangsu Liquid shaken mycelia NJNU L-01CG 7 Auricularia auricula Jiangsu Liquid shaken mycelia NJNU L-01MR 8 Flammulina velutipes Shanghai Basidiocarp NJNU L-01JZG 9 Ganoderma lucidum Jiangsu Liquid shaken mycelia NJNU L-01LZ 10 Agaricus campestris Jiangsu Basidiocarp NJNU L-01MG 11 Lentinu edodes Jiangsu Basidiocarp NJNU L-0IXG 12 Tremella fuciformis Jiangsu Liquid shaken mycelia NJNU L-01YR 13 Coriolus versicolor Jiangsu Liquid shaken mycelia NJNU L-01YZ 14 Dictyophora indusiata Jiangsu Agar cultured mycelia NJNU L-01ZS 15 Rhizopus oryzae Jiangsu Liquid shaken mycelia NJNU L-00GM 16 Diasporangium sp. Jiangsu Liquid shaken mycelia NJNU L-97LZM

Table 4. Similarity (%) of ITS Sequences (Lower Triangle) and Values of Transitions/Transversions(Upper Triangle) of Hericium

OTUs12345678

1. HTDB 1/0 0/0 1/0 5/3 6/1 2/0 3/8 2. H. erinaceus 48001 99.8 1/0 0/0 6/3 7/1 3/0 4/8 3. H. erinaceus 48002 100 99.8 1/0 5/3 6/1 2/0 3/8 4. H. coralloides 1 99.8 100 99.8 6/3 7/1 3/0 4/8 5. H. coralloides 2 98.5 98.3 98.5 98.3 7/4 3/3 1/11 6. H. abietis 98.7 98.5 98.7 98.5 97.9 6/1 1/8 7. H. americanum 99.6 99.4 99.6 99.4 98.9 98.7 6/9 8. H. alpestre 97.9 97.8 97.9 97.8 97.8 97.2 98.3 relevant fungi (Table 3) in PCR, and a DNA fragment (ap- to David S. Hibbett’s21) phylogenetic tree of Homobasid- proximately 300 bp) from the rDNA ITS gene was amplified. iomycetes generated with the data of nuc-ssu-rDNA and mt- Diagnostic PCR was performed under the conditions men- ssu-rDNA. tioned above. PCR Primer Design Seventeen species of familiar edible and medicinal mushrooms as well as 8 isolates of Heri- RESULTS cium, were chosen to test the detection primers designed on the basis of phylogenetic analysis (Fig. 2). The selected spans Sequences Analysis of Different Hericium Species ITS for specific primers were 38—61 bp and 375—397 bp, re- sequences of HTDB and H. erinaceus 48001 and 48002 was spectively. For the more sensitive detection of Hericium, the found to be almost the same, in spite of their different geo- 11th nucleotide “C” of the oligomer HT-U1 was changed graphic origins. There was only one couple of divergent to “A”. So, the specific primer pairs were HT-U1 (5Ј- Ј Ј bases between HTDB and 48001, and no sequence difference CAGGCATGTGCACGCTCCAATCTC-3 ) and HT-L1 (5 - Ј between HTDB and 48002. The ITS similarity among HTDB, CAAGACCTCCAAGTCCAAGCCCA-3 ). H. abietis, H. americanum, and H. coralloides 1 was higher When the universal primers ITS5 and ITS4 were used at than 98% (Table 4), whereas the similarity of ITS among the annealing temperature of 52 °C, 16 samples from the 13 seven species of Hericium was 97.2—100%. species of relevant mushrooms and opportunistic molds all Phylogenetic Analysis The phylogenetic tree was gener- gave an amplificon with a size between 500 and 600 bp (Fig. ated from 29 aligned sequences of Homobasidiomycetes 3A). While the specific primers HT-U1 and HT-L1 were used (Fig. 5). In the phylogenetic tree, 6 groups were assembled for PCR under the same condition, a DNA fragment with a from 29 strains of Homobasidiomycetes. Except for group 2, size of about 300 bp was clearly amplified from 4 isolates of containing strains from a different family of the same order, H. erinaceus, and there was no PCR product from Pleurotus and group 5, containing strains from a different family of a ostreatus, Vlovariella volvacea, Auricularia auricula, different order, the other four groups consisted of strains Tremella fuciformis, Coriolus versicolor, Dictyophora indu- from the same family. Our results suggested that grouping at siata, Rhizopus oryzae, or Diasporangium sp., but Auricu- a familial and generic level in ITS-based cladograms would laria auricula, Flammulina velutipes, Ganoderma lucidum, be feasible, in spite of a low confidence level supported by Agaricus campestris gave a weak signal (Fig. 3B). When the the bootstrap for the higher order. annealing temperature was increased to 54 °C, all species of As one independent branch, Hericiaceae was closely re- H. erinaceus were amplified well; however, Flammulina ve- lated to Auriscalpiaceae, Echinadontiaceae, Russulaceae, lutipes and Agaricus campestris no longer showed an ampli- Schizophyllacea and Stereaceae in the tree, which is similar fied product, while there was still faint amplification in Au- 978 Vol. 25, No. 8

Fig. 2. Hericium-Speciﬁc Primer Design Based on the ITS Region between Species of Hericium and Relevant Species Codes are the same as described in Table 2.

Fig. 4. PCR Detection of HTDB Using DNA Template from Different Re- sources with Speciﬁc Primers (HT-U1 And HT-L1) 1: Liquid cultured mycelia, 2: Fruiting body, 3: Agar cultured mycelia, 4: Solid cultures, M: DNA marker (100 bp DNA ladder), N: Negative control.

CDstitutes of medicinal fungi in the market, owing to their high price. The identification of high-quality species of economic Fig. 3. PCR Amplification of ITS Gene Region at Different Annealing Temperatures fungi tends to be of great significance. A: Positive control annealed at 52 °C with the primers ITS4 and ITS5, B, C, D: An- A molecular technique may greatly enhance detection sen- nealed at 52 °C, 54 °C and 57 °C, respectively, with the primers HT-U1 and HT-L1. M: sitivity, simplicity and rapidity. Several methods, which DNA marker (A: 100 bp DNA ladder; B, C, D: DL-2000 ), N: Negative control. Codes are the same as described in Table 3. mainly include random amplified polymorphic DNA (RAPD), arbitrary primed PCR (AR-PCR), restriction fragment length polymorphism (RFLP), PCR-RFLP and DNA ricularia auricula and Ganoderma lucidum (Fig. 3C). Once sequencing, have recently been used for the authentication of the annealing temperature was set to 57 °C, the amplified medicinal materials. Although they have been proven to be DNA band only appeared in 4 isolates of H. erinaceus, and efficient in taxonomic identification and in distinguishing there was no PCR product from the other fungi (Fig. 3D). genuine crude drugs from their substitutes or adulterants in In order to verify that various types of samples could be previous reports, the limitation of the application of these well amplified by the identification primers, we used a DNA methods is inevitable because fine quality template DNA is template that was respectively extracted from liquid cultured required in these experiments and they are relatively expen- mycelia, fruiting body, agar cultured mycelia and solid cul- sive. The reproducibility of RAPD analysis is badly affected tures of HTDB for detection PCR. A single product, which is by the quality and concentration of template DNA, the ratio identical to that in Fig. 4, could be successfully amplified at of template-to-primer, and slight fluctuations of reacting the annealing temperature of 57 °C from all types of sample components or cycling parameters. As for the PCR-RFLP by specific primers. method, the length of PCR products also confines its utiliza- tion, since the number of restriction enzyme sites is limited DISCUSSION in the DNA segments between the two primers. Although the sequence analysis of PCR products has quite good precision With increasing demands for edible and medicinal fungal and stability, the relatively high expense of DNA sequencing materials, the scarcity of some wild fungi used as Chinese and the sensitivity to contamination in the PCR reaction traditional medicine, such as Hericium erinaceus, Gano- using universal primers obstruct its wide use in the quality derma lucidum and Cordyceps sinensis, has become much control of medicinal materials.22) more serious. Moreover, there are a lot of adulterants or sub- In this study, an efficient method was presented to identify August 2002 979

Fig. 5. Phylogenetic Relationships of Homobasidiomycetes Inferred from ITS Sequences One of 29 equally parsimonious trees. The number by node is bootstrap frequency (* represent values Ͻ30%). Branches within a vertical line represent the same family. The number beside the family name indicates the group code. Tremella fuciformis was the outgroup. economic fungi. Based on the target DNA sequence analysis ment could be clearly amplified from 4 strains of H. eri- of high-quality species or original medicinal mushrooms, we naceus, whereas no PCR product from other fungi was am- designed a pair of specific primers that match exactly with a plified under the same conditions. In order to verify the qual- definite DNA sequence of Hericium, and incompletely with ity of the DNA template, all template DNAs were amplified the sequence of other species. Therefore, a high-stringency with universal primers at an annealing temperature of 52 °C, PCR reaction with the primers specific to Hericium gave a and all PCR reactions gave clear bands on agarose gel elec- positive signal only to genuine species of Hericium, but not trophoresis. These results proved that the DNA templates to other closely related fungi. Besides, the process of identifi- were suitable for PCR. It also indicated that a particular cation PCR is very simple and convenient. DNA band should be amplified from these templates if a pair It is commonly thought that ITS regions are often highly of primers can find an appropriate annealing position on the variable between morphologically distinct species or even template DNA. In addition, our experiment also proved that isolates of the same species.23) Therefore, the ITS region has specific detection PCR could be preformed with templates been generally believed a convenient target for molecular from various sources, regardless of the tissue type of the identification of fungi. However, a high similarity of ITS se- sample. quences has been found in our phylogenetic analysis of Heri- In conclusion, an accurate and practical phylogenetic cium, which is not fully in accord with previous views. This analysis would lay a theoretic foundation for defining a clas- might be due to differences in evolution sequence, variation, sified status of new edible or medicinal fungi. In addition, analytic methods, etc. Additionally, it had been demonstrated their evolution relationships could provide an important clue by preceding research that some fungus species have little di- for further exploration of the active compounds. Further- vergence in the interval gene region. This suggests that ITS more, specific detection PCR is a time-effective and low-cost may not be suitable for taxonomic analysis between some method for the authentication of edible and medicinal mush- species of intragenus and population level.24) rooms. This method will aid in the quality control of Chinese According to the high similarity of ITS sequences of medicinal materials and species identification of other eco- species in Hericium, H. erinacius was chosen as representa- nomical fungi. tive of Hericium to design detection PCR primers specific to Hericium, since it is widespread in China. To probe into the Acknowledgements We express our thanks to Prof. evolutionary history of Hericium and its relationship with Qiangtai He for providing fungi strains for this project, and other basidiomycetes, and to prepare for the specific primer to Dr. Yongxiang Zhang, who kindly read the manuscript and design, we established a phylogenetic tree of Hericium and offered a number of comments. This work was supported by some strains of correlative order/family in Homobasid- a grant from the cooperative project of Nanjing Laoshan iomycetes in a higher order taxonomic level. Based on this Pharmaceutical Co., Ltd. and Nanjing Normal University, phylogenetic tree, the ITS sequences from the nearest rela- and a grant from the Jiangsu Educational Committee (No. tives and the relevant species of Hericium were aligned, and 2000SWX0002SJ1). the results were used to design a pair of specific primers (HT- U1 and HT-L1) only for Hericium. Under a relatively high PCR annealing temperature, a signal of 300 bp DNA frag- 980 Vol. 25, No. 8

REFERENCES tor R., Mycologia, 92, 964—977 (2000). 14) Henry T., Iwen P. C., Hinrichs S. H., J. Clin. Microbiol., 38, 1510— 1) Ying J., Mao X., Ma Q., Zong Y., Wen H. (ed), “Icons of Medicinal 1515 (2000). Fungi from China,” Vol. 1, Science Press, Beijing, 1987. 15) Park D. S., Kang H. W., Kim K. T., Cho S. M., Park Y. J., Shin H. S., 2) Chang R., Nutr. Rev., 54, 91—93 (1996). Lee B. M., Go S. J., Mycobiology (Korean), 29, 7—10 (2001). 3) Liu M. S., Cheng H. Y., Sun H. B., Edible Fungi of China, 18, 24—25 16) Graham G. C., Mayer S. P., Henry R. J., Biotechniques, 16, 175—269 (1999). (1994). 4) Li J. L., Lu L., Dai C. C., Chen K., Qiu J. Y., China J. Chinese Materia 17) White T. J., Bruns T. D., Lee S. B., Taylor J. W., “PCR Protocols: A Medica, 26, 831—834 (2001). Guide to Methods and Applications,” ed. by Innis M. A., Gelfand D. 5) Saito T., Aoki F., Hirai H., Inagaki T., Matsunaga Y., Sakakibara T., H., Sninsky J. J., White T. J., Academic Press, San Diego, California, Sakemi S., Suzuki Y., Watanabe S., Suga O., Sujaku T., Smogowicz A. 1990, pp. 315—322. A., Truesdell S. J., Wong J. W., Nagahisa A., Kojima Y., Kojima N., J. 18) Jean F., Thompson J. D., Gouy M., Higgins D. G., Gibson T. J., Trends Antibiot., 51, 983—990 (1998). Biochem. Sci., 23, 403—405 (1998). 6) Mizuno T., Wasa T., Ito H., Suzuki C., Ukai N., Biosci. Biotech. 19) Kumar S., Tamura K., Nei M., Molecular Evolutionary Genetics Biochem., 56, 347—348 (1992). Analysis, version 2.0. Pennsylvania State University, University Park, 7) Xiu R. X., Moncalvo J. M., Wang H. F., Wang X. H., J. China Agri. PA 16802, 1993. Chem. Society (Taiwan), 34, 129—143 (1996). 20) Hibbett D. S., Donoghue M. J., Can. J. Bot., 73, 853—861 (1995). 8) Zervakis G. I., Venturella G., Papadopoulou K., Microbiology, 147, 21) Hibbett D. S., Pine E. M., Langer E., Langer G., Donoghue M. J., 3183—3194 (2001). Proc. Natl. Acad. Sci. U.S.A., 94, 12002—12006 (1997). 9) Bowman B. H., Taylor J. W., Brownlee A. G., Lee J., Lu S. D., White 22) Wang Y. Q., Zhou K. Y., Xu L. S., Dong T. T. X., Tsim K. W. K., Biol. T. J., Mol. Biol. Evol., 9, 285—296 (1992). Pharm. Bull., 23, 585—588 (2000). 10) Olivier C., Loria, R., FEMS Microbio. Lett., 168, 235—241 (1998). 23) Lee S. B., Taylor J. W., Mol. Biol. Evol., 9, 636—653 (1992). 11) Mullis K. B., Faloona F. A., Methods Enzymol., 155, 335—350 (1987). 24) Yu Z. D., Zhang X. Y., Cao Z. M., J. Northwest Forestry Univ. Sini., 12) Gardes M., Bruns T. D., Mol. Ecol., 2, 113—118 (1993). 15, 107—112 (2000). 13) Javier S. B., Vicente G., Oscar S., Javier A., Maria A. P., Maria J., Vic-