INTERNATIONAL JOURNAL of SYSTEMATIC BACTERIOLOGY Vol. 21, No. 1 January 1971, p. 4049 Printed in U.S.A. Copyright 0 1971 International Journal of Microbiological Societies Studies on the of the Myxobacterales III. Chondromyces and Stigmatella

HOWARD D. McCURDY

Department of Biology, University of Windsor, Windsor, Ontario

Classification of the species of the genus Chondromyces Berkeley and Curtis is considered in light of the proposal that the genus be divided into two genera: Stigmatella Berkeley and Curtis (family Cystobacteraceae), with tapered vegetative cells and encapsulated myxospores (microcysts); and Chondro myces (family Polyangiaceae), with cylindrical vegetative cells and similar myxospores. The latter genus, as redefined, would include Chondromyces crocatus (the type species), Chondromyces apiculatus, Chondro myces pedicula tus, Chondro my ces ca tenula tus, and Chondro - myces lanuginosus. Two species are recognized as belonging to the genus Stigmatella: (the type species) and Stigmatella erecta.

In a previous paper (14), the family Polyan- from the American Type Culture Collection): Chon- giaceae Jahn was redefined to include only dromyces crocatus [M38(ATCC 25 193), M204, those sporangial myxobacters with cylindrical M1201 ; Chondromyces apiculatus (M6, M32); Chon- vegetative cells and similar myxospores. Sporan- dromyces pediculatus (M 1 1 8) ; Stigmatella aurantiaca gium-forming myxobacters with tapered vegeta- [M340, M341, “cylindrica” M343, “media” M84, M88, M15 (ATCC 25190), M34] ; Stigmatella erecta tive cells were placed in a new family, Cysto- [M26 (ATCC 25191), M27 (ATCC 25192), M1621. bacteraceae. It has been noted (12, 13, 15, 17) The following were specimens from the Thaxter that the organisms presently placed in the genus collection, Farlow Herbarium, Harvard University: S. Chondromyces Berkeley and Curtis (Bergey ’s aurantiaca (acc. no. 4473 -4479); C. apiculatus (acc. Manual, 7th ed., p. 854-891) include orga- no. 4471, 4480-83; type = 4481); Chondromyces nisms of both types. It was suggested that catenulatus (acc. no. 4517 = type, 4518, 3405); C. Chondromyces (family Polyangiaceae ; reference crocatus (acc. no. 4469, 4470,4484-86,2466,6055, 14) be retained for those with cylindrical cells 5168, 601 = neotype); Chondromyces thaxteri (Chon- (1 3). Chondromyces aurantiacus (Berkeley and dromyces lanuginosus; acc. no. 4494 collected by J. H. Faull); Chondromyces sessilis (acc. no. 4505 = type); Curtis) Thaxter and Chondromyces brunneus C. pediculatus (acc. no. 4524, 4525 = type); and Krzemieniewska and Krzemieniewski, having Chondromyces erectus (Podangium erectum; acc. no. tapered cells, were placed in the genus Stigma- 4560). teUa Berkeley and Curtis (family Cystobac- teraceae), the type species of which is Stigma- tella aurantiaca Berkeley and Curtis. RESULTS AND DISCUSSION The present paper is concerned with the For the purposes of this study, a total of 16 classification of five species of Chondromyces pure cultures were identified as belonging to which were not attended to in the above the genus Berkeley and Curtis mentioned considerations. The relationship of Chondromyces according to (7th ed., p. the genus Jahn to is Bergey’s Manual Synangium Chondromyces 854-89 l), by reference to the original litera- also discussed. ture and when possible by comparison with MATERIALS METHODS herbarium specimens. Those identified as C. AND apiculatus (Fig. 6 and 7) and C. pediculatus Cultures and specimens. The media and procedures (Fig. and 9) resembled (Fig. 1-3) referred to here were described in detail elsewhere 8 C. crocatus in having cylindrical vegetative cells and myxo- (13). The following were organisms examined in pure spores of similar morphology. Furthermore, the culture (strain designations beginning with M are herbarium specimens of C. catenulatus (Fig. 4 those of the University of Windsor; ATCC strains are and 5), C. lanuginosus (C. thaxteri; Fig. lo), 40 VOL. 21. 1971 111. CHONDROMYCES AND STIGMATELLA 41

Fig. 1. Chondromyces crocatus, fruiting body. X90. Fig. 2. Chondromyces crocatus, myxospores. X 1,300. Fig. 3. Chondromyces crocatus, vegetative cells. X 1,300. Fig. 4. Chondromyces catenulatus, fruiting body. X 120. Fig. 5. Chondromyces catenulatus, myxospores. X 1,200. Fig. 6. Chondromyces apiculatus, fruiting body. X 100. Fig. 7. Chondromyces apiculatus, myxospores. XI,300. Fig. 8. Chondromyces pediculatus, fruiting body. X 90. Fig. 9. Chondromyces pediculatus, myxospores. X 1,300. 42 McCURDY INT. J. SYST. BACTERIOL. and C. sessilis (Fig. 1 la, 1 lb, and 12) were also on its close resemblance to C. apiculatus. Faull, found to have Chondromyces-type resting cells. after a careful study of the ontogeny of the The remaining isolates were members of the fruiting bodies of C. lanuginosus, concluded genus Stigmatella Berkeley and Curtis. That is, that the fruiting bodies differed from those of they had tapered vegetative cells and much- C. apiculatus only in the final stages of develop- shortened, phase-dense or refractile, rigid ment. A close relationship between the two is myxospores (microcysts). also suggested by the not infrequent occurrence As will be noted by an examination of Table in C. apiculatus of fruiting bodies with fused 1, the morphological differences between Stig- sporangia. We agree with Faull, therefore, that matella and Chondromyces are correlated with the genus Chondromyces constitutes a close a number of cultural and biochemical differ- evolutionary series in which C. lanuginosus, ences, although within the two groups the although undoubtedly the most advanced cultural and biochemical characteristics are species, is not sufficiently different from the rather uniform. As a consequence, morpholog- other species to warrant placing it in a separate ical characteristics remain of primary impor- genus. tance in distinguishing species. Chondromyces Berkeley and Curtis Chondromyces. Before giving a detailed 1874, 64. consideration of the characteristics of the (Objective synonym: Myxobotrys Zukal species of Chondromyces, it is necessary to 1896, 346.) consider the genus Synangiurn Jahn and its Vegetative cells are cylindrical, untapered relationship to Chondromyces. rods with bluntly rounded ends. Thaxter (21) described an organism whose Sporangia borne singly or in clusters on irregular sporangia were arranged in sessile simple or branched stalks. rosettes or tufts, which he named C. sessilis. Myxospores lack capsules and resemble Kofler (7) later reported a stalked organism, C. vegetative rods. lanuginosus, in which the sporangia were fused Vegetative swarms etch, erode, and pene- in the form of discoid or spherical clusters with trate agar media. Vegetative slime does not each sporangium bearing an apical tuft of hairs. adsorb Congo red dye. Shortly thereafter, Faull (4) gave the name C. Aerobic. thaxteri to what was undoubtedly the same Temperature range, 18 to 37 C; optimum 28 organism. Jahn (5), however, recognized all to 30 C. three as distinct species and erected the new Guanine plus cytosine content of species genus Synangium to contain them. Krzemie- examined is 69 to 70 moles per cent by T, niewska and Krzemieniewski (1 l), treating determinations. Jahn’s species as mere varieties, subsequently Type species: Chondromyces crocatus concluded that there was but one species, Berkeley and Curtis 1874, 64. Synangium sessile. A careful comparison of Thaxter’s descrip- Descriptions of Species of Chondromyces tion of C. sessilis with those of Faull’s and Chondromyces crocatus Berkeley and Curtis Kofler’s organisms does not at all suggest the 1857 and 1874,64. (See reference 1, Fig. 70a conclusion that they are identical. On the and 3 13.) contrary, our observations of C. lanuginosus (= (0bj ect ive synonym : Myxo bo trys varia b ilis Synangium lanuginosus = C. thaxteri; Fig. 10) Zukal 1896, 340.) and C. sessilis (Fig. 1 la, 1 lb) from the Thaxter Vegetative cells (Fig. 3) cylindrical with collection confirms that they are not at all blunt, rounded ends, 1.1 to 1.4 pm by 3 to 12 alike. From the author’s observations of the pm. specimens of C. sessilis which were available Sporangia broadly spindle-shaped, conical, (Thaxter apparently observed it only once), this or nearly spherical, 10 to 25 pm by 15 to 30 organism, although undoubtedly a chondro- pm, straw-colored initially, finally becoming mycete, should not have been regarded as a new golden-yellow or orange. Sporangia borne in species. Indeed, it might well be a variant of spherical clusters on usually branched stalks up any of the other recognized species of Chondro- to 700 pm or more in height (Fig. 1). Stalks myces. orange to brown, striated, often spirally On the other hand, C. lanuginosus is certain- twisted, tunneled internally with few cells ly a distinct species. The question is whether it within. Irregular forms with ramifying branches is necessary to assign it to a separate genus. Its and few sporangia or with secondary fruiting most distinctive characteristic is the fusion of structures arising from sporangia germinating in its sporangia. Both Kofler and Faull remarked situ may be observed in culture. VOL. 21, 1971 I 11. CHONDR OM YCES AND STIGMA TELLA 43

Fig. 10. Chondromyces lanuginosus, fruiting body. x 120. Fig. 11. Chondromyces sessile, fruiting bodies. X 120. Fig. 12. Chondromyces sessile, myxospores. X 1,100. Fig. 13. Stigmatella aurantiaca, fruiting bodies on agar. X 100. Fig. 14. Stigmatella aurantiaca, fruiting body on bark. X 90. Fig. 15. Stigmatella aurantiaca, vegetative cells. x 1,200. Fig. 16. Stigmatella aurantiaca, microcysts. X 1,400. Fig. 17. M 162 sporangia on bark. XIOO. Fig. 18. Podangium erectum herbarium specimen on bark. XlOO. Fig. 19. Stigmatella erecta microcysts. X 1,300. 44 McCURDY INT. J. SYST. BACTERIOL.

TABLE 1. Physiological characteristics of isolates

Organism Growth Liquid Agar Congo Aescu- Urease Neo- Kana- medium culture etched red lin mycin mycin

Stigmatella aurantiaca M341 SP + + + Stigmatella aurantiaca M340 SP + + + Stigmatella “cylindrica M342 SP + + + Stigmatella “media ’’ M84 SP + + + Stigmatella “media ’’ M85 SP + + + Stigmatella “media ’’ M34 SP + + + Stigmatella “media” M15 (ATCC 25190) Sp + + + Stigmatella erecta M26 (ATCC 25191) SP + + + Stigmatella erecta M27 (ATCC 25192) SP + + + Podangium erectum M162 SP + + + Chondromyces crocatus M3 8 (ATCC 25 193) SpE - - - Chondromyces crocatus M204 SPE - - - Chondromyces crocatus M120 SPE - - - Chondromyces apiculatus M6 SPE - - - Chondromyces apiculatus M36 SpE - - - Chondromyces pediculatus M118 SPE

Myxospores (Fig. 2) lack capsules and differ Streptomycete-like odor produced is dif- little from vegetative cells except for the ferent from that of myxobacters with tapered presence of conspicuous refractile granules at cells. one or both ends, 1.O to 1.3 pm by 3 to 6 pm. Source and habitat: First observed on de- Vegetative colonies on most media are cayed melons from South Carolina. Later found initially translucent, almost colorless, later be- by Thaxter (19) on old straw from Ceylon. coming yellowish-orange, and heaped at the Commonly found on dung in contact with soil, periphery to form a “front” which is particu- on bacterial streaks inoculated with soil, or on larly conspicuous in contact with masses of bark. living eubacteria. The underlying agar is pitted, Neotype: Acc. no. 601, Thaxter collection, eroded, and penetrated by columns of vegeta- Farlow Herbarium, Harvard University. tive cells. Congo red is not adsorbed. Reference strain: Windsor M38 (ATCC Growth on ECM agar (13) is poor with lysis 25 193). generally limited to the immediate area of the Chondromyces apiculatus Thaxter 1897, colony. Lysis of living requires direct 405. contact. Cultivated in pure culture on complex Vegetative rods are cylindrical with blunt, media containing enzymatically hydrolyzed rounded ends, 1.1 to 1.4 pm by 3 to 14 pm. protein and Mg*. Growth is stimulated by, and Sporangia (Fig. 6) straw-colored to bright initially requires, an extract from eubacterial orange or brownish-orange, variable in form and cells. size, cylindrical to broadly turnip-shaped, 25 to Nitrate is not reduced. Catalase and oxidase 40 pm by 35 to 50 pm, with colorless, pointed, negative. Hydrolyzes starch, Tween 80, indoxyl frequently branched, apical appendages up to acetate, ribonucleic acid, deoxyribonucleic 35 pm long. Pedicels absent or up to 30 pm acid, gelatin, and casein. Does not hydrolyze long, colorless. Sporangia borne in spherical urea, aesculin, or cellulose. Agar digestion not clusters of 60 or more, although usually fewer detected. in number than in C. crocatus. Stalk seldom Aerobic. branched, up to 700 pm in height, diameter of Temperature range, 18 to 37 C; minimum, 15 to 40 pm, longitudinally striated, tunnelled, 18 C; optimum, 28 to 30 C. and without internal cells. Forms without Antibiotic sensitivity (discs): Resistant to stalks, with basally fused sporangia, or with neomycin (10 pg), kanamycin (10 pg), penicil- large, irregular, solitary sporangia are common. lin (10 units). Inhibited by streptomycin (5 pg), Secondary fruiting body formation as in C. tetracycline ( 10 pg), chloramphenicol (1 0 pg), crocatus or sporangium formation at tips of and erythromycin (5 pg). appendages is often observed. VOL. 21, 1971 111. CHONDROMYCESAND STIGMATELLA 45

Myxospores similar to vegetative cells, 1.O Myxospores are nonrefractile cylindrical to 1.3 pm by 3 to 6 pm (Fig. 7). rods with blunt, rounded ends, 1.2 to 1.4 pm In all other characteristics, it is identical to by 3 to 6 pm (Fig. 5). C. crocatus. Cultivated only on original source, not Source and habitat: Originally isolated from reported in pure culture. antelope dung from Liberia. Commonly ob- Source and habitat: Decaying poplar wood served on rabbit dung in contact with soil. from New Hampshire. Guanine plus cytosine content is 69.3 moles Type: Acc. no. 45 17, Thaxter collection, per cent by T, determination. Farlow Herbarium, Harvard University. Type: Acc. no. 4481, Thaxter Herbarium, Chondromyces lanuginosus Kofler 19 13, Harvard University. 861. Chondromyces pediculatus Thaxter 1904, [ 0b j e ct ive synonym : Syna ngiu m Zan ugin 0sus 410. (Kofler) Jahn 1924, 79.1 Vegetative cells cylindrical with blunt, [Subjective synonyms: Chondromyces thax- rounded ends, 1.1 to 1.3 pm by 3 to 16 pm. teri Faull 1916, 226 and Synangium thaxteri Sporangia (Fig. 8) pale-yellow to orange, (Faull) Jahn 1924, 79.1 when dry orange-red, nearly spherical to long Vegetative cells cylindrical with blunt, cylindrical, club-shaped or pyriform, usually rounded ends 0.9 to 1.0 pm by 3 to 8 pm. broader and truncate at distal end, surface Sporangia (Fig. 10) fused at their bases to rough, 25 to 40 pm by 35 to 60 pm. Borne in form discoid or nearly spherical clusters con- groups up to 60 in umbel-shaped heads on taining up to 80 sporangia each with an apical slender pedicels 20 to 40 pm long. Stalk tuft of hairs. Diameter of clusters variable (40 unbranched, up to 750 pm in height, straited, to 250 pm) as is the length of the apical hairs (7 sometimes twisted, colorless initially, becoming to 30 pm). Stalks simple or occasionally orange to brown when dry. branched, bearing from 1 to 30 clusters. Myxospores lack capsules, resemble vegeta- Sporangia initially white changing to yellow , tive cells, 1.0 to 1.2 pm by 3 to 7 pm (Fig. 9). light-pink, and eventually orange. Stalks are at Vegetative colonies orange to reddish-orange first colorless but become yellow. Sometimes and resembling those of C. crocatus. Congo red the sporangial clusters give rise to secondary is not adsorbed. stalks which are thinner than the primary ones Cultivated on dung pellet agar and living and which are tipped with smaller clusters. bacteria. Growth is slow. Obtained once in pure Myxospores do not differ from vegetative culture on Escherichia coli extract -enriched cells, only slightly smaller, 0.6 pm to 1.0 by 2.6 Casitone-Mg* agar. Pm- Cellulose is not hydrolyzed. Urease negative. Cultivation: Grown in laboratory culture on Source and habitat: Originally isolated on hay (Krzemieniewska and Krzemieniewski goose dung from South Carolina. Obtained on 1946, 37). Pure cultures not obtained. rabbit dung in contact with soil. Source and habitat: Found on the dung of herbivores in Canada (Faull 1916, 226) and Type: Acc. no. 4524, Thaxter collection, Farlow Herbarium, Harvard University. Austria (Kofler 19 13, 861); also found in soil in Poland (Krzemieniewska and Krzemieniewski C hondromyces catenulatus Thaxter 19 04, 1946, 37). 410. Neotype: Acc. no. 4494 collected by J. H. Rods in rising spore mass, 1.0 to 1.3 pm by Faull, Thaxter collection, Farlow Herbarium, 4 to 6 pm. Cells in vegetative colonies not Harvard University. described. Sporangia (Fig. 4) light-yellow to orange, Species incertae sedis fusiform, long elliptical or irregular in shape, Chondromyces sessilis Thaxter 1904, 4 1 1. dimensions 18 pm by 20 to 50 pm, united in Vegetative rods not described. chains up to 300 pm long which may be Sporangia (Fig. 1 la,b) yellow to reddish- branched once or twice. Sporangia separated by orange, forming a sessile rosette or tuft on the shrivelled, membranous isthmuses. Stalk simple, substrate without a clearly differentiated stalk, orange to rust-colored, up to 400 pm in height, although a poorly developed stalk is said broad at base, tapering above, and several times occasionally to occur. cleft into swollen, tapering parts, each bearing Sporangia quite variable in shape, irregularly one or several sporangial chains. and broadly fusiform, often subapiculate, with 46 McCURDY INT. J. SYST. BACTERIOL. a wrinkled surface, variable in size, coherent at Manual, 7th ed.) species of StigmatelEa. Table 2 base or more or less completely confluent in summarizes the characteristics used by irregular masses. Dimensions of sporangia, 18 to Krzemieniewska and Krzemieniewski to dis- 35 ym by 25 to 75 ym; dimensions of rosettes, tinguish the species and permits a comparison 100 to 250 pm. with those of our isolates. It will be noted that Myxospores are cylindrical with blunt, great emphasis is placed on cyst shape, size, rounded ends, 0.8 to 1.0 ym by 3 to 5 ym (Fig. color, and the presence or absence of pedicels. 12). The Krzemieniewskis were not very specific Source: Rotten wood from Florida. Cultiva- about the media or substrates employed, and tion not reported. Observed only on natural there is no indication that they observed pure substrate. cultures. However, both the nature of the Type: Acc. no. Thaxter collection, substrate and the presence of contaminants Farlow Herbarium, Harvard4505, University. (especially molds) can have a profound effect Stigmatella Berkeley and Curtis. on precisely those characteristics that the The distinguishing features of species of Krzemieniewskis emphasized. Stigmatella employed by the Krzemieniewskis It was not surprising, therefore, that there (8-11) seemed arbitrary and not always were differences (Table 1) among the pure consistent. However, it was possible on cultures identified as Stigmatella aurantiaca, S. the basis of their descriptions and of the media, and S. cylindrica in cultural charac- appearance of our isolates on the natural teristics, fruiting bodies, and biochemical substrates on which they were first encountered properties; these were trivial, however, and in to assign tentatively each of our isolates to one any case uncorrelated with their “species” of the four presently recognized (Bergey ’s assignments. On the basis of these results, we

TABLE 2. Comparison of isolates of Stigmatella with original descriptions

Color of mature Shape of Sporangial Organisma sporangia sporangia dimensions Pedicels

S. aurantlhca Orange-red to Almost spherical 14-54 X Present or (1 1) yellow-brown or elongated 24-72 pm absent M340 Orange-red Oval or spherical 13-40 X Absent 30-50 pm M34 1 Orange Oval or nearly 20-30 X Absent spherical 30-50 pm

S. “cy lindr ica ” Brilliant orange Usually cylindrical 20-30 X 30 pm (1 1) sometimes brownish but often oval or 90 pm spherical M342 Orange Cylindrical 20-30 X 40 pm 40-70 pm S. “media” (1 0) Orange-red, reddish- Variable, oval or 26-93 X 40 um orange or light brown tapering to base 24-78 pm

M84 Bright orangish-red Oval or spherical 25-40 X 40 pm 40-50 pm

M85 Bright orange-red Oval or spherical 25-40 X 40 pm 30-50 pm S. brunneus (1 1) Dark chestnut-brown, Spherical or oval X 28-83 30 pm but almost black 37-102 pm settling down M26 Dark chestnut-brown, Spherical or oval 40-80 X 20-30 pm almost black 30-50 pm settling M27 Dark chestnut-brown Spherical or oval Avg 40 X 20-30 pm 55 pm settling

Numbers in parentheses refer to references. VOL. 21, 1971 111. CHONDROMYCES AND STIGMATELLA 47 conclude that the diagnostic characteristics Myxospores are short, rigid, phase-dense or used by Krzemieniewska and Krzemieniewski refractile rods surrounded by a definite slime to distinguish S. cylindrica and S. media fall capsule. well within the range of variation of S. auran- Vegetative colonies do not etch, erode, or tiaca, not only as observed among our isolates penetrate agar media. Congo red is adsorbed. and in the Thaxter specimens but also as was The minimal nutritional requirements are previously described (5, 11, 17, 19). We con- not known, but the organism is easily cultivated clude, therefore, that s. media and s. cylindrica on media containing enzymatically hydrolyzed are merely varieties of S. aurantiaca. protein. Reichenbach and Dworkin (1 7) previously Urea is usually hydrolyzed. stated the opinion that all of the “species” of Aerobic. Stigmatella are varieties of S. aurantiaca. We Temperature range, 18 to 37 C; optimum believe, however, that Stigmatella brunnea is 30 C. sufficiently different morphologically to retain Guanine plus cytosine content of species it as a separate species. We previously expressed examined is 68.5 to 68.7 moles per cent by T, the opinion (15) that S. brunnea and Podan- determination (1 6). gium erectum are identical. Various authors Type species: Stigmatella aurantiaca have cited the close similarity between P. Berkeley and Curtis 1874,97. erectum and the stigmatellae and in fact the Descriptions of Species of Stigmatella Krzemieniewskis (8) made special reference to Stigmatella aurantiaca Berkeley and Curtis the very close resemblance of S. brunnea to P. 1874, 97. In the present study, isolate M162 was erectum. [Objective synonym: Chondromyces auran- initially identified as P. erectum (9) because, tiacus (Berkeley and Curtis) Thaxter 1892, when first observed on bark (Fig. 17), it had 4011. prod ced no chondromyces-type fruiting u Vegetative cells are rods with tapered ends, bodies and it appeared to be identical to the 0.6 to 1.0 pm by 4 to 10 pm (Fig. 15). Thaxter herbarium specimens (Fig. 18). How- ever, once obtained in pure culture, it was On bark (Fig. 14) sporangia are spherical, oval, pear-shaped, or cylindrical; yellowish- indistinguishable from our cultures of S. orange to bright orange-red or reddish-brown; brunnea. variable in size, 16 to 70 pm by 25 to 102 pm. The name Schroeter Cystobacter erectus Pedicels may be absent at maturity; when 1 8 8 6 antedates Chondromyces brunneus present, they are up to 40 pm long. Stalks up to Krzemieniewska and Krzemieniewski 1946, and the specific epithet erectus has priority over 400 pm long, usually unbranched, sometimes brunneus. Consequently, the name Stigmatella arising from a common origin in a fascicled erecta (basonym: Cystobacter erectus arrangement, granular, not striated, of hardened Schroeter) is proposed for this organism. The slime containing some cells, colorless to the characteristics distinguishing S. erecta from S. color of the sporangia. Morphology of the aurantiaca are the greater frequency of mono- fruiting bodies is variable especially in culture; sporangial fruiting bodies, the consistently dark forms (Fig. 13) with very long (to 900 pm) and color of the sporangia, the larger average size of irregularly branched, white to orange stalks the sporangia, and the “settling down” of the lacking sporangia or tipped by one or few stalks at maturity of the former. sporangia are often observed on plain agar in Thus, the genus Stigmatella is to be regarded the presence of microbial contaminants; as consisting of a range of closely related archangium-like forms are also common. When morphological types with the chondromyces- induced on Ca*-water agar, the fruiting bodies like S. aurantiaca at one extreme and S. erecta bear one or few sporangia. Microcysts, 0.9 to 2 pm by 1.6 to 4.0 of the “Podangium ” morphological type at the pm other. (average 1.O pm by 2.8 pm; Fig. 16). Genus Stigmatella Berkeley and Curtis Vegetative colonies are thin, flat with 1874,97. numerous radiating and concentric ridges, edge (Objective synonym: Polycephalum Kalch- more or less definite or thin, filamentous and brenner and Cooke 1880,22.) poorly delimited, light yellow or flesh-colored, Vegetative cells are rods with tapered ends. occasionally producing a yellowish or brownish Sporangia borne singly or in clusters on discoloration of agar media. Orange aggregates stalked fruiting bodies (the stalks often occur- often form in old cultures on 0.1% casitone or ring in groups arising from a common hypothal- E. coli media, but fruiting bodies fail to lus). develop. Continued laboratory cultivation 48 McCURDY INT. J. SYST. BACTERIOL. yields variants which produce mucoid colonies hundreds together. On rabbit dung pellets, the but which are unable to form fruiting bodies. fruiting bodies may appear as archangium-like Easily cultivated on agar or liquid media masses or coralloid accumulations of pinkish- containing 0.1 to 0.2% hydrolyzed protein, white or salmon-colored, finger-like projections starch, and 0.01 M Mg' or on agar media without clear-cut delimitation of sporangia. The containing dead bacterial cells. tips eventually turn dark brown, ;he remainder Nitrate is not reduced. Catalase positive, becoming yellowish-white. On Ca -water-agar, oxidase negative. Hydrolyzes starch (3 days), fruiting bodies with one to four sporangia on Tween 80, indoxyl acetate, ribonucleic acid, sparingly branched stalks (30 to 50 pm by 80 deoxyribonucleic acid, gelatin, casein, urea, and to 200 pm) are produced. aesculin. Microcysts are straight, curved or somewhat Aerobic. fusiform, short rigid, phase-dense or refractile Temperature range, 18 to 37 C; optimum, rods, 0.8 to 1.5 pm by 1.5 to 3.5 pm (Fig. 19). 30 C. Vegetative colonies are at first thin and Optimum pH 7.0 to 7.2. transparent , later yellow or light flesh-colored Antibiotic sensitivity (discs): Resistant to with numerous radiating ridges. The edge is thin penicillin (1 0 units), sensitive to streptomycin and indefinite. Continued laboratory cultiva- (5 pg), tetracycline (1 0 pg), chloramphenicol tion selects variants with yellow or orange (10 pg), kanamycin (10 pg), and erythromycin mucoid colonies. In old cultures the swarm (5 pg). Response to neomycin is variable. turns dark brown, and the surrounding medium Source and habitat: Originally observed on also becomes darkened. lichen. Most commonly observed on bark kept Easily cultivated on media containing in a moisture chamber. Also from soil inoc- enzymatically hydrolyzed protein, starch, 0.0 1 ulated on streaks of living bacteria on filter M Mg', and salts. Growth has also been ob- paper over water agar. tained on a medium containing 17 amino acids, Guanine plus cytosine content is 68.5 to thiamine, 0.01 M Mg', and starch (16). Strictly 68.7 moles per cent by T, determination. respiratory, utilizing complex amino acids as Neotype strain: Windsor M 34 1. energy source. Stigmatella erecta (Schroeter) comb. nov. Nitrate is not reduced. Catalase positive. [Objective synonyms: Cystobacter erectus Oxidase negative. Schroeter 1886, 170 Hydrolyzes starch (3 days), Tween 80, Chondromyces erectus (Schroeter) Thaxter indoxyl acetate, deoxyribonucleic acid, ribo- 1897,407 nucleic acid, gelatin, casein, urea, and aesculin. Podangium erectum (Schroeter) Jahn 1924, Aerobic. 80.1 Temperature range, 18 to 37 C; optimum, [ Subjective synonyms: Chondromyces 28 to 30 C. aurantiacus var. Jjutescens Krzemieniewska and Optimum pH 7.0 to 7.2 Krzemieniewski 1927,9 1 Antibiotic sensitivity (discs): Resistant to Chondromyces brunneus Krzemieniewska penicillin (10 units), sensitive to streptomycin and Krzemieniewski 1946,44 (5 pg), tetracycline (1 0 pug), chloramphenicol Stigmatella brunnea (Krzemieniewska and (10 pg), kanamycin (1 0 pg), and erythromycin Krzemieniewski) McCurdy 1969, 1460.1 (5 pg). Response to neomycin is variable. Vegetative cells are slightly tapering, flexible Source and habitat: Obtained on the dung rods, 0.7 to 0.8 pm by 5 to 10 pm. of herbivores placed in contact with soil or bark On bark the sporangia are spherical (Fig. 17, incubated in a moist chamber. 18), oval or elongated, at first flesh-colored Guanine plus cytosine content is 68.7 moles becoming orange-red and finally dark chestnut- per cent by T, determination (1 6). brown or almost black at maturity, 30 to 90 Neotype strain: Windsor M26 (ATCC pm by 35 to 140 pm. Sometimes (especially on 25191). rabbit dung) borne singly on opaque white, later yellowish, stalks arising in groups from a ACKNOWLEDGMENT common hypothallus. In other forms, several This investigation was supported by a National sporangia are arranged in clusters on yellowish- Research Council of Canada grant. white to orange-red stalks. The sporangia may or may not be borne on pedicels. In both LITERATURE CITED forms, the stalks (and pedicels) commonly 1. Berkeley, M. J., and Curtis. 1857. In M. J. wither, depositing the cysts on the substrate; Berkeley (ed.), Intr. Crypt. Bot., p. 1-604. H. thus, they appear sessile in masses of fifty to Baillere, London. VOL. 21,1971 111. CHONDROMYCES AND STIGMA TELLA 49

2. Berkeley, M. J., and Curtis. 1874. In M. J. myces crocatus. Arch. Mikrobiol. 65:380-390. Berkeley (ed.), Notices of the North American 13. McCurdy, H. D. 1969. Studies on the taxonomy fungi. Grevillea 3: 1- 17. of the Myxobacterales. I. Record of Canadian 3. Enderlein, G. 1924. Bemerkungen zur Systematik isolates and survey of methods. Can. J. der Chondromyciden. Walter de Gruyter and Microbiol. 15: 1453 -1461. Co., Berlin. 14. McCurdy, H. D., Jr. 1970. Studies on the taxonomy 4. Faull, J. 1916. Chondromyces thaxteri, a new of the Myxobacterales. 11. Polyangium and the Myxobacterium. Bot. Gaz. 62: 226-232. demise of the Sorangiaceae. Int. J. Syst. Bacteriol. 20:283-296. I 5. Jahn, E. 1924. Beitrage zur botanischen Protis- tologie. I. Die Polyangiden, p. 1-107. Gebriider 15- McCurdy, H. D., and B. T. Khouw. 1969. Studies Born traeger ,Leipzig . on Stigmatella brunnea. Can. J. Microbiol. 6. Kalchbrenner, C., and M. C. Cooke. 1880. South 15 :73 1-7 38. McCurdy, D., and S. Wolf. 1967. Deoxyribo- f African Fungi. Grevillea 9: 17 -34. 16. H. 7. Kofler, L. 1913. Die Myxobacterien der Umge- nucleic acid base compositions of fruiting bung von Wien. SB Akad. Wiss. Abt. I Wien Myxobacterales. Can. J. Microbiol. 122:845 -876. 13:1707 -1 708. 8. Krzemieniewska, H., and S. Krzemieniewski. 7* Reichenbach, H., and M. Dworkin. 1969. Studies 1926. Miksobakterje Polski. Acta SOC.Bot. Pol. on Stigmatella aurantiaca (Myxobacterales) J. 4:l-54. Gen. Microbiol. 58:3-14. 9. Krzemieniewska. H., and S. Krzemieniewski. 1927. 18* Schroeter, J. 1886. Pilze, in Cohn, Ferdinand, Miksobakterje Pol. Uzupelnienie. Acta SOC.Bot. Kryptogamen-Flora von Schlesien. J. U. Kern. Pol. 5:79-98. Breslau 3:l-814. 10. Krzemieniewska, H., and S. Krzemieniewski. 19. Thaxter, R. 1892. On the Myxobacteriaceae, a 1930. Miksobakterje Polski. CzeS6 Trzecia. Acta new order of Schizomycetes. Bot. Gaz. SOC.BOt. Pol. 7:250-273. 17: 389 -406. 11. Qzemieniewska, H., and s. ~~~~~i~~i~~~ki.20. Thaxter, R. 1897. Further observation on the 1946. of the species Chondro- Myxobacteriaceae. Bot. Gaz. 23:385-411. myces Berkeley and Curtis. Bull. Int. Acad. 21. Thaxter, R- 1904. Notes on the Myxobacteria- Cracovien (Acad. Pol. Sci.) Ser. B. Sci. Nat. ceae. Bot. Gaz. 37:405-416. 1~31-48. 22. Zukal, H. 1896. Myxobotrys variabilis Zuk., als 12. McCurdy, H. D. 1968. Light and electron micro- Reprasen tant einer neuen Myxomyceten- scope studies on the fruiting bodies of Chondro- Ordnung. Ber. Deut. Bot. Ges. 14:340-347.