System. Appl. Microbiol. 17, 620-624 (1994) © Gustav Fischer Verlag, Stuttgart · Jena . New York

New Strains from Israel in the Aspergillus niger Group

REGINE STEIMAN, PASCALE GUIRAUD, LUCILE SAGE and FRANCOISE SEIGLE-MURANDI*

Groupe pour I'Etude du devenir des Xenobiotiques dans l'Environnement (GEDEXE), Universite J. Fourier, UFR Pharmacie, BP 138; Avenue de Verdun, 38043 Meylan cedex, France. Fax: (33 ) 76418571

Received June 6, 1994

Summary

In the course of a survey of the mycoflora of the Dead Sea area, rwo new species of Aspergillus have been isolated. The species are considered as members of the niger group, but did not correspond to any other described in this group up to now. A description and some physiological properties of these species, named Aspergillus homomorphus and Aspergillus pseudo-heteromorphus are reported.

Key words: Aspergillus homomorphus - Aspergillus pseudo-heteromorphus - Aspergillus niger group -Dead Sea - Aspergillus helicothrix - Aspergillus heteromorphus

Introduction

During investigations of mycoflora from soil samples of Dead Sea area, two new species of Aspergillus were iso­lated. These strains have been included into the Aspergil­lus niger group. They resembled mostly Aspergillus heteromorphus but differed sufficiently from it to justify new taxa. To our knowledge, no report exists concerning such species neither in the general literature concerning the niger group (Raper and Fennel, 1973; Samson, 1979; Al­Musallam, 1980) nor in previous reviews about the myco­flora of South Israel (Rayss, 1950; Rayss and Borut, 1958; Borut, 1960). The species were named Aspergillus homomorphus and Aspergillus pseudo-heteromorphus, their description and some of their physiological proper­ties are provided.

Materials and Methods

Strain isolation. The soil sample was collected in Israel, 2 kilometers away from the Dead Sea. The isolation of the strains was accomplished by using the soil plates method of Warcup (Parkinson and Waid, 1960): the soil sample was dispatched into Petri dishes (90 mm diameter) and sterile malt extract (1.5%)­agar (1.5%) with chloramphenicol (0.05 %) (MEA) was poured over. After solidification, dishes were incubated at 22 °C and strains were isolated as soon as they appeared.

Soil analysis. 1 g of dry soil was suspended in 5 ml of distilled

* Corresponding author

water by vigourous shaking. After centrifugation (5 min. at 3000 g) andlor filtration, pH of the supernatant was measured and the ionogram (Na+, K+, Cl-, Ca2 +, Mg2+) was established with an automated analyzer BMIHitachi 717 (Boehringer Mannheim, France) using the methods adapted by Boehringer Mannheim. Results were expressed as mmol/g of dry soil.

Cultural needs. The species were grown at 22 °C for 14 days on Czapek agar (1.5%) (Smith, 1949) and malt extract agar (MEA).

Enzyme assays. The methods used for the detection of lipase, protease and amylase production have been described by Hankin and Anagnostakis (1975) and were previously employed in the laboratory to characterize other new species (De Hoog et aI., 1985). Cellulases were detected according to Smith (1977); the same method was adapted to the detection of amylolytic activity according to Rinderknecht et at. (1967) . Results of these assays were only qualitative. Extracellular phenoloxidases (POx) pro­duction was researched on MEA as previously described (Guiraud et aI., 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 at 2ZOC on 10 to 15 day-old cultures.

Antimicrobial activity. For antibacterial and anti human pathogens assays, the fungi (mycelium + spores) were inoculated in 250 ml Erlenmeyer flasks, containing 70 ml of malt extract (1.5%) medium and grown under shaking or static conditions at 22 °C for 8 days. For anti phytopathogens and anti entomopatho­gens assays, cultures run in yeast extract (2°/o)-saccharose (4%) medium (pH 6.5 ), under static conditions for 5 days (previous experiments showed that these conditions allowed optimal activi­ty) (Pujol et aI., 1990). Media were separated from the mycelia by

filtration and extracted with 2 x 35 ml ethyl acetate. Organic phases were pooled and dried at 40°C under reduced pressure. Crude extracts were dissolved in 3 ml ethyl acetate. Target pathogenic strains were 5 bacteria strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus penicillin resistant, S. aureus penicillin sensitive, Streptococcus faecalis ), 7 yeasts strains (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, T. 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 aI., 1992).

Polysaccharide production. Presence of extracellular polysac­charide in culture medium was researched at 25°C according to Aouadi et al. (1992). The strains were grown in liquid malt ex­tract medium under static and shaking conditions for 7 days. If present, precipitation of the polysaccharide was obtained by ad­ding 2% NaCI 5M and 1.5 volume of absolute ethanol to the filtered culture medium.

All assays were repeated four times.

Results and Discussion

Aspergillus homomorphus sp. nov. and Aspergillus pseudo-heteromorphus sp. nov., collected August 1992, are deposited in the Collection Mycologie Pharmacie Gre­noble (collection of our laboratory).

Table 1. Comparison Aspergillus between the new ISO-

lated Aspergilli and A. helicothrix

helicothrix or A. hete-romorphus.

Growth (Czapek, mm)

7 days 20 14 days 42

Reverse Greyish green to black

Conidial head Blackish brown to carbon black

Conidiophore 430-900 (!-tm ) x 8.5-13 .5

Vesicle (!-tm) 38-60

Metulae (!-tm) 20-40 x 7-11

Phialides (!-tm) 8.5-12 x 5-7

Conidia (et> !-tm) 6.5-8 echinate

Sclerotia + Heteromorphism

New Aspergillus in the niger group 621

Cultural and morphological characteristics were estab­lished from colonies grown on Czapek-Dox and MEA media. On Czapek medium, the strains were compared to Aspergillus helicothrix and Aspergillus heteromorphus (Table 1).

Description of Aspergillus homomorphus (Fig. 1 a, b, c, d)

Aspergillus homomorphus, Steiman, Guiraud, Sage and Seigle-Murandi, sp. nov.

In agaro Czapek coloniae veteres unius hebdomadis 40 mm diam; mycelium inconspicuum, album, tenue; conidiophora 700-900 x 12-14.5 (18) !-lm, parietibus laevibus; vesicula 50-65 !-lm; metulae 8-10 x 3 !-lm; phialides 1-2 per metula 6-7 x 2.5-3.5 !-lm; conidia spherica 5-7 !-tm diam, echinulata, portatis in column is longis; capita nigra, globosa vel radiata 200-250 !-lm diam.

Colonies on Czapek agar attaining 4 cm diameter in 7 days, 5 cm in 14 days, carbon black (5H2, Kornerup and Wanscher, 1978), plane or slightly wrinkled in the center, submerged mycelium thin covered with a white felt, re­verse pale greenish grey becoming greyish buff, clear ex­udate produced centrally. Conidial heads carbon black, globose to radiate, splitting into defined columns, 200 to 250 [lm diameter at 7 days. Conidiophore stalks smooth and thick walled, sinuous and reddish brown pigmented over most of the length, 700-900 [lm long, 12-14.5 (18) [lm wide. Vesicles brown, globose to subglobose, 50-65 [lm, bearing densely crowded metulae over the entire sur­face. Metulae hyaline, 8-10 x 3 [lm. Phialides erratically

Aspergillus Aspergillus Aspergillus heteromorphus homomorphus pseudo-

heteromorphus

20 40 45 35 50 50

Uncolored Greenish grey Pale greyish buff to dull green to greyish buff

Greenish olive Carbon black Carbon black with to greyish brown slightly olive tinge

(500)-980-1000 700-900 up to 1500 x 12-18.5 x 12-14.5-(18) x 12-16-(25)

48-80 50-65 40-60

12-16 x 4.5-5.5 8-10 x 3 10-13 x 5

8-10 x 3-4 6-7 x 2.5-3.5 7-10 x 3.5-4 1-2 per metula 1-2 per metula

3.5-5 5-7 5-6 echinate echinate echinate ( occasionally (spines up to (spines up to smooth) 1 !-tm) 1.5 !-tm) or smooth

+ +

622 R. Steiman et al.

,

• : I

•

•

A

I ..... "1-

Fig. 1. Aspergillus homomorphus (a) Conidial heads, 60 x. (b) Conidial head, 600 x. (c) Metulae, phialides and conidia, 600 x. (d) Conidia, 1500 x.

produced only 1 or 2 per metula, hyaline, flask-shaped, 6-7 x 2.5-3.5 !lm. Conidia dark brown, conspicuously echinate, globose, 5-7 !lm diameter, with spines up to 1 !lm.

Colonies on MEA attaining 2.5 cm diameter in 7 days, 4 cm in 14 days, basal mycelium hyaline, thin, submerged. Conidial heads blackish brown to black, radiate, splitting into 2-4 well defined divergent columns, 360 x 120 !lm. Conidiophore stalks 400-800 !lm long, 15-17 !lm wide, thick walled. Vesicles 40-65 !lm diameter. Metulae 7 x 5 !-lm. Phialides 6 x 3 !-lm.

Description of Aspergillus pseudo-heteromorphus (Fig. 2 a, b, c, d)

Aspergillus pseudo-heteromorphus, Steiman, Guiraud, Sage and Seigle-Murandi, sp. nov.

In agaro Czapek coloniae veteres unius hebdomadis 45 mm diam, sulcatae; mycelium inconspicuum, album vel eburneum, tenue; conidiophora usque 1500 x 12-16 (25) ~m, parietibus laevibus; vesicula 40-60 ~m, multae vesiculae portatis ex metu­les; metulae 10-13 x 5 ~m; phialides 1-2 per metulae 7-10 x 3.5-4 ~m; conidia spheric a 5-6 ~m diam, echinulata (spina us­que 1.5 ~m), portatis in columnis longis; capita nigra, globosa vel radiata 150-200 ~m diam.

Colonies on Czapek agar attaining 4.5 cm diameter in 7 days,S cm in 14 days, carbon black (5H2, Kornerup and Wanscher, 1978), wrinkled, submerged mycelium thin covered with a yellow whitish felt, reverse pale greyish buff, exudate produced as colorless droplets. Conidial heads carbon black with slightly olive tinge, globose, 150-200 !-lm diameter at 7 days, numerous aberrant forms present consisting of metulae ended by vesicles bearing phialides and conidia. Conidiophore stalks slightly undu­late, pigmented at the level of the vesicle, up to 1500 !-lm long, 12-16 !lm wide, larger at the base (25 !-lm). Vesicles globose, pitted when metulae are detached, 40-60 !-lm diameter, bearing densely crowded metulae over the entire surface. Metulae hyaline 10-13 X 5 !-lm. Phialides errati­cally produced, only 1 or 2 per metulae, 7-10 x 3.4-4 !-lm, sometimes 3 stages. Conidia dark brown, conspicu­ously echinate, 5-6 !-lm diameter, spines up to 1.5 !-lm, sometimes conidia hyaline, smooth,S .5-6 !-lm diameter on the same head.

Colonies on MEA attaining 2.5 cm diameter in 7 days, 4 cm in 14 days, mycelium hyaline, thin submerged, reverse greenish grey becoming salmon. Conidial heads blackish brown, radiate, becoming columnar, 600 X 220 !-lm. Con­idiophore stalks reddish brown at the apex, up to 1500 !-lm

New Aspergillus in the niger group 623

B

o

Fig. 2. Aspergillus pseudo-heteromorphus (a) Conidial heads, 150 x. (b) Metulae, phialides and conidia, 600 x. (c) Metulae, phialides and conidia, 1500 x. (d) Conidia, 1500 x.

diameter. Vesicle reddish brown, 34-42 !lm diameter. Metulae slightly pigmented, 8-10 x 5 !lm. Phialides 7 x 4!-tm.

The species described were both closely related to As­pergillus heteromorphus and only punctually to A. helicothrix, however none of them exhibited the olive green color becoming greyish brown typical of the young colonies of A. heteromorphus mentionned by Raper and Fennel (1973) (Table 1). Heteromorphism reported by Al­Musallam (1980) in A. heteromorphus was also found in one of the new Aspergilli. Numerous metulae ended by a vesicle bearing phialides and conidia were systematically observed. Conidia exhibited the same spaced and promi­nent spines but were occasionally smooth. Moreover, only one phialide was sometimes found on metulae as if they were bicellular structures (septate), this feature was re­ported by Raper and Fennel (1973) in their description of A. heteromorphus. The main differences were the color of reverse and of conidial heads, smaller hyaline metulae and phialides, larger conidia and longer conidiophores. These points justified the new species A. pseudo-heteromorphus. For the other Aspergillus strain, an additional difference was the lack of heteromorphism, so the name of A. homo­morphus is proposed.

41 System. App!. Microbio!. Vo!'17/4

It must be pointed out that these 2 species were found in the same soil sample. The pH of the suspension of soil was 7.4. Analysis of the soil revealed the following composi­tion in mmollg: Na 32, K 27, C187, Ca 15, Mg 8, suggest­ing the presence of various chlorides, however the global salt level was low when compared to number of other samples from the Dead Sea area.

Concerning the extracellular enzymatic activities, both 2 species were found to be lipolytic and proteolytic, while they did not exhibit any cellulolytic, amylolytic or Pox activity.

No polysaccharide was produced by the strains. Antifungal and antibacterial tests revealed neither an

activity against bacteria nor against phytopathogenic or entomopathogenic fungi. Yeasts were not inhibited except Cryptococcus neoformans 1, which was sensitive to the extract of culture medium of Aspergillus homomorphus grown under static conditions. Among Dermatophytes, extracts from Aspergillus homomorphus were efficient against Epidermophyton (loccosum, Microsporum canis, M. gypseum, Trichophyton mentagrophytes 1, 2 and var. interdigitale. Most powerful activities were obtained under static conditions except in the case of Microsporum canis which was only sensitive to shaked-culture extracts.

624 R. Steiman et al.

Aspergillus pseudo-heteromorphus was only active against E. f/occosum and M. gypseum when grown under static conditions.

In conclusion, the isolated strains from soil of Israel were classified into new taxa among Aspergillus niger group because of some morphological differences with the well described species of this group. Description of the strains was coupled with determination of some phy­siological features. These strains did not exhibit strong extracellular enzymatic activities. Their ability to produce antimicrobial exometabolites was also limited.

Acknowledgements. Authors wish to thank]. L. Lafond from the Laboratoire de Biochimie C, CHRU A. Michallon, Grenoble, France for his contribution to this work.

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F. Seigle-Murandi, Groupe pour I'Etude du devenir des Xenobiotiques dans l'Environnement (GEDEXE), Universite ]. Fourier, UFR Pharmacie, BP 138, Avenue de Verdun, 38043 Meylan cedex, France. Fax: (33) 7641 85 71