Mycologia ISSN: 0027-5514 (Print) 1557-2536 (Online) Journal homepage: https://www.tandfonline.com/loi/umyc20 Black spot disease of Lentinula edodes caused by the Hyphozyma synanamorph of Eleutheromyces subulatus Akihiko Tsuneda, Shigeyuki Murakami, Warwick M. Gill & Nitaro Maekawa To cite this article: Akihiko Tsuneda, Shigeyuki Murakami, Warwick M. Gill & Nitaro Maekawa (1997) Black spot disease of Lentinula�edodes caused by the Hyphozyma synanamorph of Eleutheromyces�subulatus, Mycologia, 89:6, 867-875, DOI: 10.1080/00275514.1997.12026857 To link to this article: https://doi.org/10.1080/00275514.1997.12026857 Published online: 28 Aug 2018. Submit your article to this journal Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=umyc20 Mycologia, 89(6), 1997, pp. 867-875. © 1997 by The New York Botanical Garden, Bronx, NY 10458-5126 Black spot disease of Lentinula edodes caused by the Hyphozyma synanamorph of Eleutheromyces subulatus1 1993; Tsuneda et al., 1995b). Tsuneda et al. ( 1995a) found that numerous yeastlike cells occurred in black spot lesions on fresh L. edodes fruiting bodies developing outdoors on Quercus bedlogs, and they identified the fungus as the Hyphozyma synanamorph of Eleutheromyces sp. Later, the present authors carried out further taxonomic and pathological studies of this fungus and found that it can be accommodated in E. subulatus (Tode: Fr.) Fuckel and that it is highly pathogenic to L. edodes fruiting bodies on which it causes the black spot symptom. Eleutheromyces subulatus is a coelomycete fungus which has been recorded from North America and Europe on decayed fleshy fungi (Seeler, 1943; Malloch, 1974; MorganJones, 1977; Sutton, 1980; Sigler, 1990). The hyphomycete genus Hyphozyma de Hoog & M.Th. Smith forms slimy, yeastlike colonies that later become mycelial (de Hoog and Smith, 1981; Hutchison et al., 1993). This is the first report on the pathological activities of Hyphozyma on fresh basidiomata and on the ultrastructural process of pycnidial conidium formation in Eleutheromyces. Akihiko Tsuneda2 Shigeyuki Murakami Warwick M. Gill Nitaro Maekawa Tottori Mycological Institute, 211 Kokoge, Tottori 689ll,japan Abstract: The Hyphozyma synanamorph of Eleutheromyces subulatus was found to be the cause of black spot disease of Lentinula edodes fruiting bodies. Multiplication ofyeastlike cells and hyphae of Hyphozyma resulted in blackening and mild lysis of the infected host cap tissues where partially degraded skeletal microfibrils of host cell walls were abundant. In general, host tissue lysis remained superficial; however in some cases, Hyphozyma caused severe lysis and formed deep cavities containing remnants of highly dissolved host cell walls and numerous yeastlike cells of the pathogen. Enzyme assays demonstrated the ability of Hyphozyma to produce both chitinase and 13-(1,3) glucanase enzymes, requirements for hyphal wall degradation. On agar, conidiogenesis is enteroblastic in the Hyphozyma morph, whereas it is apparently holoblastic in the Eleutheromyces morph. No evidence of phialidic conidiogenesis was obtained. Key Words: chitinase, electron microscopy, hyphomycete, pathogen, shiitake mushroom MATERIALS AND METHODS Fungal isolates and culture conditions.-Two strains were used throughout this study: TMIC (Tottori Mycological Institute Culture Collection) 32748 (= UAMH 8225, University of Alberta Microfungus Collection and Herbarium, Edmonton, Canada) and TMIC 32749 ( = UAMH 8226) isolated from black spot lesions developed on L. edodes fruiting bodies formed on Quercus serrata Thunb. at Shinji-cho, Yatsuka-gun, Shimane prefecture and at Oki-gun, Shimane pref., respectively. Two further strains were included to examine their pathogenicity to L. edodes and to compare cultural characteristics, particularly morphology of pycnidia and pycnidial conidia: UAMH 5529 (isolated from a decaying russulaceous fruiting body, Canada); UAMH 5671 (isolated from a decaying agaric, Sweden). All of these strains were maintained either on potato dextrose agar (PDA, Difco, Detroit), or on 1.5% malt extract agar (MEA, Difco). Pycnidium formation was induced on potato sucrose agar (PSA; 1 L extract of 200 g fresh potatoes supplemented with 20 g sucrose and 20 g agar) at 20 C in the dark. INTRODUCTION Lentinula edodes (Berk.) Pegler, or shiitake mushroom, is a wood decaying basidiomycete cultivated widely in Asia, Europe, North America and Australia, using either cut oak logs or saw dust as the substrate (Tsuneda, 1994). Today, shiitake mushroom enjoys a flourishing international market and its world production is rapidly expanding: 320 000 tons in 1986 to 526 000 tons in 1991 (Chang, 1993). No pathogenic fungi have been found to cause diseases of L. edodes fruiting bodies, although a few reports are available on bacterial diseases (Komatsu and Goto, 1974; Nakai et al., 1982; Suyama and Fujii, Accepted for publication June 26, 1997. 1 Paper no. 319 of the Tottori Mycological Institute. 2 Email: i90080@sinet.adJp 867 Published online 28 Aug 2018 868 MYCOLOGIA Inoculation tests.-Budding cells of Hyphozyma were collected from one week old PDA cultures and washed twice by suspending them in sterile distilled water and centrifuging them at 2000 rpm for 5 min. Both young, healthy caps of fruiting bodies and cap tissue blocks were used as inoculation materials. Fresh fruiting bodies of L. edodes (TMIC 800) were harvested and stipes removed. The cap surfaces were washed with running water and air dried before use. Tissue blocks (ca. 0.5 X 1.0 em) were cut from caps after removing the outer tissues. For inoculation, one loopful of cell suspension (ca. 105 cells/mL) was placed both on a cap surface in four different locations and on the cut surface of each tissue block. Sterile distilled water was applied to controls instead of the cell suspension. The inoculated caps and tissue blocks were placed on sterilized glass microscope slides in glass Petri dishes lined with moistened filter paper. They were then incubated at 5, 10, 15, 20 or 25 C in the dark for 24 to 72 h and examined for the occurrence of typical black spot lesions. Inoculation tests were repeated three times. Reisolations of the fungus from black spot lesions induced by artificial inoculations were made and the lesions were examined by scanning electron microscopy (SEM). Microscopic observations.-The methods used in preparation of materials for light (LM), scanning and transmission electron microscopy (TEM) were those described by Tsuneda and Murakami ( 1985) and Tsuneda et al. (1991). SEM and TEM micrographs were taken with a Hitachi S-800 field-emission type microscope and with a JEOL JEM-lOOB microscope, respectively. Hyphal wall degradative enzyme assay.-The ability of Hyphozyma to degrade host fungal walls was investigated by assaying for the presence of both chitinase and glucanase enzymes. The production of chitinase enzymes was indicated by the formation of a clear halo around Hyphozyma (TMIC 32749) colonies growing on chitin overlay plates, resulting from the degradation of suspended chitin. The overlay plates were prepared by a modification of a standard method (Lorraine Bolger, Plant and Microbial Sciences, University of Canterbury, Christchurch, New Zealand, personal communication). The chitin suspension was prepared by dissolving 5 g chitin (Wako Pure Chemical Industries. Ltd., Tokyo, Japan) in 200 mL of 85% phosphoric acid for 72 h at 4 C. The chitin was then precipitated with approximately 2.5 L distilled water and allowed to settle. The water was removed after 12 h and the washing procedure was repeated three times. The pH of the suspension was adjusted to 5.8 with NaOH, and washed a further four times. Following removal of water from the final wash, the precipitated chitin was centifuged, supernatant discarded and the pellet was resuspended in 0.075M sodium phosphate buffer (pH 5.8) to a concentration of 1%. The prepared chitin was stored at 4 C until required. The overlay plates were prepared by pouring 20 mL of single-strength PDA base, pH 5.8, into sterile petri dishes and allowing to set. Fifty mL of quadruple-strength PDA and 150 mL of chitin suspension were autoclaved separately and gently mixed while still warm. The PDA base was overlaid with 10 mL of chitin-agar suspension and allowed to dry before inoculation. The dry chitin overlay plates were inoculated with Hyphozyma by streaking colonies exhibiting yeast morphology from half-strength PDA plates (48 h, 25 C in darkness) onto the overlay plates and incubating for 10 d at 25 C in darkness. The ability of the chitin overlay method to detect chitinase was confirmed by inoculating overlay plates (results not shown) with a known chitinase producer Serratia liquefaciens Hennerty & Grimes (CANU-PMS 179; culture collection of the Department of Plant and Microbial Sciences, University of Canterbury, Chistchurch, New Zealand), which was isolated from chitin-amended soil (Gill, 1994). A halo of clearing surrounding colonies, detectable against a background of transmitted white light, was considered as a positive result. The ability of Hyphozyma (TMIC 32749) to produce glucanase enzymes was tested against 7 commercially available glucan substrates exhibiting a variety of linkage and branching patterns; curdlan, a straight chain 13-(1,3) glucan (Sigma, St. Louis); laminarin, a 13-(1,3) glucan (Sigma); lichenan, a 13-(1,3) (1,4) glucan (Sigma); nigeran, an c:x-(1,3) (1,4) glucan (Sigma); pachyman, a 13-(1,3) glucan (Calbiochem, San Diego); pullulan, an c:x-(1,4) (1,6) glucan (Sigma); and pustulan, a 13-(1,6) glucan (Calbiochem). Stock solutions of the above glucans, 4% (w/v), were prepared in distilled water and sterilized. The freely dissolving glucans, laminarin and pullulan, were filtered through 0.22 J.tm cellulose nitrate membranes (Advantec Toyo, Japan) and the remainder autoclaved (20 min, 121 C, 15 psi). Each glucan was added to each of 6 test tubes containing 3.4 mL of sterilized 0.3% yeast extract broth (Wako, Japan) to yield a final glucan concentration of 0.6%. To 3 of the tubes, 0.1 mL of Hyphozyma suspension (ca. 105 cfu/mL) was added and, as a control, 0.1 mL sterile distilled water was added to each of the remaining 3 test tubes. All tubes were then incubated in stationary culture at 20 C for 10 d in darkness. Following incubation, the test tubes were placed in a water bath (80 C, 30 min) to stop enzymatic activity, and 1 mL aliquots of culture fluid from each test tube were clarified by centrifugation (12 000 rpm, 5 min). TSUNEDA ET AL.: BLACK SPOT DISEASE OF LE.VTJNULA FJJODES Glucan degradation was subsequently determined by the presence of D-glucose which was detected by a colorimetric method utilizing a food analysis UV-test kit, following the instructions of the manufacturer (Boehringer Mannheim, Germany) on an Hitachi U-1100 spectrophotometer at a wavelength of 334 nm. The resultant glucose levels are the means of three culture fluids from separate test tubes. As not all glucanases yield glucose as the direct end product of glucan degradation, a second assay was performed on the same aliquot by which reducing sugars, various intermediate oligosaccharides such as laminaribiose from laminarin (Reese and Mandels, 1959), were detected colorimetrically based on the jrhydroxybenzoic acid hydrazide (HBH) method of Lever (1973) as described by Jarvis (1992). RESULTS Development of black spot symptom in field collected material.-Black spot lesions of various sizes and differing degrees of cap tissue lysis were found on L. edodes fruiting bodies formed on Quercus bedlogs (FIGS. 13). In the early stages of development, black spots on fruiting bodies had no visible lysed areas (FIG. 1), but a creamy colored, indented area developed in the center of each spot as the fungus multiplied (FIG. 2). Under SEM and TEM, numerous yeastlike budding cells and scattered hyphae of the Hyphozyma morph were seen in lesions with relatively mild lysis (FIGS. 4, 10, 11). Hyphae penetrated into the host cap tissue, growing laterally within the one or two surface cell layers (FIGS. 5, 10), and reemerged to form budding cells at their tips (FIG. 4, arrows). Microfibrillar elements of host cell walls were clearly visible under SEM (FIG. 6) in the vicinity of the budding cells or hyphae of Hyphozyma. While host tissue lysis usually remained superficial, in some cases lysis was rapid and severe, resulting in the formation of deep cavities that pervaded the gill region. The gills below the cavities turned reddish brown in color. Such cavities contained a slimy or fluid material in which numerous yeastlike cells and remnants of degraded host cell walls were present (FIGS. 8, 9, 12, 13). Remaining host walls were highly dissolved, suggesting all wall components had been degraded more or less simultaneously (FIG. 13, asterisk). Yeastlike cells of Hyphozyma are capable of multiplying by budding within the host tissues (FIGS. 11, 13, arrows), and lysing the host tissues without making direct physical contact (FIGS. 12, 13). Inoculation tests.-In the inoculation tests, all four strains tested were equally pathogenic to L. edodes caps, developing the typical black spot symptom with 869 mildly lysed central regions. None of the tested strains produced severe lysis within 3 d. SEM observations of the black spot lesions induced by artificial inoculations did not reveal any differences from natural ones in terms of the growth of Hyphozyma and the mode of host tissue degradation, and no contaminants were found. Cultures made from artificially induced black spot lesions were identical with the original ones used to obtain inoculum cell suspensions. Inoculated tissue blocks were also blackened and subsequently collapsed. Hypha[ wall degradative enzyme assay.-On chitinPDA overlay plates, the yeastlike colony morphology was retained by Hyphozyma. Surrounding both individual, discrete colonies and coalescent colonies, a halo of clearing was discernible in the medium (FIG. 14), indicating the degradation of suspended chitin, thus confirming the production of chitinase by the black spot organism. The analysis of D-glucose production indicated that Hyphozyma was capable of hydrolyzing laminarin and liberated 2. 7 mM glucose from the substrate, indicating the production of a ~-(1,3) glucanase enzyme. Minute amounts of glucose were detected from pustulan and lichenan (272 J..LM and 67 J..LM respectively). From the remaining glucans, glucose was recorded in trace amounts, however the recorded absorbance was below the manufacturer's recommended threshhold of precision. It is interesting to note that Hyphozyma liberated a detectable amount of glucose from all glucans, with the exception of curdlan. Both spontaneous dissociation of the substrates in water and partial degradation by autoclaving can be eliminated as a possible source of this glucose as the control tubes failed to yield glucose. The reducing sugar assay supported the above findings, confirming the production of a ~-(1,3) glucanase by Hyphozyma. The higher concentration of sugars detected by the HBH method (7.5 mM) suggests that the Hyphozyma ~-(1,3) glucanase also generates intermediate smaller chain polysaccharides from laminarin and not solely glucose. Neither assay indicated the significant degradation of curdlan or pachyman, both ~-(1,3) glucans. Growth in the inner bark of L. edodes bedlogs.-FIGURES 15, 16 show typical yeastlike cells of Hyphozyma occurring in inner bark samples cut from bedlogs of L. edodes. These samples were taken from regions adjacent to the attachment areas of the stalks of heavily infected fruiting bodies. Hyphozyma was readily isolated from such inner bark samples by means of a dilution plate method (Booth, 1971). The arrow in FIG. 16 points to disarticulating cells, one ofwhich is budding out a new cell: this is a characteristic feature 870 MYCOLOGIA FIGS. 1-9. Black spot lesions on Lentinula edodes fruiting bodies caused outdoors by the Hyphoz.yma synanamorph of Eleutheromyces subulatus. 1. Small black spots on a young cap. X 10. 2. Mild lysis of infected cap tissue in the center of a black spot (arrow). X 15. 3. Deep cavities (arrows) resulted from severe lysis. X 0.8. 4. Abundant yeastlike cells and some hyphae that have emerged from host tissue to form terminal bud cells (arrows). 5. Superficial penetration of cap tissue by a hypha (arrow). 6. Mild-type lysis showing exposed skeletal microfibrils of the host cell wall. 7. Initial stage of severe-type lysis forming a cavity. 8,9. Advanced stages of severe lysis showing remnants of host cell walls (arrows) and numerous yeastlike cells of Hyphoz.yma. Scale bars: FIG. 4 = 30 f.Lm; 5 = 5 f.Lm; 6 = 0.5 f.Lm; 7 = 500 f.Lm; 8 = 10 f.Lm; 9 = 5 f.Lm. of Hyphozyma. The occurrence of Hyphozyma in the inner bark was sporadic but, where present, cells were usually found in high numbers. Hyphae of L. edodes were abundant and often covered with a slimy material (FIG. 15). Furthermore, numerous rodshaped bacteria coexisted with L. edodes and Hyphozyma in some inner bark samples (FIG. 16). Cultural characteristics.-Colonies on agar media were initially dominated by yeastlike budding cells that gradually became mycelial (FIG. 17) and had a strong fragrant odor. Conidiogenesis in the Hyphozyma morph was sympodial, annellidic or intermediate between the two modes (Tsuneda et al., 1995a). FIGURE 18 clearly shows the enteroblastic mode of TSUNEDA ET AL.: BLACK SPOT DISEASE OF LE.VTINULA EDODES 871 FIGS. 10-14. Transmission electron micrographs of field collected material. 10. Hypha of Hyphoz.yma growing laterally within the surface layer(s) of host cap tissue (arrow). 11. Yeastlike cells in host cap tissue. The arrow points to a budding cell and the arrowheads indicate affected host cells. 12. Severely degraded host cells. 13. Transverse section of a cavity caused by Hyphoz.yma, showing yeastlike cells including a budding one (arrow) and highly dissolved host cells (asterisk). Scale bars: FIG. 10 = 5 f.Lm; 11 = 10 f.Lm; 12, 13 = 5 f.Lm. 14. Distinct halo of clearing surrounding each individual or coalesced colony of Hyphoz.yma (TMIC 32749) grown on chitin-PDA overlay plate. X 1.2. 872 MYCOLOGIA FIGS. 15-23. Hyphozyma and its Eleutheromyces morph. 15-18. Hyphozyma in inner bark of Quercus serrata and on agar. 15, 16. Yeastlike cells occurring in inner bark. The arrow in FIG. 16 points to a budding cell. Numerous bacteria are present together with Hyphozyma and L. edodes hyphae. 17. Part of a colony on PDA showing intermixed hypha! and yeastlike forms (TMIC 32748). 18. Enteroblastic budding (arrow) (TMIC 32748). Scale bars-FIG. 15, 20 j.Lm; 16, 3 j.Lm; 17, 7 j.Lm; 18, 1 fLm. 19-23. Eleutheromyces morph (TMIC 32748). 19. Pycnidium formed on PSA. 20. Pycnidial conidia discharged from a pycnidium. Note truncate bases (arrows). 21. Pycnidial wall, conidiophores and conidia. Conidiogenous cells usually arise from immediately below septa (arrows). 22. Holoblastic conidiogenesis (arrow). 23. Released conidium. Appendages of mature conidia are devoid of cytoplasm (arrows). CA = conidial appendage; CB = conidial body; CP = conidiophore; PE = peridium. Scale bars: FIG 19 = 50 j.Lm; 20 = 3 j.Lm; 21 = 5 j.Lm; 22 = 2 j.Lm; 23 = 1 j.Lm. TSUNEDA ET AL.: BlACK SPOT DISEASE OF TABLE I. 873 LENTINULA EDODES Conidial dimensions (f.Lm) in different strains of Eleutheromyces subulatuS' Strain No. TMIC 32748 TMIC 32749 UAMH 5529 UAMH 5671 Seeler (1943) Nag Raj (1993) Main Body-L Main Body-W Apical Append Basal Append Total L/ Average (SD) 6.5-10 5-8.5 5-8 4.5-5 4-4.5 5-7 1-1.5 1-1.5 2-2.5 1.5-2 1.5-2 2-2.5 11-16 5-11 6-12 4.5-9 3-7 2.5-5 6-10 2.5-8 2.5-4 2-4.5 2-3 5-8 25.5-33.5/28.3(1.6) 13-24.5/19.2(2.6) 13.5-21/18.0(1.7) 11.5-18/13.9(0.9) 15.9 (average) • L = length; W = width; Append = appendage; data based on 20 measurements (a result of three repetitions); SD = standard deviation. conidiogenesis occurring in a budding cell. Pycnidia formed in culture were yellowish brown, oval to subglobose, and half embedded in agar (FIG. 19). The pycnidial wall consisted of several layers of relatively thick-walled cells containing electron-dense bodies (FIG. 21). Mature pycnidia discharged numerous one-celled conidia, possessing long slender appendages at both ends (FIGS. 20-23). Basal appendages were shorter than apical ones and were characterized by having truncate bases (FIG. 20, arrows). The appendages were initially filled with cytoplasm (FIGS. 21, 22, arrowheads) but those discharged were devoid of cytoplasm (FIG. 23, arrows). Conidiogenous cells were terminal, or more commonly arose as a minute stalk from immediately below the septum of conidiophores (FIG. 21, arrows) which extended longitudinally with occasional branching. Pycnidial conidia developed holoblastically from tips of conidiogenous stalks, many of which were slightly swollen (FIG. 22, arrows). There was no evidence of phialidic conidiogenesis. None of the strains used in this study showed any significant differences in their cultural characteristics except for the size of discharged pycnidial conidia which differed markedly between strains. As shown in TABLE I, the total length of pycnidial conidia of TMIC 32748 was more than double that of UAMH 5671. Even the two strains isolated from L. edodes fruiting bodies exhibited a significant difference in their dimensions. Conidia of TMIC 32749 and UAMH 5529 were similar in total length but differed considerably in width of their main bodies and length of basal appendages. DISCUSSION We consider that both strains of Eleutheromyces from L. edodes fruiting bodies (TMIC 32748, 32749) should be accommodated in E. subulatus because major characteristics other than the size of pycnidial conidia agree well with the descriptions for this species (Seeler, 1943; Malloch, 1974; Morgan:Jones, 1977; Sutton, 1980; Sigler, 1990) and differ from another species, E. mycophila (Hohnel) Nag Raj (Nag Raj, 1993) which forms darkly pigmented globose conidiomata. Furthermore, both the Canadian and Swedish strains were also capable of creating the typical black spot symptom on L. edodes fruiting bodies. However, it is striking that conidial dimensions differ widely between different strains, including those described by Seeler (1943) and Nag Raj (1993) (TABLE I). Since the variation is so wide, conidial size alone cannot justify the separation of species in this genus. The present study demonstrated that the Hyphozyma synanamorph of E. subulatus is the cause of the black spot disease of L. edodes. No fungus had been known to be pathogenic to L. edodes fruiting bodies prior to this study. Although the occurrence of this disease is so far limited to western parts of Japan, once it becomes epidemic, the consequences could be extremely serious as this disease degrades the crop quality considerably. Removal of infected fruiting bodies from bedlogs at early stages of infection and transfer of the infected bedlogs to relatively dry and well ventilated conditions should be effective in preventing a further increase in pathogen population in the laying yard. However, once the Hyphozyma morph colonizes the inner bark of infected bedlogs, subsequent crops from these bedlogs have a high risk of being infected, and this is exactly what is happening in the yards of some growers. We surmise that this pathogen is able to utilize the products of inner bark breakdown by L. edodes and, in fact, E. subulatus is known to assimilate cellobiose and xylose (Sigler, 1990). According to Malloch (1974), the spore drop in E. subulatus probably plays a role in spore dispersal by arthropods. Likewise, Sigler (1990) stated that the strong fragrant odor characteristic of the Hyphozyma morph might be expected to serve as an attractant to soil invertebrates. Our field surveys on the black spot disease for the last few years also strongly indicated the presence of insect vectors whose range of activity is not very wide. We consider that certain my- 874 MYCOLOGIA cophagous collembolans, which occur commonly in shiitake yards (Tsuneda and Arita, 1982), are the most probable candidates because E. subulatus is known to be one of the most common fungi associated with the collembolan Onychiurus subtenuis (Folsom) in the litter layer of aspen woodlands (Visser et al., 1987). Further investigation on this aspect is important to establish proper control measures of this disease. Macro- and microscopic observations of black spot symptom development revealed two types of cap tissue lysis; i.e. mild type characterized by the gradual degradation of the microfibrils, and severe type showing more or less simultaneous degradation of all wall components. Similarly, these two types of cell wall degradation were observed in three different degradation systems as described by Tsuneda and Thorn (1995): (i) sapwood wall degradation by wood decay fungi; (ii) hyphal wall degradation by mycoparasitic Trichoderma; and (iii) hyphal wall degradation by pathogenic bacteria. The simultaneous-type wall degradation in systems (i) and (ii) usually occurred in the vicinity of hyphal tips where activities of various extracellular enzymes are the highest (Archer and Wood, 1995). However, no such explanation is available for the Hyphozyma-L. edodes interaction because the severe lysis is primarily caused by the yeastlike cells (FIGS. 9, 13). Enzyme assays revealed that Hyphozyma produced both chitinase and a 13-(1,3) glucanase among its enzyme complement. It is widely accepted that organisms purporting to lyse fungal cells must possess both 13-glucanase and chitinase that work sequentially to digest first the outer glucan and then the inner chitin (Potgieter and Alexander, 1966; Michalenko et al., 1976), which is embedded in a 13-glucan matrix (Novaes-Ledieu et al., 1987). The fact that the two components of the 13-glucan/ chitin matrix are inextricably covalently linked (Sietsma and Wessels, 1977, 1979; Mol et al., 1988) confers a degree of resistance against exogenous hydrolytic enzymes (Mahadevan and Tatum, 1967). As demonstrated by Gill (1994), chitinase and 13-glucanase individually are unable to lyse hyphal walls. However, in unison, these enzymes were able to digest large tracts of hyphal wall. The ability of Hyphozyma to produce both these hydrolytic enzymes, and perhaps, as suggested by the assay results, other glucanases, renders this organism a potent pathogen of L. edodes and by virtue of its demonstrated enzymatic mode of pathogenesis, a potential pathogen of other cultivated mushrooms. We have clarified the details of conidiogenesis in both Hyphozyma and Eleutheromyces morphs. We previously reported that conidiogenesis in the Hyphozyma morph was sympodial, annellidic or intermediate between the two modes but wall relations between mother and daughter cells remained unclear (Tsuneda et al., 1995a). This time, we have clearly shown by TEM the presence of enteroblastic conidiogenesis in the Hyphozyma morph (FIG. 18). As to pycnidial conidium formation in E. subulatus, it has been described as phialidic (Sutton, 1980; Sigler, 1990) based on light microscopy (LM). In our LM observations, however, we could not determine the exact mode of conidiogenesis due to the extremely small size of the conidiogenous pegs. We therefore carried out TEM and found that formation of pycnidial conidia are not phialidic but holoblastic from the tips of conidiogenous cells (FIG. 22, arrows). ACKNOWLEDGMENT We wish to thank Prof. Lynne Sigler, University of Alberta, for fungal strains (UAMH 5529, 5671) used in this study and valuable discussions. LITERATURE CITED Archer, D. B., and D. A. Wood. 1995. Fungal exoenzymes. Pp. 137-162. In: The growing fungus. Eds., N. A. R Gow, and G. M. Gadd. Chapman and Hall, London. Booth, C. (ed.). 1971. Methods in microbiology. Vol. 4. Academic Press, London. 795 pp. Chang, S. T. 1993. Mushroom biology: the impact on mushroom production and mushroom products. Pp. 3-20. In: Mushroom biology and mushroom products. Eds., S. T. Chang, J. A. Buswell, and S. W. Chiu. The Chinese University Press, Hong Kong. de Hoog, G. S., and Th. M. 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