Sabouraudia(1977), 15, 1-10
MICROSPOR UM MAGELLANICUM AND CUNNINGHAMELLA ANTARCTICA, N E W SPECIES I S O L A T E D F R O M A U S T R A L I C A N D A N T A R C T I C SOIL OF CHILE
G. CARETTA AND E. PIONTELLI*
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Cattedra di Micologia Medica e Centro di Micologia Medica "R. Ciferri e P. Redaelli," Universita di Pavia, Italy Keratinophilic fungi were isolated from samples of soil collected in the extremity of Chile, in Southern Shetland and Antarctic continent. Two new species, Microsporum magellanicum and Cunninghamella antarctica are described. Microsporum magellanicum is characterized by verrucose or verruculose clavate macroconidia predominantly uniseptate, 4"8-7-2/t× 14.4-21-6B. Cunningharnella antarctica has conidiophores usually verticillately, pseudoverticillately and sympodially branched; and globose conidia with evident spines, 12-8-16 B in diameter.
Taxonomic studies of keratinophilic and other fungi isolated from soil samples collected in Southern Chile, in Southern Shetland and in the Antarctic were studied. The fungi were isolated by using keratinoceous material as bait (9, 7); two unusual species of the genera Microsporum Gruby and Cunni@amella 1V[atruchot were discovered for the first time
MATERIAL AND M~ETHODS
Collection of Soil Samples The collections were made from 6 places (see Figure) as follows:
1, Desolaci6n Island: soil temp. 11-2°C, samples collected on Jan. 2, 1974 near the sea and in green zone. There were domestic animals and a few inhabitants in the region.
2, Puerto Williams in Navarino Island: soil temp. 12.7°C; soil collected on Jan. 4, 1974 near the port entrance. There were about 900 inhabitants.
3, Rey Jorge Island (South Shetland Island) : samples collected on the air base E. Frey on Jan. 7, 1974, soil temp. 4°C, loamy soil, lichens present.
4, Rey Jorge Island: samples taken 1 km from E. Frey base, soil temp. 2°C. 5, Greenwich Island (South Shetland Island) : base A. Prat, soil collected near the base on Jan.-7, 1974, no vegetation present, sandy soil, temp. --2°C. 6, O'Higgins base (Antarctic Continent) : soil collected near the base on Jan. 18, 1974, temp. --4°C. There were 30 Eskimo dogs in the vicinity. All samples were collected superficially, the depth not exceeding 5 cm, in sterile plastic bags and received in the laboratory within 3 weeks. Sterile Petri dishes, halffilled with the soil samples, were moistened with sterile distilled water. Sterile human or horse hair and feathers were added and incubation was at 25°C for 6-8 weeks. The colours of these new species are those of Ridgway (1912). * Present address: Area de Ciencias de la Salud., Dpto Pathologia, Seccion Microbiologia, Universidad de Chile, Valparaiso, Chile.
2
O. C A R E T T A
A N D E. P I O N T E L L I
70 °
75 °
/ ~J~,
60°
6~ ",-J
I
55°
Med Mycol Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
r~e \ \
\ \
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\ \
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+---H6~
\ \, .....'"" ....,~'"'
F i g u r e - - M a p of Antarctic sector of Chile s h o w i n g location of soil samples collected : 1 : Desolaci6n Island ; 2 : N a v a r i n o Island ; 3 a n d 4 : R e y J o r g e Island ; 5 : G r e e n w i c h Island ; 6 : O ' H i g g i n s base (Antarctic continent).
RESULTS
The distribution of keratinophilic fungi was different in the 2 continents (see Table). Chrysosporium keratinophilum, C. asperatum, Trichophyton ajelloi and T. terrestre were isolated from soil in the Austral zone of Chile ; C. indicum and C. keratinophilum from South Shetland Islands and Antarctic continent. Most of the other fungi isolated from both the continents, belonged to the Fungi Imperfecti. Two fungi believed to be new species of genera Microsporum and Cunninghamella were found. Microsporum magellanicum was isolated only in 2 soil samples from the zones 1 and 2. Cunninghamella spp. were recovered from 3 sites (zones 3, 4, 6). The new species was present only in 1 sample (zone 6). Since the first isolation was from soil of the Straits of Magellano, we propose the
MICROSP. MAGELLANICUM AND CUNNINGHAMELLA ANTARCTICA TABLE.--FUNGI ISOLATED FROM CHILEAN SOIL
Austral zone of Chile 1
2
Antarctic continent
Southern islands 3
4
5
6
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Base Desolacidn Puerto Base Base Base General Island [~Villiams E. Frey E. Frey A. Prat O'Higgins Cunninghamella antarctica sp. nov. C. echinulata (Thaxter) " Thaxter Mierosporum magellanicum sp. nov. Chrysosporium asperatum Carmichael Ch. indicum (Randhawa & Sandhu) Garg. Ch. keratinophilum (Frey) Carmichael Trichophyton ajelloi (Vanbreuseghem) Ajello T. terrestre Durie & Frey Alternaria sp. Aspergillus sp. Botryotrichum sp. Diheterospora catenulata Kamyschko D. chlamydosporia (Goddard) Barron & Onions Fusarium sp. HumicoIa sp. Mucor sp. Penicillium chrysogenum Thorn P. lilacinum Thom P. variabile Sopp Penicillium sp. StemphyIium sp. Vertwillium sp.
Number of isolates
1 2 1
2
1
1
3 2 1
2 l 1 1
2 1 1 1
1
3
4
l
!
1 I 1
1 2
1 1 1
2
2 2
9
2 1 2
1
l
2
2 i1
2
2
1
1 1
ll
4
1
4 3 5
9
58
1
1 1
3
1
15
10
4
n a m e Microsporum magellanicum a n d t h e n a m e Cunninghamella antarctica for t h e species i s o l a t e d f r o m O ' H i g g i n s soil.
Microsporum
magellanicum
sp. nov.
F u n g u s t e r r e s t r i s , m y c e l i o s e p t a t o , h y a l i n o , 2 - 4 # l a t o et r a m o s o ; r a m i s e c u n d a r i i flexuosi c u m spiris t e r m i n a l i b u s . Macroconidia p l u r i m a h y a l i n a , o v a t a , v e l c l a v a t a , r a r o c y l i n d r i c a , 1 - 2 septis p e r r a r o 4 - 6 septis, a m p l i t u d i n e 4 . 8 - 7 . 2 x 1 4 . 4 - 2 1 - 6 # c u m e p i s p o r i o v a l d e v e r -
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4
G. CARETTA AND E. PIONTELLI
rucoso aliquando ornato pedicellato, sed etiam macroconidia verrucolosa; ab hyphis et ramis lateralibus terminaliter aut lateraliter oriuntur et singula plerumque pedunculata sed etia m sessilia. Microconidia vera absunt; adsunt conidia piriformia aut clavata basi truncata, leviter verrucolosa, amplitudine 2"4- 3 x 4.8-5.8#, sessilia aut pedunculata, saepe uniseptata, quae macroconidia incipiente formatione sunt. Chlamidosporae rarae intercalares, singulae et acrogenae, rotundae vel ovatae, 6-8/~ diam. Colonia in agaro Sabouraudii 20°C celeriter crescens; post 12 dies 4 cm diametero, temperatura optima 25°C. Post 30 dies, 4°C, 0"5 cm diametero cum abundantibus macroconidiis. Non crescit ad t e m p e r a t u r a m 37°C. Aspectus subtiliter granulatus, pulverulentus, flos lactis flavescens; pars reversa flava, maturitate ochracea. Species keratinophila cum destructione pili et formatione organorum perforantium in trine. Isolatur ex terra australica Cilense, insula Desolacidn et insula Navarino. Typus conservatur in micoteca Cathedrae Micologiae Medicae, Universitatis Papiae, No. 1051; isotipus apud C.M.I. No. 195827 sub Microsporum verruculosum nomen nudum. Terrestrial fungus, septate mycelium, hyaline, 2-4 # large and branched; secondary branches flexuous with spiral coils. Abundant macroconidia, hyaline, ovate or clavate, rarely cylindrical, 1-2 septa, rarely 4-6 septa, measuring 4.8-7-2 x 14.4-21.6/~, verrucous and pedicellate walled; but also verrucolose. Solitary terminal and lateral macroconidia arising on lateral hyphae, often pedunculate but also sessile. T r u e microconidia absent; pyriform or clavate conidia present, with truncate base, lightly verrucoIose, 2-4-3 x 4.8-5.8 #, sessile or pedunculate, often with septa, that are young macroconidia. Rare intercalary solitary and terminal chlamydospores, spherical and ovate, 6-8/~ diam. The colony on Sabouraud's glucose agar (SGA) at 20°C attains a diameter of 4 cm in 12 days; optimum growth at 25°C; the colony at 4°C attains a diameter of 0.5 cm in 30 days, with numerous macroconidia. There is no growth at 37°C. T h e colony is finely granular, powdery, cream-yellowish; reverse yellow, becoming ochre at maturity. The fungus is keratinophilic with destruction of hair; perforating organs present. Microsporum magellanicurn has been isolated from australic soil of Chile, in the Desolacidn and Navarino Islands. The type is preserved in the collection of Medical Mycology of the University of Pavia, No. 1051; isotype deposed in the Commonwealth Mycological Institute (Kew), No. 195827 as Microsporum verruculosumnomen nudum.
Appearanceof Colony O n SGA the moderately fast growing colonies (40 m m in diameter after 12 days at 25°C), were cream-yellow and finely granular, flat with a central umbo (P1. 1, A). Light yellow-ochre reverse, buckthorn brown in the center. O n malt agar (MA) it grows to a diameter of 35 m m after 12 days, changing to finely granular with concentric rings. Reverse buff-yellow to honey yellow in the centre. The development on potato carrot agar is 3-5 cm in diameter after 12 days at 25°C; fluffy in the center
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MICROSP. MAGELLANICUM AND CUNNINGHAMELLA ANTARCTICA
Plate 1
A--~dicrosporum magellanicum ~)n S.G,A. after 14 days at 27°C. B--Macroconidia and spiral coils in lactofuchsin x 400. C--Perforating body in human hair, macroconidia X 400. D--Verrucose macroconidia x 1000. E--Verruculosity of typical monoseptate macroconidia and cylindrical macroconidia in Czapek Dox Agar. F--Echinulated young macroconidia in S.G.A. × 1000.
5
6
G. CARETTA AND E. PIONTELLI
and granular in the margin, with some zonate growth. Surface white to cream at the periphery; reverse buff-yellow. On Czapek agar growth is similar to that on potato carrot (PCA), with the production of abundant verrucose macroconidia and spiral coils. O n MA at 4°C the growth is very slow, attaining a diameter of 5 m m after 30 days, with a cottony colony.
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Microscopic Appearance The hyphae are septate, hyaline, branched, 2-4 # diam. Spiral coils have been observed (P1. 1,B). Arthrospores scanty on SGA and MA. Intercalary chlamydospores have been observed on Czapek and PCA, and occasionally on MA and SGA racquet hyphae are present. Macroconidia are produced abundantly; hyaline, mostly clavate, arising terminally or laterally from conidiophores mostly single (P1. 1, D). They are not very. thick-walled, markedly verrucose on Czapek and PCA, and verrucolose on SGA and MA. They are variable in length and in number of septa on the following cultural media: SGA macroconidia abundant, 4.8-7-2 x 1421-6 #, mostly verrucose, verrucolose, predominantly 1-2 septa; occasionally macroconidia up 30-40/~ with 4-5 septa. O n MA, abundant, 3-6-6 x 11-20 #, predominantly verrucolose with 1-3 septa. O n PCA and Czapek agar, macroconidia abundant, 4-7.2 x 14.4-19-5 ~, clearly verrucose, predominantly 1 septum (PI. 1, E). True microconidia are absent. Unicellular conidia are present, verruculose, thinwalled, subclavate and pyriform with truncate base (P1. 1, F). These are initial forms of macroconidia. In SGA, PCA, 1VIA and Czapek agar 2"4-3"4 x 4.8-7.2 #, on corn meal agar 2"4-3 x 4-6 p. O n human or horse hair the growth is luxuriant with production of macroconidia and perforating organs (P1.1, C). Cleistothecia were not observed.
Cunninghamella antarctica sp. nov. Colonia in agaro tubero solani dextrosi 25°C celeriter crescit post 6 dies ad 1-1.5 cm, hyphis assurgentibus, ebore flavescens, granulosa; pars reversa flavobubalina, tenuiter ochracea. Mycelium hyalinum, coenocyticum, pseudoseptatum, 16 # latum; rhizoidi et chlamidosporae adsunt; conidiofora erecta, 14/z lata, cum ramis verticillatis, pseudoverticillatis et sympodialibns, et vesiculis globosis terminalibus; w'siculae conidioforarum 30-40 p diam. cum parvis sterigmatibus; et conidiis globosis, 12.8-16/t, valde spinosis; vesiculae ramorum globosae, 16-24 # diam. cum conidiis globosis aut ovatis, spinosis, 9.6-13 # diam. Conidia magna 14-21 ~ diam. absunt. Fungus keratinophilus crescit 9-40°C; optimum 25°C. Isolatur ex terra antarctica, loeo nominato Base O'Higgins. Typus conservatur in micoteca Cathedrae Micologiae Medicae, Universitatis Papiae, No. 1060. Colonies on PDA at 25°C growing rapidly up to 1-1.5 cm high in 6 days, granulated in appearance, ivory-yellow, reverse cream-buff. Mycelium hyaline, finely granular and smooth walled, hyphae up to 16 # in diam.; rhizoids present; chlamydospores 1-2 # diam., conidiophores erect, up to 14 # in diam., being usually verticillately (P1.2, A), pseudoverticillately and sympodially branched (PI. 2; B, D). Conidiophores and lateral branches have terminal vesicles (Pl. 2, E) ; these are usually spherical, average 30-40 # in diam. with minute sterigmate-bearing globose conidia
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MICROSP. MAGELLANICU~tI A N D CUNNINGHAMELLA ANTARCTICA
Plate 2
A--Cunninghamella antarctica in surface of P.D.A. × 160. B - D - - S y m p o d i a l ramifications on hair baiting, and M.A. × 100. C--Conidiophores and conidia in P.D.A. × 400. E - - T y p e s of conidiophores on hair baiting × 100. F--Cunninghamella echinulata, types of eonidiophores × 100.
7
8
G. CARETTA AND E. PIONTELLI
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average 12-8-16 # in diam. covered with true spines, 4-6 # in length (PI. 3; A, C); lateral verticillated branches, in number up to 6, have spherical or subspherical vesicles average 16-24 # in diam., with globose or ovoidal conidia, 9-6-13 #, with true spines, spinescent giant conidia are present (PI. 3, B), 14-21/~ diam. Growth appears at 9°C and 40°C, optimum 25°C. O n MA good growth similar to that on PDA; on corn meal agar growth is poor. Development of the hair bait appears in 3 days at 22°C. Zygospores not formed. Isolated from antarctic Continent soil, O'Higgins base. The type is preserved in the collection of Medical Mycology of the University of Pavia, No. 1060. DiscussioN
Among the 15 species of Microsporum considered to be valid members of the genus (1), M. mageIlanicum shows some similarity to M. nanum Fuentes. Microsporum nanum presents small, 2 or 3 celled macroconidia; the wall is finely verrucose or occasionally smooth. Microsporum mageIlanicum has verrucose or verruculose clavate macroconidia and true microconidia are absent. It does not produce infections in h u m a n and animal skin.
Plate 3
A--Cunninghamella antarctica,normal conidia in P.C.A. × 400, direct view. B--Giant conidia in P.D.A. × 400, C--Conidia in lactofuehsin ×400.
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MICROSP. MAGELLANICUM AND CUNNINGHAMELLA ANTARCTICA
9
Microsporium ripariae (5) has verruculose a n d verrucose m a c r o c o n i d i a , b u t some o f t h e m are smooth, a n d relatively v a r i a b l e in shape, fusiform or c y l i n d r i c a l ; t h e y h a v e 3-6 septa a n d m e a n length 36.5/~, a n d a m e a n w i d t h 9.33 #; it grows well a t 37°C on feathers. Microsporum magellanicum does not g r o w a b o v e 35°C b u t there is some g r o w t h w i t h p r o d u c t i o n o f a b u n d a n t m a c r o c o n i d i a at 4°C. I t grows p o o r l y o n feathers, r a r e l y fructifying. T h e m e a n length o f m a c r o c o n i d i a o f M. mageltanicum is 4-8-7.2 x 14-421.6 # w i t h dense verruculose surface, usually uniseptate, r a r e l y to 4 - 5 septate. Those w i t h m o r e t h a n 4 septa a r e present in S G A a n d 1VIA cultures. O n horse a n d h u m a n h a i r culture, t h e y are p r e v a l e n t l y uniseptate. O n these substrates spiral coils a n d p e r f o r a t i n g organs a r e present. T h e confused s y n o n y m y a n d o v e r l a p p i n g specific descriptions (2, 4, 3, 6) m a d e it difficult to identify Cunninghamella antarctica. S a m s o n (8) presented the results of a revision of the Cunni@amella strains in the collection o f the C e n t r a a l b u r e a u voor S c h i m m e l c u l t u r e s ; this w o r k contains a k e y description a n d illustrations of cultures g r o w n at 25°C on b e e r w o r t - a g a r . Cunninghamella antarctica is similar to C. phaeospora a n d C. elegans. Cunninghamella antarctica differs from C. phaeospora b y h a v i n g very spiny a n d h y a l i n e conidia. I t is also different from C. echinulata owing to the spinosity o f conidia, the true s y m p o d i a l r a m i f i c a t i o n a n d characters of colony. I t shows a g r e a t affinity w i t h C. elegans b u t the c o n i d i a of the l a t t e r are smooth, verruculose or shortly echinulate. Considering the easy p o l y m o r p h i s m of these fungi, the v a r i a b l e synonymies proposed b y various workers a n d the specific c h a r a c t e r s which our isolate p r e s e n t e d , we think it a d v i s a b l e to create a new species. Cunninghametla echinutata ( T h a x t e r ) , was isolated from soil samples collected from South S h e t l a n d Islands a n d A n t a r c t i c c o n t i n e n t (zone 3, 4, 6 o f the Figure). i t grows well at 9°C a n d 40°C; shows g o o d d e v e l o p m e n t at 25°C. T h e r e were no significant m o r p h o l o g i c a l differences on SDA, P D A , P C A m e d i a . I n 6 days, there was r a p i d growth u p 2-2-5 c m high, at first white, l a t e r p a l e olive buff, floccose. T h e conidiophores, l a t e r a l verticillate or pseudo-verticillate b r a n c h i n g , vesicles globose to subglobose, c o n i d i a e c h i n u l a t e d , g i a n t c o n i d i a (P1.2,F) c o r r e s p o n d to the description a n d figures given b y S a m s o n (8). Zygospores were n o t f o r m e d b y this strain. T h e other species of k e r a t i n o p h i l i c fungi isolated from the different samples, Ch. indicum, Ch. keratin@hilum are w o r t h special a t t e n t i o n , since t h e y h a v e n o t y e t been isolated from the a n t a r c t i c region. RIASSUNTO Vengono presentati i funghi cheratinofili isolati da eampioni di terra raecolti nell'estremit~ Sud de Cile, helle isole Shetland del Sud e nel continente Antartico. Sono descritte due nuove specie, Microsporumm~gellanicum e Cunninghamdlaantarctica. Microsporum magellanicum ~ caratterizzato da macroconidi clavati, verrucosi o verrucolosi, prevalentemente unisettati, 4,8-7,2/t × 14,4-21,6/t. Cunninghamellaantarctica ~ caratterizzata da conidiofori generalmente verticillati, pseudoverticiilati e a ramificazioni simpodiali, conidi globosi con vere spine, t2,8-16 It in diam. ACKNOWLEDGEMENTS
The authors wish to thank Drs. R. A. Samson, G. A. De Vries and P. M. Stockdale fur the examination of these strains and for helpful suggestions and criticisms; the Navy of Chile for facilities to take the samples; and Mr. L. Tarifefio who took the soil samples.
l0
G. CARETTA AND E. PIONTELLI
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REFERENCES 1. AJELLO, L. (1974). Natural history of the dermatophytes and related fungi. Mycopathologia e Mycologia Applicata, 53, 93-110. 2. ALCORN,G. D. & YEAGER,C. C. (1938). A monograph of the genus Cunninghametlawith additional descriptions of several common species. Myeologia, 30, 653-658. 3. BOEmJN, K. B. (1958). Notes on the Mucorales of Indonesia. Sydowia, 12, 321-362. 4. CUTTEr, Jr. V. M. (1946). The genus Cunninghamella (Mucorales). Farlowia, 2, 321-343. 5. HUBALEg, Z. & RUsH-MuNRo, F. M. (1973). A dermatophyte from birds: Microsporum ripariae sp. nov. Sabouraudia, 11, 287-292. 6. MIL'KO, A. A. & BELYAKOVA,L. A. (1967). The genus Cunninghamella Matruchot and taxonomy of the Cunninghamellaceae. Mikrobiologiya, 36, 684-699. 7. ORR, G. F. (1969). Keratinophilic fungi isolated from soils by a modified hair bait technique. Sabouraudia, 7, 129-134. 8. SAMSON,R. A. (1969). Revision of the genus Cunninghamella (Fungi, Mueorales). ProceedingKoninkl. Neederl. Akademie van Wetenschappen--Amsterdam, series C, 72, 322-335. 9. VANBREUSEG~EM,R. (1952). Technique biologique pour l'isol~ment des dermatophytes du sol. Annales de la Socidtd Belge de Mgdecine Trop#aI, 30, 173-178.
MICROSPOR UM MAGELLANICUM AND CUNNINGHAMELLA ANTARCTICA, N E W SPECIES I S O L A T E D F R O M A U S T R A L I C A N D A N T A R C T I C SOIL OF CHILE
G. CARETTA AND E. PIONTELLI*
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Cattedra di Micologia Medica e Centro di Micologia Medica "R. Ciferri e P. Redaelli," Universita di Pavia, Italy Keratinophilic fungi were isolated from samples of soil collected in the extremity of Chile, in Southern Shetland and Antarctic continent. Two new species, Microsporum magellanicum and Cunninghamella antarctica are described. Microsporum magellanicum is characterized by verrucose or verruculose clavate macroconidia predominantly uniseptate, 4"8-7-2/t× 14.4-21-6B. Cunningharnella antarctica has conidiophores usually verticillately, pseudoverticillately and sympodially branched; and globose conidia with evident spines, 12-8-16 B in diameter.
Taxonomic studies of keratinophilic and other fungi isolated from soil samples collected in Southern Chile, in Southern Shetland and in the Antarctic were studied. The fungi were isolated by using keratinoceous material as bait (9, 7); two unusual species of the genera Microsporum Gruby and Cunni@amella 1V[atruchot were discovered for the first time
MATERIAL AND M~ETHODS
Collection of Soil Samples The collections were made from 6 places (see Figure) as follows:
1, Desolaci6n Island: soil temp. 11-2°C, samples collected on Jan. 2, 1974 near the sea and in green zone. There were domestic animals and a few inhabitants in the region.
2, Puerto Williams in Navarino Island: soil temp. 12.7°C; soil collected on Jan. 4, 1974 near the port entrance. There were about 900 inhabitants.
3, Rey Jorge Island (South Shetland Island) : samples collected on the air base E. Frey on Jan. 7, 1974, soil temp. 4°C, loamy soil, lichens present.
4, Rey Jorge Island: samples taken 1 km from E. Frey base, soil temp. 2°C. 5, Greenwich Island (South Shetland Island) : base A. Prat, soil collected near the base on Jan.-7, 1974, no vegetation present, sandy soil, temp. --2°C. 6, O'Higgins base (Antarctic Continent) : soil collected near the base on Jan. 18, 1974, temp. --4°C. There were 30 Eskimo dogs in the vicinity. All samples were collected superficially, the depth not exceeding 5 cm, in sterile plastic bags and received in the laboratory within 3 weeks. Sterile Petri dishes, halffilled with the soil samples, were moistened with sterile distilled water. Sterile human or horse hair and feathers were added and incubation was at 25°C for 6-8 weeks. The colours of these new species are those of Ridgway (1912). * Present address: Area de Ciencias de la Salud., Dpto Pathologia, Seccion Microbiologia, Universidad de Chile, Valparaiso, Chile.
2
O. C A R E T T A
A N D E. P I O N T E L L I
70 °
75 °
/ ~J~,
60°
6~ ",-J
I
55°
Med Mycol Downloaded from informahealthcare.com by University of Otago on 12/27/14 For personal use only.
r~e \ \
\ \
\
\
\\ oCE
N
\
\ \
\
\ '\
\
+---H6~
\ \, .....'"" ....,~'"'
F i g u r e - - M a p of Antarctic sector of Chile s h o w i n g location of soil samples collected : 1 : Desolaci6n Island ; 2 : N a v a r i n o Island ; 3 a n d 4 : R e y J o r g e Island ; 5 : G r e e n w i c h Island ; 6 : O ' H i g g i n s base (Antarctic continent).
RESULTS
The distribution of keratinophilic fungi was different in the 2 continents (see Table). Chrysosporium keratinophilum, C. asperatum, Trichophyton ajelloi and T. terrestre were isolated from soil in the Austral zone of Chile ; C. indicum and C. keratinophilum from South Shetland Islands and Antarctic continent. Most of the other fungi isolated from both the continents, belonged to the Fungi Imperfecti. Two fungi believed to be new species of genera Microsporum and Cunninghamella were found. Microsporum magellanicum was isolated only in 2 soil samples from the zones 1 and 2. Cunninghamella spp. were recovered from 3 sites (zones 3, 4, 6). The new species was present only in 1 sample (zone 6). Since the first isolation was from soil of the Straits of Magellano, we propose the
MICROSP. MAGELLANICUM AND CUNNINGHAMELLA ANTARCTICA TABLE.--FUNGI ISOLATED FROM CHILEAN SOIL
Austral zone of Chile 1
2
Antarctic continent
Southern islands 3
4
5
6
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Base Desolacidn Puerto Base Base Base General Island [~Villiams E. Frey E. Frey A. Prat O'Higgins Cunninghamella antarctica sp. nov. C. echinulata (Thaxter) " Thaxter Mierosporum magellanicum sp. nov. Chrysosporium asperatum Carmichael Ch. indicum (Randhawa & Sandhu) Garg. Ch. keratinophilum (Frey) Carmichael Trichophyton ajelloi (Vanbreuseghem) Ajello T. terrestre Durie & Frey Alternaria sp. Aspergillus sp. Botryotrichum sp. Diheterospora catenulata Kamyschko D. chlamydosporia (Goddard) Barron & Onions Fusarium sp. HumicoIa sp. Mucor sp. Penicillium chrysogenum Thorn P. lilacinum Thom P. variabile Sopp Penicillium sp. StemphyIium sp. Vertwillium sp.
Number of isolates
1 2 1
2
1
1
3 2 1
2 l 1 1
2 1 1 1
1
3
4
l
!
1 I 1
1 2
1 1 1
2
2 2
9
2 1 2
1
l
2
2 i1
2
2
1
1 1
ll
4
1
4 3 5
9
58
1
1 1
3
1
15
10
4
n a m e Microsporum magellanicum a n d t h e n a m e Cunninghamella antarctica for t h e species i s o l a t e d f r o m O ' H i g g i n s soil.
Microsporum
magellanicum
sp. nov.
F u n g u s t e r r e s t r i s , m y c e l i o s e p t a t o , h y a l i n o , 2 - 4 # l a t o et r a m o s o ; r a m i s e c u n d a r i i flexuosi c u m spiris t e r m i n a l i b u s . Macroconidia p l u r i m a h y a l i n a , o v a t a , v e l c l a v a t a , r a r o c y l i n d r i c a , 1 - 2 septis p e r r a r o 4 - 6 septis, a m p l i t u d i n e 4 . 8 - 7 . 2 x 1 4 . 4 - 2 1 - 6 # c u m e p i s p o r i o v a l d e v e r -
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G. CARETTA AND E. PIONTELLI
rucoso aliquando ornato pedicellato, sed etiam macroconidia verrucolosa; ab hyphis et ramis lateralibus terminaliter aut lateraliter oriuntur et singula plerumque pedunculata sed etia m sessilia. Microconidia vera absunt; adsunt conidia piriformia aut clavata basi truncata, leviter verrucolosa, amplitudine 2"4- 3 x 4.8-5.8#, sessilia aut pedunculata, saepe uniseptata, quae macroconidia incipiente formatione sunt. Chlamidosporae rarae intercalares, singulae et acrogenae, rotundae vel ovatae, 6-8/~ diam. Colonia in agaro Sabouraudii 20°C celeriter crescens; post 12 dies 4 cm diametero, temperatura optima 25°C. Post 30 dies, 4°C, 0"5 cm diametero cum abundantibus macroconidiis. Non crescit ad t e m p e r a t u r a m 37°C. Aspectus subtiliter granulatus, pulverulentus, flos lactis flavescens; pars reversa flava, maturitate ochracea. Species keratinophila cum destructione pili et formatione organorum perforantium in trine. Isolatur ex terra australica Cilense, insula Desolacidn et insula Navarino. Typus conservatur in micoteca Cathedrae Micologiae Medicae, Universitatis Papiae, No. 1051; isotipus apud C.M.I. No. 195827 sub Microsporum verruculosum nomen nudum. Terrestrial fungus, septate mycelium, hyaline, 2-4 # large and branched; secondary branches flexuous with spiral coils. Abundant macroconidia, hyaline, ovate or clavate, rarely cylindrical, 1-2 septa, rarely 4-6 septa, measuring 4.8-7-2 x 14.4-21.6/~, verrucous and pedicellate walled; but also verrucolose. Solitary terminal and lateral macroconidia arising on lateral hyphae, often pedunculate but also sessile. T r u e microconidia absent; pyriform or clavate conidia present, with truncate base, lightly verrucoIose, 2-4-3 x 4.8-5.8 #, sessile or pedunculate, often with septa, that are young macroconidia. Rare intercalary solitary and terminal chlamydospores, spherical and ovate, 6-8/~ diam. The colony on Sabouraud's glucose agar (SGA) at 20°C attains a diameter of 4 cm in 12 days; optimum growth at 25°C; the colony at 4°C attains a diameter of 0.5 cm in 30 days, with numerous macroconidia. There is no growth at 37°C. T h e colony is finely granular, powdery, cream-yellowish; reverse yellow, becoming ochre at maturity. The fungus is keratinophilic with destruction of hair; perforating organs present. Microsporum magellanicurn has been isolated from australic soil of Chile, in the Desolacidn and Navarino Islands. The type is preserved in the collection of Medical Mycology of the University of Pavia, No. 1051; isotype deposed in the Commonwealth Mycological Institute (Kew), No. 195827 as Microsporum verruculosumnomen nudum.
Appearanceof Colony O n SGA the moderately fast growing colonies (40 m m in diameter after 12 days at 25°C), were cream-yellow and finely granular, flat with a central umbo (P1. 1, A). Light yellow-ochre reverse, buckthorn brown in the center. O n malt agar (MA) it grows to a diameter of 35 m m after 12 days, changing to finely granular with concentric rings. Reverse buff-yellow to honey yellow in the centre. The development on potato carrot agar is 3-5 cm in diameter after 12 days at 25°C; fluffy in the center
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MICROSP. MAGELLANICUM AND CUNNINGHAMELLA ANTARCTICA
Plate 1
A--~dicrosporum magellanicum ~)n S.G,A. after 14 days at 27°C. B--Macroconidia and spiral coils in lactofuchsin x 400. C--Perforating body in human hair, macroconidia X 400. D--Verrucose macroconidia x 1000. E--Verruculosity of typical monoseptate macroconidia and cylindrical macroconidia in Czapek Dox Agar. F--Echinulated young macroconidia in S.G.A. × 1000.
5
6
G. CARETTA AND E. PIONTELLI
and granular in the margin, with some zonate growth. Surface white to cream at the periphery; reverse buff-yellow. On Czapek agar growth is similar to that on potato carrot (PCA), with the production of abundant verrucose macroconidia and spiral coils. O n MA at 4°C the growth is very slow, attaining a diameter of 5 m m after 30 days, with a cottony colony.
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Microscopic Appearance The hyphae are septate, hyaline, branched, 2-4 # diam. Spiral coils have been observed (P1. 1,B). Arthrospores scanty on SGA and MA. Intercalary chlamydospores have been observed on Czapek and PCA, and occasionally on MA and SGA racquet hyphae are present. Macroconidia are produced abundantly; hyaline, mostly clavate, arising terminally or laterally from conidiophores mostly single (P1. 1, D). They are not very. thick-walled, markedly verrucose on Czapek and PCA, and verrucolose on SGA and MA. They are variable in length and in number of septa on the following cultural media: SGA macroconidia abundant, 4.8-7-2 x 1421-6 #, mostly verrucose, verrucolose, predominantly 1-2 septa; occasionally macroconidia up 30-40/~ with 4-5 septa. O n MA, abundant, 3-6-6 x 11-20 #, predominantly verrucolose with 1-3 septa. O n PCA and Czapek agar, macroconidia abundant, 4-7.2 x 14.4-19-5 ~, clearly verrucose, predominantly 1 septum (PI. 1, E). True microconidia are absent. Unicellular conidia are present, verruculose, thinwalled, subclavate and pyriform with truncate base (P1. 1, F). These are initial forms of macroconidia. In SGA, PCA, 1VIA and Czapek agar 2"4-3"4 x 4.8-7.2 #, on corn meal agar 2"4-3 x 4-6 p. O n human or horse hair the growth is luxuriant with production of macroconidia and perforating organs (P1.1, C). Cleistothecia were not observed.
Cunninghamella antarctica sp. nov. Colonia in agaro tubero solani dextrosi 25°C celeriter crescit post 6 dies ad 1-1.5 cm, hyphis assurgentibus, ebore flavescens, granulosa; pars reversa flavobubalina, tenuiter ochracea. Mycelium hyalinum, coenocyticum, pseudoseptatum, 16 # latum; rhizoidi et chlamidosporae adsunt; conidiofora erecta, 14/z lata, cum ramis verticillatis, pseudoverticillatis et sympodialibns, et vesiculis globosis terminalibus; w'siculae conidioforarum 30-40 p diam. cum parvis sterigmatibus; et conidiis globosis, 12.8-16/t, valde spinosis; vesiculae ramorum globosae, 16-24 # diam. cum conidiis globosis aut ovatis, spinosis, 9.6-13 # diam. Conidia magna 14-21 ~ diam. absunt. Fungus keratinophilus crescit 9-40°C; optimum 25°C. Isolatur ex terra antarctica, loeo nominato Base O'Higgins. Typus conservatur in micoteca Cathedrae Micologiae Medicae, Universitatis Papiae, No. 1060. Colonies on PDA at 25°C growing rapidly up to 1-1.5 cm high in 6 days, granulated in appearance, ivory-yellow, reverse cream-buff. Mycelium hyaline, finely granular and smooth walled, hyphae up to 16 # in diam.; rhizoids present; chlamydospores 1-2 # diam., conidiophores erect, up to 14 # in diam., being usually verticillately (P1.2, A), pseudoverticillately and sympodially branched (PI. 2; B, D). Conidiophores and lateral branches have terminal vesicles (Pl. 2, E) ; these are usually spherical, average 30-40 # in diam. with minute sterigmate-bearing globose conidia
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MICROSP. MAGELLANICU~tI A N D CUNNINGHAMELLA ANTARCTICA
Plate 2
A--Cunninghamella antarctica in surface of P.D.A. × 160. B - D - - S y m p o d i a l ramifications on hair baiting, and M.A. × 100. C--Conidiophores and conidia in P.D.A. × 400. E - - T y p e s of conidiophores on hair baiting × 100. F--Cunninghamella echinulata, types of eonidiophores × 100.
7
8
G. CARETTA AND E. PIONTELLI
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average 12-8-16 # in diam. covered with true spines, 4-6 # in length (PI. 3; A, C); lateral verticillated branches, in number up to 6, have spherical or subspherical vesicles average 16-24 # in diam., with globose or ovoidal conidia, 9-6-13 #, with true spines, spinescent giant conidia are present (PI. 3, B), 14-21/~ diam. Growth appears at 9°C and 40°C, optimum 25°C. O n MA good growth similar to that on PDA; on corn meal agar growth is poor. Development of the hair bait appears in 3 days at 22°C. Zygospores not formed. Isolated from antarctic Continent soil, O'Higgins base. The type is preserved in the collection of Medical Mycology of the University of Pavia, No. 1060. DiscussioN
Among the 15 species of Microsporum considered to be valid members of the genus (1), M. mageIlanicum shows some similarity to M. nanum Fuentes. Microsporum nanum presents small, 2 or 3 celled macroconidia; the wall is finely verrucose or occasionally smooth. Microsporum mageIlanicum has verrucose or verruculose clavate macroconidia and true microconidia are absent. It does not produce infections in h u m a n and animal skin.
Plate 3
A--Cunninghamella antarctica,normal conidia in P.C.A. × 400, direct view. B--Giant conidia in P.D.A. × 400, C--Conidia in lactofuehsin ×400.
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MICROSP. MAGELLANICUM AND CUNNINGHAMELLA ANTARCTICA
9
Microsporium ripariae (5) has verruculose a n d verrucose m a c r o c o n i d i a , b u t some o f t h e m are smooth, a n d relatively v a r i a b l e in shape, fusiform or c y l i n d r i c a l ; t h e y h a v e 3-6 septa a n d m e a n length 36.5/~, a n d a m e a n w i d t h 9.33 #; it grows well a t 37°C on feathers. Microsporum magellanicum does not g r o w a b o v e 35°C b u t there is some g r o w t h w i t h p r o d u c t i o n o f a b u n d a n t m a c r o c o n i d i a at 4°C. I t grows p o o r l y o n feathers, r a r e l y fructifying. T h e m e a n length o f m a c r o c o n i d i a o f M. mageltanicum is 4-8-7.2 x 14-421.6 # w i t h dense verruculose surface, usually uniseptate, r a r e l y to 4 - 5 septate. Those w i t h m o r e t h a n 4 septa a r e present in S G A a n d 1VIA cultures. O n horse a n d h u m a n h a i r culture, t h e y are p r e v a l e n t l y uniseptate. O n these substrates spiral coils a n d p e r f o r a t i n g organs a r e present. T h e confused s y n o n y m y a n d o v e r l a p p i n g specific descriptions (2, 4, 3, 6) m a d e it difficult to identify Cunninghamella antarctica. S a m s o n (8) presented the results of a revision of the Cunni@amella strains in the collection o f the C e n t r a a l b u r e a u voor S c h i m m e l c u l t u r e s ; this w o r k contains a k e y description a n d illustrations of cultures g r o w n at 25°C on b e e r w o r t - a g a r . Cunninghamella antarctica is similar to C. phaeospora a n d C. elegans. Cunninghamella antarctica differs from C. phaeospora b y h a v i n g very spiny a n d h y a l i n e conidia. I t is also different from C. echinulata owing to the spinosity o f conidia, the true s y m p o d i a l r a m i f i c a t i o n a n d characters of colony. I t shows a g r e a t affinity w i t h C. elegans b u t the c o n i d i a of the l a t t e r are smooth, verruculose or shortly echinulate. Considering the easy p o l y m o r p h i s m of these fungi, the v a r i a b l e synonymies proposed b y various workers a n d the specific c h a r a c t e r s which our isolate p r e s e n t e d , we think it a d v i s a b l e to create a new species. Cunninghametla echinutata ( T h a x t e r ) , was isolated from soil samples collected from South S h e t l a n d Islands a n d A n t a r c t i c c o n t i n e n t (zone 3, 4, 6 o f the Figure). i t grows well at 9°C a n d 40°C; shows g o o d d e v e l o p m e n t at 25°C. T h e r e were no significant m o r p h o l o g i c a l differences on SDA, P D A , P C A m e d i a . I n 6 days, there was r a p i d growth u p 2-2-5 c m high, at first white, l a t e r p a l e olive buff, floccose. T h e conidiophores, l a t e r a l verticillate or pseudo-verticillate b r a n c h i n g , vesicles globose to subglobose, c o n i d i a e c h i n u l a t e d , g i a n t c o n i d i a (P1.2,F) c o r r e s p o n d to the description a n d figures given b y S a m s o n (8). Zygospores were n o t f o r m e d b y this strain. T h e other species of k e r a t i n o p h i l i c fungi isolated from the different samples, Ch. indicum, Ch. keratin@hilum are w o r t h special a t t e n t i o n , since t h e y h a v e n o t y e t been isolated from the a n t a r c t i c region. RIASSUNTO Vengono presentati i funghi cheratinofili isolati da eampioni di terra raecolti nell'estremit~ Sud de Cile, helle isole Shetland del Sud e nel continente Antartico. Sono descritte due nuove specie, Microsporumm~gellanicum e Cunninghamdlaantarctica. Microsporum magellanicum ~ caratterizzato da macroconidi clavati, verrucosi o verrucolosi, prevalentemente unisettati, 4,8-7,2/t × 14,4-21,6/t. Cunninghamellaantarctica ~ caratterizzata da conidiofori generalmente verticillati, pseudoverticiilati e a ramificazioni simpodiali, conidi globosi con vere spine, t2,8-16 It in diam. ACKNOWLEDGEMENTS
The authors wish to thank Drs. R. A. Samson, G. A. De Vries and P. M. Stockdale fur the examination of these strains and for helpful suggestions and criticisms; the Navy of Chile for facilities to take the samples; and Mr. L. Tarifefio who took the soil samples.
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G. CARETTA AND E. PIONTELLI
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REFERENCES 1. AJELLO, L. (1974). Natural history of the dermatophytes and related fungi. Mycopathologia e Mycologia Applicata, 53, 93-110. 2. ALCORN,G. D. & YEAGER,C. C. (1938). A monograph of the genus Cunninghametlawith additional descriptions of several common species. Myeologia, 30, 653-658. 3. BOEmJN, K. B. (1958). Notes on the Mucorales of Indonesia. Sydowia, 12, 321-362. 4. CUTTEr, Jr. V. M. (1946). The genus Cunninghamella (Mucorales). Farlowia, 2, 321-343. 5. HUBALEg, Z. & RUsH-MuNRo, F. M. (1973). A dermatophyte from birds: Microsporum ripariae sp. nov. Sabouraudia, 11, 287-292. 6. MIL'KO, A. A. & BELYAKOVA,L. A. (1967). The genus Cunninghamella Matruchot and taxonomy of the Cunninghamellaceae. Mikrobiologiya, 36, 684-699. 7. ORR, G. F. (1969). Keratinophilic fungi isolated from soils by a modified hair bait technique. Sabouraudia, 7, 129-134. 8. SAMSON,R. A. (1969). Revision of the genus Cunninghamella (Fungi, Mueorales). ProceedingKoninkl. Neederl. Akademie van Wetenschappen--Amsterdam, series C, 72, 322-335. 9. VANBREUSEG~EM,R. (1952). Technique biologique pour l'isol~ment des dermatophytes du sol. Annales de la Socidtd Belge de Mgdecine Trop#aI, 30, 173-178.
