Bution of the Microascus Species in Japan. in Compar

Home , Graphium (fungus), Microascaceae, Microascus, Petriella, Petriella setifera

J. Gen. Appl. Microbiol. Vol. 9, No. 2, 1963.

MICROASCACEAE IN JAPAN

SHUN-ICHI UDAGAWA National Institute of Hygienic Sciences Tamagawa-yoga-machi, Setagaya-ku, Tokyo Received January 30, 1963

In an earlier paper (1), an account was given of the occurrence and distri- bution of the Microascus species in Japan. In comparing morphological characteristics of these isolates with those of some European specimens, it was realized that the perithecial or conidial characteristic is not satisfactorily reliable as taxonomic criteria and therefore the characters of the ascospores were used as the key to the primary separation of the Microascus species. Continued search for the Microascaceae has brought to light the need for more extensive studies of this group of fungi from the standpoint of their developmental morphology, physiology, biochemistry, and ecology. However, for the present, only taxonomic aspect of three species of Microascus and a Petriella is noted, particularly with regard to a Graphium stage of P. setifera. Recently two comprehensive reports concerned with this family were made by BARRONet al. (2, 3) in which they recognized fourteen species of Microascus and five species of Petriella as valid, including two new species and new combinations, and a new variety. Nevertheless, no concerted effort has been made to treat the fungi on a world-wide basis, especially such as covering the Asian areas.

METHODS

The experimental procedures and the types of media employed were similar to those described in detail in the earlier paper (1).

DESCRIPTION AND DISCUSSION

Microascus cirrosus Cuazi, in Boll. regia staz, patol. vegetale, 10: 302 (1930) ; BARRON,CAIN and GILMAN, in Can. J. Botany, 39: 1628 (1961) ; UDAGAWA, in J. Gen. Appl. Microbiol., 8: 43 (1962). Habitat : On soil, Tokyo, Japan, 1961, NHL 2277.

Microascus doguetii MOREAU,in Rev. Mycol., 18: 174 (1953) ; BARRON,CAIN and GILMAN,in Can. J. Botany, 39: 1628 (1961). (Figs. 1, 5-7) Conidial stage : Scopulariopsis. Colonies on potato-dextrose agar growing slowly, dark brownish gray to dark gray, tough-textured, with black perithecia arranged in more or less 137 138 S. UDAGAWA VOL. 9 conspicuous concentric zones, overgrown by loose aerial mycelium and conidial apparatus, reverse pale yellowish to dark-colored. Perithecia immersed or partially superficial, scattered spherical to ovate, 130-340 in diam.; neck papillif orm to short cylindrical, 30-130 ,u in length ; peridium carbonaceous, nearly bare or with fine, short hairs. Asci ovate to elongate, 9-15>( 7-10.5 ,u, 8-spored. Ascospores pale reddish brown, concavo-convex, usually heart-shaped, sometimes ovate, 4.5-6 (7.5) <3-5.5 rounded at the ends, smooth. Conidial fructifications varying from sessile sterigmata (annellophores) borne as per- pendicular projections on aerial hyphae and ropes of hyphae to irregular branching systems. Conidiophores often wanting, when present, very short, up to 15 x 1.5-2.5 p, arising as lateral branches to the hyphae. Sterigmata (annellophores) short, singly or in groups of 2-3, cylindrical, 4.5-13.5>< 2.5-3 ~, frequently septate, strongly contorted. Conidia olive brown, ovate to elliptical, truncate at the base, rounded or slightly pointed at the apex, 4.5-7x 3-4.5 ,u, with walls ornamented 2-3 (5) longitudinal bands, in chains showing a short

Fig. 1. M. doguetii Moreau, NHL 2297. a. Perithecia b. Asci c. Ascospores d. Conidial fructifications e. Conidia 1963 Microascaceae in Japan 139 connective. On corn meal agar growing restrictedly, thin, producing perithecia in limited numbers. At 37°, growth is nil. Habitat : A laboratory contaminant, Tokyo, Japan, 1962, NHL 2297. M. doguetii appears to be a rare fungus, having been reported only once (BARRON,CAIN and GILMAN,3) since its original isolation by DOGUET(MOREAU, 5) as a plate contaminant, Caen, France. Unfortunately, no information regarding its true occurrence in nature is available. This unique species is easily distinguished from all others of the genus by the dark-colored and striate nature of the conidial wall. The Japanese isolate was compared in detail with a subculture of the European material, obtained from the Museum National d'Histoire Naturelle, Paris, France; and a close similarity in both cultural and morphological features was observed. The only significant difference noted was in the ascospores, those of the Japanese form being predominantly heart-shaped and slightly smaller in their dimentions, whereas the European specimen produces more or less broadly lunate and large spores.

Microascus longirostris ZUKAL, in Verhandl. zoolo, botan. Ges. Wein, 35: 339 (1885) ; BARRON, CAIN and GILMAN, in Can. J. Botany, 39: 1624 (1961). (Figs. 2, 8-10) Microascus variablis Massee et Salm., 1901(7). Conidial stage : Scopulariopsis. Colonies on potato-dextrose agar growing very slowly, brownish black to grayish black, fairly tough, producing numerous perithecia in concentric rings at the agar surface, loosely overgrown by white aerial hyphae appearing slightly floccose, reverse uncolored. Perithecia mostly superficial, often gre- garious, black, spherical to subspherical, 130-260 p in diam., ripening late ; neck rather long, cylindric, 180-300><25-30 p ; peridium carbonaceous, with olivaceous, straight, septate hairs 2.5-3 p wide at the base. Asci borne in short chains, globose to subglobose, 7-7.5 (9) p in diam., 8-spored. Ascospores pale yellowish brown, definitely concavo-convex with rounded ends, small, 3-4.5>< 2.5-4 p, smooth, discharged at maturity in the form of a cirrhus. Conidial fructifications simple, usually reduced to single detached sterigmata (annellophores) occurring irregularly along aerial hyphae. Conidiophores not well differentiated, 1.5-2 p in diam., arising as lateral branches. Sterigmata (annellophores) continuous with conidiophores, cylindrical, 6-45 x 1.5-2 p, straight to sinuous, often swollen at the apex. Conidia hyaline, ovate to lemon-shaped or pyrif orm, 3-5 x 2.5-3 p, with truncate base, smooth or sometimes roughened, in short chains. On corn meal agar thin-growing, restricted as on potato-dextrose agar, perithecia sparsely produced, not associated with conidial structures. At 37°, growth is nil. 140 S. UDAGAWA VOL. 9

Fig. 2. M. longirostris Zukal, NHL 2296. a. Perithecia b. Asci C. Ascospores d. Conidial fructifications e. Conidia

Habitat : On soil, Tokyo, Japan, Feb. 1962, NHL 2296. M. longirostris is common and cosmopolitan, and occurs on a wide variety of substrata, including : dung, rotting wood, stored corn, wasp's nest, and pea seeds, etc. Nevertheless, so far as is known, no reports of this fungus have appeared outside Europe or America, except an isolation from Indian soil (8). The gelatinous ascospore sheath which ZUKAL(6) originally regarded as characteristic of the species and which used as a basis for separating it from M, variablis Massee et Salm. (7) may be an artifact, as already pointed out by BARRONet al. (3). In M. longirostris, as in other widely distributed species of Microascus, individual strains show appreciable variation in fruiting appearance and in the finer details of morphology. Gradations, however, are encountered with the result that such variants do not stand apart as forms clearly separable. For example, in the Japanese isolate the ascospores are 3-4.5x 2.5-4 p, in the culture from the Centraalbureau voor Schimmelcultures, 1963 Microascaceae in Japan 141

Baarn, Netherlands, 3.5-4.5 x 2.5-3.5 u, and in the description of BARRONet al., 3-4x 2.5 rc. Likewise, other morphological variability of the isolate observed in the present study reveals no reliable bases for separation from M. longirostris in the broad sense that M. variables is considered here.

Microascus manginii (LOUBIERE)CURZI, in Boll. regia staz. patol, vegetale, 11: 60 (1931) ; BARRON,CAIN and GILMAN,in Can. J. Botany, 39: 1627 (1961). (Figs. 3, 11, 12) Nephrospora manginii Loub., 1924 (10) ; Scopulariopsis albo-flavescens Zach, 1934 (11). Conidial stage : Scopulariopsis. Colonies on potato-dextrose agar spreading broadly, white, thin-textured, with abundant growth of aerial hyphae appearing loosely floccose, quickly developing black perithecia with an admixture of conidial structures, often zonate, giving the colony appearance a grayish color, reverse uncolored to

Fig. 3. M. manginii (Lonb.) Curzi, NHL 2278. a. Perithecia b. Asci C. Ascospores d. Conidial f ructifications e. Conidia f, Chlamydospores 142 S. UDAGAWA VOL. 9 pale yellowish brown. Perithecia commonly superficial spherical or subspherical, 80-200 i in diam., papillate ; peridium carbonaceous, glabrous or with scanty hairs. Asci 8-spored, subglobose to ovate or broadly clavate, 10.5-15 x 7.5-12 i, evanescent. Ascospores pale reddish brown, markedly concavo-convex, rarely planoconvex or oval, 5.5-6x4-5.5 p, with ends rounded, smooth, extruded as a gelatinous ball at the ostiole or in the form of a long cirrhus. Conidial f ructifications simple and unbranched, consisting of sterigmata (annellophores) scattered along aerial hyphae or bundles of hyphae ; conidiophores lacking. Sterigmata (annellophores) solitary, cylindrical, 10.5-20 (27) x 2.5- 4,1, often septate, sometimes curved or sinuous. Conidia hyaline, variable in shape and size, usually broadly ovate to lemon-shaped, 4-8 x 3.5-6.5 p, smooth, with basal ends more or less flattened, borne in short chains. Chlamydospores infrequently present, terminal, subglobose. On the medium containing arsenious acid (As203) stinging odor pronounced, suggestive of leeks. On malt agar growing rather slowly, perithecia as described above but tardily producing. At 37°, restrictedly growing. Habitat : On milled rice from Burma, 1954, NHL 2278. This species appears to be widely but not abundantly distributed in nature. LoUBIERE's original isolate was from cheese in France. ZACH(11) described it as isolated from skin infection in Austria. Furthermore, according to BARRON, CAIN and GILMAN(3), there are two recent records for its terrestrial habitat, one from the U.S.A. and the other from England. M. manginii is characterized by the white color of its conidial f ructifications and the fairly large, entirely heart-shaped ascospores ; in these respects it differs from other species of the genus.

Petriella setifera (SCHMIDT)CURZI, in Boll. regia staz. patol. vegetale, 10: 411 (1930) ; BARRON,CAIN and GILMAN,in Can. J. Botany, 39: 842 (1961). (Figs. 4, 13-15) Microascus setif er Schm., 1912 (13). Conidial stages : Sporotrichum and Graphium. Colonies on potato-dextrose agar spreading broadly, light brownish gray to brownish gray, consisting of a fairly tough basal felt bearing abundant conidial structures, conspicuously f uniculose, densely crowded in a radial pat- tern, perithecia abundantly produced throughout the whole colony or in localized sectors, mostly obscured by the overlying aerial growth, reverse pale yellowish brown to dark-colored. Perithecia superficial or semi-embedded, dark olive drown, spherical, 100-150 (200) 4a in diam.; neck short, 15-20 e in length, with os- tiolar fringes; peridium membranous, covered on upper part with long, straight to flexuous, pale olive, smooth-walled, septate hairs which measure up to 115 long and 1-1.5 N wide. Asci 8-spored, ovate to clavate, 19-24 x 10.5- 15 p, evanescent. Ascospores reddish brown, guttulate when young, asym- 1963 Microascaceae in Japan 143 metrically convex, subapiculate at both ends, 7.5-11.5 x 4.5-6 p, in side view depressed, smooth, with terminal germ pores, at maturity emerging in a long cirrhus. Conidial f ructifications in two types. Sporotrichum stage consisting of very numerous blastophores as short lateral branches of aerial hyphae or fascicles of hyphae. Conidiophores hyaline, simple, unbranched, gradually tapering and sinuous at the tips, 10-25>< 1-1.5 p, smooth-walled, with conidia bearing as blownout ends of the apex. Conidia (blastospores) hyaline, oblong elliptical to pyrif orm, 4.5-9>< 3-4 p, with rounded top and narrow truncated base, smooth, forming loose clusters in mucus. Graphium stage produced abundantly on malt agar, arising from trailing hyphae, usually aggregated into coremia, from which the individual conidial structures diverge in the terminal portions. Coremia dark brown, stalks consisting of a variable number of Conidiophores, up to 500 p long, 5-25 p in diam., heads of hyaline, peni-

Fig. 4. P. setifera (Schm.) Curzi, NHL 2279. a. Perithecium b. Asci c. Ascospores d. Conidial fructifications and conidia of Sporotrichum stage e. Conidial head of Graphium stage f. Conidia of Graphium stage 144 S. UDAGAWA VOL. 9 ciliate branches producing conidia on terminal phialides. Conidia similar in shape to the Sporotrichum type, 6-10.5>' 3-4 p, embedded in mucus to form a large rounded head, 15-50 i in diam. At 37°, restrictedly growing, immature. Habitat : On soil, Tokyo, Japan, 1962, NHL 2279. As referred to the earlier paper (1), the genus Petriella was established by CuRzI (9, 12) to include four species of fungi with Microascus-like perithecial and ascosporic characters, but differing from that genus in the hairly appearance of the perithecium. In the recent complete treatment, BARRON et al. (2) confirmed CuRzI's separation of Petriella, indicating, however, that Petriella must be distinguished from Microascus by the size and color of ascospores, the conidial stages, and the cultural morphology. BARRONet al., following their revised concept, regarded M. sordidus Zukal as conspecific with P. asymmetrica Curzi and reduced the latter name to a later synonym under the combination P. sordida (Zukal) Barro et Gilman. They also added a new species, P, guttulata Barron et Cain. P. setif era appears to be very rare in nature, and only two records can be traced. SCHMIDT(13) originally found it on horse dung in Germany. SHIGO(14) isolated it from wilted oak tree in West Verginia, U.S.A. On account of the failure to observe a Graphium stage in the American material, its finding in the Japanese isolate is of interest as the first description, tending to confirm the suggestion made by BARRONet al, that all species of Petriella would have both Graphium and Sporotrichum as conidial stages.

Microascus stysanophorus In 1931, CURZI(9) first referred MATTIROLO'sfungus " Melanospora stysano- phora " to synonymy under an incorrect name " Microascus stysanopporus (Matt.) Curzi." Because of this, BARRONet al. (3) more recently proposed M. stysanophorus (Matt.) Barron, Cain et Gilman as a new combination. However, the validity of their proposal is still in question, since CuRzI later used the correct name M. stysanophorus (Matt.) Curzi in his paper (15) on " Dematophora glomerata Viala e Vialina n. gen. (pp. 235, 249, 250, 256 and 257)." The writer does not wish to judge its final nomenclature without having a chance to survey the acceptable literature.

KEY TO THE SPECIES OF MICROASCUS (A REVISION)

A slight revision to the key (1) is needed to accommodate the additional inf ormations obtained up to date. The following should be substituted for the previous one : A. Ascospores light-colored, mostly small; asexual reproduction (when present) by annellated characteristic of the form genera Scopulariopsis and Stysanus (except in the case of the Sporotrichum stage of M. stysanophorus)...... B A. Ascospores dark-colored; asexual reproduction (when present) forming 1963 Microascaceae in Japan 145

blastospores and phialospores of the form genera Sporotrichum and Graphium ....Genus Petriella (P. sordida, P. guttulata, P. setifera, P. lindforsii, and P. boulangeri) B. Ascospores essentially triangular ...... C B. Ascospores strictly concavo-convex ...... E B. Ascospores commonly piano-convex to asymmetrically convex ...... H B. Ascospores ovoid ...... L C. Ascospores 3.5-5.5 x 2.5-4.5/1 ...... M. trigonosporus C. Ascospores 5.5-7 (7.5) x 5-6.5p ...... D D. Ascospores with broadly rounded ends...... M. trigonosporus var. macrosporus D. Ascospores with tapering ends...... M. pyramidus E. Conidial structures persistently white...... F E. Conidial structures in shades of dark gray ...... G F. Ascospores 5-6 x 4-5.5p, heart-shaped ...... M. manginii F. Ascospores 3-5 x 2-4p, abruptly concave ...... M. longirostris G. Ascospores heart-shaped to broadly concavo-convex, 4.5-7.5x3-7 p; conidia striate ...... M. doguetii G. Ascospores broadly concavo-convex, 4-6x2-5.5 p; conidia not striate .M. cirrosus H. Ascospores less than 6.5p in length...... I H. Ascospores more than 6.5 p in length ...... J I. Scopulariopsis stage unknown ...... M. intermedius I. Scopulariopsis stage present; conidial structures hyaline ...... M. albo-nigrescens I. Scopulariopsis stage present; conidial structures dark gray...... M. cinereus J. Ascospores narrow, less than 2 p in diameter ...... M. nidicola J. Ascospores thick, 2.5-6p in diameter...... K K. Ascospores plano-convex to slightly concavo-convex, conidial stage entirely lacking ...... M. schumacheri K. Ascospores asymmetrically convex ...... M. stysanophorus L. Ascospores 4-5 x 3p ...... M. desmosporus L. Ascospores 7 x 5 p...... M. niger

SUMMARY

Microascus doguetii Moreau, M. longirostris Zukal, M. manginii (Loub.) Curzi and Petriella setifera (Schm.) Curzi were recorded for Japan; a Graphium stage was first recognized for P. seti f era. On the basis of some new obser- vation made during the study, a revision of the key to the Microascus species was proposed. The writer wishes to express his gratitude to Prof. S. Hirayama, Mie University, Dr. Y. Kobayasi, National Science Museum, and Dr. H. Iizuka, Institute of Applied Microbiology, University of Tokyo, for their guidance. The writer is grateful to Mr. T. Matsushima, National Institute of Hygienic Sciences, for supplying some cultures. Thanks are also due to Dr. H. Kurata, National Institute of Hygienic Sciences, and Dr. K. Tubaki, Institute for Fermentation, for their advice. 146 S. UDAGAW.A VoL. 9

Figs. 5-7. M. doguetii; 5, ascospores, x 1000; 6, conidial fructifications, x650; 7, conidia, x 1100. Figs. 8-10. M. longirostris; 8, perithecial initials, x2000; 9, ascospores, x 1000; 10, a portion of peritheeial neck, x 600. 1963 Microascaceae in Japan 147

Figs. 11 and 12. M. manginii; 11, ascospores, x l000; 12, conidial fructifications, x700. Figs. 13-15. P. seti f era; 13, perithecium, x200; 14, ascospores, X1000; 15, conidial f ructifications of Graphium stage, x200. 148 S. UDAGAWA VOL. 9

REFERENCES

(1) S. UDAGAWA: J. Gen. Appl. Microbiol., 8, 39 (1962). (2) G. L. BARRON, R. F. CAIN and J. C. GILMAN: Can. J. Botany, 39, 837 (1961). (3) G. L. BARRON, R. F. CAIN and J. C. GILMAN: ibid., 39, 1609 (1961). (4) M. CURZI: Boll. regiat staz. patol. vegetale, 10, 302 (1930). (5) F. MOREAU and Mme. MOREAU: Rev. Mycol., 18, 165 (1953). (6) H. ZUKAL: Verhandl. zoolo. botan. Ges. Wien, 35, 333 (1885). (7) G. MASSEE and E. S. SALMON: Ann. Botany, 15, 313 (1901). (8) V. AGNIHOTHRUDU: J. Indian Botan. Soc., 37, 422 (1958). (9) M. CURZI: Boll. regia staz. patol. vegetale, 11, 55 (1931). (10) A. LOUBIERE: Theses Fac. Sci. Paris, France, Ser. A, 982, 1 (1924). (11) F. ZACH: Oesterr. Botan. Z., 82, 173 (1934). (12) M. CuRzI: Boll. regia staz. patol. vegetale, 10, 380 (1930). (13) A. SCHMIDT: Diss. Breslau, W. C. Korn, Germany, 1912, pp. 81 (Cited by P. A. SACCARDO: Syll. Fung., 17, 610 (1905).) (14) A. L. SHIGO: Mycologia, 50, 757 (1958). See also BARRON et al. (2). (15) M. CURZI: Boll. regia staz. patol. vegetale, 15, 235 (1935).