Podospora Lautarea sp. nov. from Southern Alps (France): description and physiological properties

Anwnie van Leeuwenhoek 66:351-355, 1994. (g) 1994KluwerAcademicPublishers. Printedin the Netherlands.

351

Podospora Lautarea sp. nov. from Southern Alps (France): description and physiological properties Pascale Guiraud, Lucile Sage, Fran~oise Seigle-Murandi & Rdgine Steiman Groupe pour l'Etude du devenir des Xdnobiotiques dans l'Environnement (GEDEXE), Universitd J. Fourier, UFR Pharmacie, BP 138, Avenue de Verdun, 38043 Meylan cddex, France Accepted 13 May 1994

Key words: Podospora Lautarea sp. nov., Southern French Alps

Abstract A taxonomic description of Podospora Lautarea sp. nov. is provided. This species is characterized by a reddish brown peridium, and by its large, asymmetric ascospores, small, hyaline, unique primary appendage, absence of secondary appendage and cylindrical asci. Due to the size of its appendage, this species may be related to Podospora minicaudaFaurel et Locquin-Linard. Asci and ascospores are close to those of P. fimbriata (Bayer) Cain, but the dimensions and ornamentation of perithecia are quite different. To summarize, this species belongs to the small group of Podospora exhibiting only one appendage (such as P. minicauda, P. carpinicola Mouchacca or P. horridula (Sacc.) Francis and Sparrow) but can not be assimilated to one of the described species in this group. The main cultural characteristics and physiological properties of this species are described.

Abbreviations: CMPG - Collection Mycologie Pharmacie Grenoble, MEA - Malt Extract Agar, PDA - Potato Dextrose Agar, POx - Phenoloxidases

Introduction During the determination of the populations in soil samples from an especially cold place in the area of the Col of Lautaret (France), an unusual Podospora species was isolated. Since this species was not described in reviews concerning the genus Podospora (Cain 1956; Cain 1962; Cailleux 1969; Mirza & Cain 1969; Malloch & Cain 1971a, 1971b; Furuya & Udagawa 1972; Lundqvist 1972; Malloch & Benny 1973; Garcia-Zorron 1977; Locquin-Linard 1978; Mouchacca 1986; Sultana 1987; Krug & Khan 1989; Rao & Mani Varghese 1989; Guarro et al. 1991; Khan & Krug 1991) it was considered as a new taxon. Our purpose was to describe the new species and to give some of its physiological properties.

Methods All assays were repeated 4 times.

Strain isolation Soil samples were collected in Bois de Prmrant, near the Col of Lautaret in the Southern French Alps. Isolation of the strains was accomplished by using the soil plate method of Warcup (Parkinson & Waid 1960): the soil sample was placed into Petri dishes (90 mm diameter) and sterile malt extract (MEA) (malt extract, 1.5%; agar, 1.5%; chloramphenicol, 0.05%) medium was poured over it. After solidification, dishes were incubated at 220 C and strains were isolated as soon as they appeared. Cultural conditions Podospora sp. was grown on MEA and PDA (potato dextrose agar) at 22 and 30 ° C for 10 days. Growth was followed up by measuring the diameter of colonies every day. Enzyme assays The methods used for the detection of lipase, protease and amylase production have been described by

352 Hankin & Anagnostakis (1975) and were previously employed to characterize a new species of Embellisia (De Hoog et al. 1985). Cellulases were detected according to Smith (1977); the same method was adapted to the detection of amylolytic activity according to Rinderknecht et al. (1967). The results of these assays were only qualitative. Extracellular phenoloxidases (POx) production was investigated on PDA and MEA at 220 C as previously described (Guiraud et al. 1992). Ten reagents were used and following their intensity, the colored reactions observed were noted from 0 to 4. Results were given as a POx index corresponding to the sum of the different reactions obtained. These assays were performed with 10 to 15 day-old cultures.

Antimicrobial activity The strain was grown in different liquid media. For antibacterial and anti-human pathogenic fungi assays, the fungus (mycelium + spores) was inoculated in 250 ml Erlenmeyer flasks, containing 70 ml of malt extract (1.5%) and grown under shaking or static conditions at 22 o C for 8 days. For anti-phytopathogens and anti-entomopathogens assays, cultures run in yeast extract (2%)-saccharose (4%) pH 6.5 medium, under static conditions for 5 days (Pujol et al. 1990). Media were separated from the mycelia by filtration and extracted by 2 x 35 ml ethyl acetate. Organic phases were pooled and dried at 400 C under reduced pressure. Crude extracts were dissolved in 3 ml ethyl acetate. Target pathogenic strains were 5 bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus penicillin resistant, S. aureus penicillin sensitive, Streptococcus faecalis), 7 yeasts (Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. tropicalis amphotericin resistant, Cryptococcus neoformans 1, C. neoformans 2), 8 Dermatophytes (Epidermophyton floccosum, Microsporum canis, M. Gypseum, Trichophyton mentagrophytes 1, T. mentagrophytes 2, 7:. mentagrophytes var. interdigitale, T. rubrum, T. tonsurans), 5 phytopathogens ( Colletotrichum musae, Drechslera spicifera, Fusarium oxysporum, Geotrichum candidum, Pyricularia oryzae), 4 entomopathogens (Beauveria bassiana, B. brongniartii, Metarrhizium anisopliae, Verticillium lecanii). Assessment of antimicrobial activity of crude extracts was performed using the disk diffusion method previously described (Okeke et al. 1992).

Polysaccharide production Presence of extracellular polysaccharide in culture medium was investigated at 220 C according to Aouadi et al. (1992). The strain was grown in malt extract (1.5%) liquid media under static and shaking conditions for 8 days.

Results and discussion

Podospora Lautarea Guiraud, Sage, Seigle-Murandi and Steiman sp. nov. (Fig. 1A, B, C, D) Coloniae in agaro-malto dicto 220 C post 7 dies 6062 mm diametrum, crassae, subnigrae, inversa obscure brunneus, maturitatis serae peritheciis, forma conidialis absens. Peritheciis superficialibus, piriformibus, obscure brunneis, 600-800 x 300-450 #m, collo 150--200 x 250-300 #m; vestitis omnino cure pilis comatis praecipus circa ostiolum (aliquando peritheciis glabrae). Pili ascocarparum olivaceobrunnei, 170300 x 2-3 #m, ad recti subcurvati, cum cellula hyalina apicali rotunda. Ascis octosporis cylindraceis, 170220 x 15-17 #m, cum elongatus basis (20-30 #m), annulo apicali distincto. Paraphysiae hyalinae septatae. Ascosporis uniseriatis, ellipsoideis, levis, unilateraliter applanateis, obscure brunneis maturitate, 22-25 x 1517 #m, primaria appendice hyalina3-4 x 4 #m, secundaria appendice non visa. Holotypus: ex terra sub Pinus uncinata Ramond separatus, silva P6m6ant, Col du Lautaret, France, 22 Sept. 1993, CMPG 1203. In fungi herbarium, Grenoble, France. Colonies on MEA at 220 C attaining 60-62 mm in 7 days, thick, dark greyish with a brownish tinge, reverse dark brown, perithecia late developing, conidial structures not observed. Perithecia superficial, scattered, pyriforme, 600-800 x 300-450 #m, dark brown to black, covered on the exposed part with long septate olivaceous brown hairs, straight or slightly flexuous at the top, 170-300 x 2-3 #m, with rounded subhyaline tip, or sommetimes perithecia nearly glabrous. Peridium thick, dark reddish brown. Neck papillate, usually 150-200 x 250-300 #m. Asci 8-spored, cylindric, 170-220 x 15-17 #m, with evident ring-like thickening at the apex, tapering below into a slender stipe, 2030 #m long. Paraphyses hyaline, thread-like, septate. Ascospores obliquely uniseriate, hyaline, hardly guttulate and clavate when young, becoming dark brown-

353 B

A Q

C

D

Fig. 1, PodosporaLautarea. (A), Perithecium, 60 x ; (B), Perithecium releasing ascospores, 60 x ; (C), Asci, 300 x ; (D), Asci and ascospores, 600 ×,

354 Table 1. Comparison between Podospora Lautarea sp. nov. and related species, according to literature data (dimensions are in/~m).

Perithecia Neck Hairs Asci Ascospores Primary appendage Conidialstage Origin

Podospora Lautarea

Podospora fimbriata

Podosporaminicauda

600-800 × 300-450 150-200 × 250-300 170-300 × 2-3 Sometimes absent 8-spored 170-220 × 15-17 22-25 × 15-17

350-550 × 200-240 Up to 80 in length Up to 60 in length

250-500 x 140-250 Not reported Absent

8-spored 110-150 x 9-10 18-20 x 8-9.5

8-spored 110-120 x 9-11 (11) 12-14(15)

3-4 × 4

4-6 × 2

× (6) 7 (8) 3-5 × 1.5-2

Absent Soil underPinus

Present Goat dung Japan

Absent Gazelle dung No~hAffica

uncinata South France

ish black at maturity, inequilaterally ellipsoid, flattened on one side, 22-25 x 15-17 #m, more or less apiculate at both ends, primary appendage hyaline, reduced to a small apiculum, 3-4 x 4 #m attached to the base of spore, secondary appendage not observed. Primary appendage generally oriented to the stipe of the ascus. Holotype: from pine forest soil, Bois de P6m6ant, Col du Lautaret, France, 22 Sept., 1993; CMPG 1203. Subcultures of CMPG 1203 have been deposited with the American Type Culture Collection (ATCC). This species is characterized by its large, asymmetric ascospores, small, hyaline, unique primary appendage, absence of secondary appendage, cylindrical asci and a dark reddish brown peridium. Due to the size of its appendage, this species may be related to Podospora minicauda Faurel et Locquin-Linard (Locquin-Linard 1978). Asci and ascospores are close to those of P. fimbriata (Bayer) Cain, as described by Furuya and Udagawa (1972), but the dimensions and ornamentation of perithecia are quite different (Table 1). To summarize, this species belongs to the small group of Podospora exhibiting only one appendage (Locquin-Linard 1978) but can not be assimilated to one of the described species in this group. To our knowledge no report of new species in this group has been published since 1986 (P. carpinicola Mouchacca).

The main plant present in the forest of P6m6ant was Pinus uncinata Ramond, but other species were also abundant and linked to cold climate and sometimes to

the chalky nature of the soil (*): Bellidiastrum michelii* Cass, Calamagrostis villosa (Chaix) Gmel., Carex ferruginea* Scop. ssp. Tendae W. Dietrich, Deschampsia flexuosa (L.) Nees, Homogyne alpina Cass, Luzula maxima DC, Rhododendron ferrugineum L., Sesleria leucocephala* DC, Sideritis scardica Griseb., Vaccinium myrtiIlus L., Vaccinium vitis-idaea L., Valeriana montana* L. Details concerning the other fungi found in this peculiar region will be the object of another paper. It must be emphasized that the present Podospora was found in soil under Pinus uncinata, but it was not clearly shown to be linked to this plant. On the other hand, the new species seemed not linked to animal dung, although the majority of Podospora spp. are usually coprophilous species whose distribution is relatively unaffected by the type of vegetation. Some of previously described species have been found in soil or plant debris, for instance Podospora carpinicola, isolated from dead leaves of Carpinus (Mouchacca 1986), P. austroamericana (Speg.) Mirza and Cain, P. fimbriata, P. horridula (Sacc.) Francis and Sparrow and P. inaequalis (Cain) Cain, which constitute a group of species very close to Zopfiella (Guarro et al. 1991). The new isolated species may be possibly included in this group. Apart from their origin, one of the most important feature in these species is the lack of gelatinous appendages which are considered as a particularity of coprophilous Podospora. The name of Podospora Lautarea is proposed for the described species, owing to the origin of the soil

355 s a m p l e in w h i c h it was found. O n l y one strain o f P

Lautarea was isolated, it is maintained in the collection o f our laboratory ( C o l l e c t i o n M y c o l o g i e Pharmacie G r e n o b l e ; C M P G 1203). T w o culture m e d i a (PDA, M E A ) and two temperatures (220 C, 300 C) o f growth w e r e tested. O n l y m i n o r differences w e r e o b s e r v e d in both comparisons. G r o w t h rate was a little decreased at 30 o C since the dishes w e r e totally covered after 10 days against 9 days at 22 ° C. This last observation is not surprising since the strain was isolated f r o m a b r o w n l i m e s t o n e soil in a c o l d region at an altitude o f 2 0 2 0 meters. P Lautarea was not found to be c e l l u l o l y t i c or proteolytic, but was strongly lipolytic and arnylolytic. Phen o l o x i d a s e activity d e p e n d e d on the culture m e d i u m . On P D A , a reaction was o b s e r v e d with most o f the reagents (benzidine, guaiacol, gallic acid, pyrogallol, R 5 6 and c~-naphtol; P O x index = I0) but no tyrosinase activity was detected. O n M E A , o n l y guaiacol gave p o s i t i v e results ( P O x index = 2). T h e s e results indicate that p h e n o l o x i d a s e s are i n d u c i b l e in this strain. N o p r o d u c t i o n o f polysaccharides was obtained in our e x p e r i m e n t a l conditions. Organic extracts o f culture m e d i a o f P lautarea w e r e not toxic towards the various p a t h o g e n i c m i c r o o r g a n i s m s tested. These p h y s i o l o g i c a l properties are often intraspecific rather than intrageneric; for example, a species o f Podospora (P. appendiculata) has recently been shown to prod u c e three new antifungal c o m p o u n d s (Wang et al. 1993).

R e f e r e n c e s

Aouadi S, Heyraud A, Seigle-Murandi F, Steiman R & Foumet B (1992) Structural analysis and rheological behaviour of an extracellular polysaccharide from Drechslera spici[era. Carbohyd. Polym. 17:177-183 Cailleux R (1969) Champignons stercoraux de R6publique Centrafricaine. III - Podospora nouveaux. Cah. Mabok6 7:87-102 Cain RF (1956) Studies of coprophilous ascomycetes. II. Phaeotrichum, a new cleistocarpous genus in a new family, and its relationships. Can. J. Bot. 34:675-687 - (1962) Studies of coprophilous ascomycetes VIII. New species of Podospora. Can. J. Bot. 40:447-490 De Hoog GS, Seigle-Murandi E Steiman R & Eriksson KE (1985) A new species of Embellisia from the North Sea. Antonie van Leeuwenhoek 51: 409-413

Furuya K & Udagawa SI (1972) Coprophilous pyrenomycetes from Japan I. J. Gen. Appl. Microbiol. 18:433-454 Garcia-Zorron N (1977) Una nueva especie de Podospora (Sphaeriales/Ascomycetes). Bol. Soc. Arg. Bot. XVIII: 173-175 Guarro J, Cannon PF & van der Aa HA (1991) A synopsis of the genus Zopfiella (Ascomycetes, Lasiosphaeriaceae). Systema Ascomycetum 10:79-112 Guirand P, Seigle-Murandi E Steiman R & Benoit-Guyod JL (1992) Extracellular phenoloxidases activity of micromycetes from various taxonomic groups. Microbiologica 15:367-390 Hankin L & An agnostakis SL (1975) The use of solid media for the detection of enzyme production by fungi. Mycologia 67:597-607 Khan RS & Krug JC (1991 ) Podosporafibrinocaudata, a new species from California. Mycologia 83:817-821 Krug JC & Khan RS (1989) New records and new species of Podospora from East Africa. Can. J. Bot 67:1174-1182 Locquin-Linard M (1978) Un nouvel ascomycete coprophile de la famille des Sordariaceae: Podospora minicauda Faurel et Locquin-Linard. Rev. Mycol. 42:341-345 Lundqvist N (1972) Nordic Sordariaceae s. lat. Symb. Bot. Upsal. 20:1-374 Malloch D & Benny GL (1973) California ascomycetes: four new species and a new record. Mycologia 65:648-660 Malloch D & Cain RF (1971a) Four new genera of cleistothecial ascomycetes with hyaline ascospores. Can. J. Bot. 49:847-854 -(1971b) New cleistothecial Sordariaceae and a new family, Coniochaetaceae. Can. J. Bot. 49:869-880 Mirza JH & Cain RF (1969) Revision of the genus Podospora. Can. J. Bot. 47:1999-2048 Mouchacca J (1986) Podospora carpinicola spec. nov., un ascomycete isol6 de feuilles mortes de Carpinus, et deux esp6ces du m~me genre. Persoonia 13:107-112 Okeke B, Steiman R, Seigle-Murandi F & Benoit-Guyod JL (1992) Assessment of microbial metabolites active against Pyricularia oryzae using microtitration and disk diffusion methods. Proc. Intern. Syrup. Environ. Aspects Pestic. Microbiol. August 17-21, Sigtuna, Sweden, pp 332-337 Parkinson D & Waid JS (Eds) (1960) The ecology of soil fungi. Liverpool University Press, England Pujol V, Seux V & Villard J (1990) Recherche de substances antifongiques s6cr6t6es par les champignons sup6rieurs en culture. Ann. Pharm. Fr. 48:17-22 Rao VG & Mani Varghese KI (1989) Forest micro-fungi-VII. Two new taxa of ascomycetes from India. Int. J. Mycol. Lichenol. 4: 155-159 Rinderknecht H, Wilding P & Haverback BJ (1967) A new method for the determination of a-amylase. Experientia 23:805 Smith RE (1977) Rapid tube test for detecting fungal cellulase production. Appl. Environ. Microbiol. 33:980-981 Sultana N (1987) Some Pyrenomycetous fungi from Lahore. Biologin 33:265-270 Wang Y, Gloer JB, Scott JA & Malloch D (1993) Appenolides A-C: Three new antifungal furanones from the coprophilous fungus Podospora appendiculata. J. Nat. Prod. 56:341-344