Mycopathologia 115: 83-88, 1991. 9 1991 KluwerAcademic Publishers. Printedin the Netherlands.
Comparison between antifungal and antibacterial activities of several strains of Epicoccum purpurascens from the Mediterranean area M. Mallea, 1 D. Pesando, 2 P. Bernard 2 & B. Khoulalene t 1Laboratoire de Microbiologie, Facultd des Sciences de Saint Jdr6rne, 13397 Marseille Cedex 13, France 2Institut National de la Santd et de la Recherche Mddicale, INSERM, Unitd 303, 06230 Villefranche-surMer, France Received 17 July 1990; accepted in revised form 10 February 1991
Key words: Antimicrobial activity, cultural characteristics, Epicoccum purpurascens, intraspecies comparison, metabolites, thin layer chromatography
Abstract
The antimicrobial activities of seven Epicoccum purpurascens strains isolated either from evergreen oak leaves (Quercus ilex) collected over a period of one year, or from the atmosphere were compared in vitro. Two strains sporulated and conspicuously inhibited the growth of Staphylococcus aureus and Trichophyton mentagrophytes. Thin-layer chromatographic studies showed the existence of some compounds, such as flavipin, which were common to all the strains. Epicorazine B was present in the extracts of only the two most active strains.
Introduction
Epicoccum purpurascens Link ( = E . nigrum Ehrenb. ex Schlecht) is an ubiquitous hyphomycete which grows saprophytically on many substrates, particularly decaying plants. It has also been isolated from a wide variety of soils. This fungus produces the slightly phytotoxic substance flavipin, which is capable of inhibiting the germination of Botrytis allii conidia at pH = 3.5 [1]. It also has a pH-dependent fungistatic action [2, 3]. The A and B pigments, which have antibacterial properties, were isolated by Burge et al. [4]. These yellow alcohol soluble pigments (epirodins) have antifungal effects [5]. Moreover epicorazines A and B, which are also produced by Epicoccum purpurascens, inhibit the growth of
Staphylococcus aureus [6] and that of plant pathogenic fungi [7, 8]. The metabolic products include a new compound, triornicin, which has anti-tumor properties [9]. The purpose of this study was to compare the activities of seven strains of Epicoccum purpurascens from various biotopes on several opportunist pathogens, and to observe the compounds which are present in these strains.
Material and m e t h o d s
Origin of the strains. The strains (El to E7) were isolated either from the litter (E5 and E7) or from oak leaves, Quercus ilex at different times of year
84 ( E l in May; E2 in October; E3 and E4 in December 1988), or from the atmosphere (E6). All of these strains came from the state forest of the Gardiole de Rians, Var, France. They were cultured on 2% malt extract agar (MEA) at p H = 6 and incubated at 25~ in daylight. Maintainance was on M E A at 4~
Test micro-organisms. Epicoccum purpurascens strains were tested against some fungal species that are opportunist pathogens or pathogenic to humans (capable of causing mycosis). Two yeasts, Candida aIbicans (IPC, 1283), C. pseudotropicalis (IPC, 842), and three dermatophytes, Microsporum canis, M. gypseum (IPC, 1296), and Trichophyton mentagrophytes (IPC 1273), were obtained from the Pasteur Institute Collection in Paris. Aspergillus fumigatus was obtained from Sainte Marguerite hospital in Marseille. They were cultured on Sabouraud medium (20 g Merck glucose, 10 g Difco peptone, 20 g Difco agar, l l distilled water; p H = 6.0-6.3). Tests were also performed against three bacteria, Staphylococcus aureus (IPC, 53146), Micrococcus luteus (IPC, 5345) and Escherichia coli (Sainte Marguerite hospital), maintained on trypticase soy agar (Bio-Merieux; pH = 7.3).
Fractionation of compounds. Each strain of Epicoccum purpurascens was cultured on 100 ml of MEA. After 10d, the agar was finely crosssectioned with a scalpel and extracted in an Erlenmeyer flask with 250 ml of ethanol on a magnetic stirrer for 3h at 4 ~ The ethanol was removed and retained, and the residue was extracted again overnight at 4 ~ in an equal volume of alcohol. The combined ethanol mixture (100 ml of medium containing mould and 500 ml of alcohol) was filtered on a porosity 3 fritted disk and the filtrate vacuum-dried at 35 ~ (Rotavapor). The residue was dissolved in ethanol to yield fraction I at 1 0 m g m l i Part of fraction I was further dried and extracted with a mixture of chloroformmethanol ( 9 5 : 5 , v/v) to obtain fraction II (10 mg ml 1). the chloroform-methanol insoluble
residue was once again dissolved in ethanol to obtain fraction III, which was concentrated to 10 mg ml i using the same procedure. With this method it was possible to separate non-polar compounds (fraction II) from more polar ones (fraction III). Chromatography of the extracts was on 0.2 mm thick silica gel 60 F25o (Merck) plates in chloroform-methanol ( 9 5 : 5 , v/v) [6]. After migration, the plates were examined under uv light at 254 nm and 360 nm and were then sprayed with 2 - 4 dinitrophenylhydrazine and examined under visible light. Partial purification of detected metabolites was performed using preparative thin-layer chromatography (TLC) silica gel 60 F254 0 . 5 m m (Merck), previously washed with methanol. Aliquots of the chloroform fraction, 1 ml, were deposited on the plate (200 • 200 ram), which was developed using the same solvent system as above. Each band, localized under uv light (254 and 360 nm), was scraped off, eluted with methanol, and the methanol removed in a vacuum. The residue was redissolved with 1 ml of methanol and tested for antibacterial and antifungal activities.
Anti-microbial tests. Anti-microbial tests were carried out by measuring the growth inhibition zone of each micro-organism, using two techniques. First, cylinders, 8 mm diam, were punched out of 6, 10 and 20 d old cultures of Epicoccum purpurascens on agar, and transferred into a well of the same diameter in a nutritive agar, the surface of which had been previously seeded with a suspension of bacterial and fungal spores or mycelial fragments. The spore suspensions, prepared in distilled water, were adjusted to 1 • 103, 1 x 105 and 1 • 106 cells ml * with a Thoma chamber and homogenized with a vortex mixer. Suspensions of filamentous fungi were homogenized (Tenbroeck homogenizer; Kontes Glass Co, Vineland N.J.). Cultures of dermatophytes, Microsporum canis, M. gypseum and Trichophyton mentagrophytes, were incubated at 25~ those of Candida
albicans, C. pseudotropicalis, Aspergillus fumi-
85
Table 1. Activity of 7 strains Epicoccum purpurascells against some pathogenic micro-organisms. to
o
E1
~
6 i0 20
. .
6 10 20
6 6 6
6 10 20
. .
E4
6 10 20
.
E5
6 10 20
.
E6
6 10 20
2 4 2
+ + .
E7
6 10 20
2 4
+ +
E2
E3
.
.
.
.
.
.
.
.
.
+ + . .
.
. .
+
.
-
.
.
2 2
24 21 2O
-
3
. .
10 11 4
.
.
.
.
.
+ +
-
.
2 +
8 7 10
.
.
4
.
.
.
.
.
. .
.
. . .
.
.
-
. . .
.
12 8
.
-
.
+ +
.
. -
.
-
+ + +
.
. + +
+ +
-
.
.
.
.
-
.
-
.
.
.
9 14.5 8
. -
-
Average diameter of growth inhibition zone (ram) around an 8 mm cylinder. ( - ) No Epicoccum effect on the growth of the micro-organisms tested; (+) Zone around the Epicoccum cylinder with weak microbial growth. Results obtained on test microorganism suspensions of 1 x 106 cells ml ~.
gatus, Staphylococcus aureus, Escherichia coli a n d Micrococcus luteus a t 37 ~
bacteria.
S. aureus b e i n g m o r e s e n s i t i v e t h a n M .
500/~g) of the fungal extracts and set on surfaces
luteus. T h e a n t i f u n g a l a c t i v i t y o f t h e s e v e n E . pupurascens s t r a i n s w a s p a r t i c u l a r l y strong a g a i n s t T. rnentagrophytes. A l l t h e r e s u l t s w e r e n e g a t i v e w i t h M. canis b u t a t a h i g h e r d i l u t i o n , a
previously
zone
Second,
cellulose disks 6 mm in diameter
impregnated
with seeded
test suspension
25/xl with
and lml
50/zl
were
( 2 5 0 p,g a n d
of a homogeneous
of the micro-organism.
A l l t e s t s w e r e c a r r i e d o u t i n t r i p l i c a t e in P e t r i d i s h e s (14 c m d i a m ) .
of partial
inhibition
was observed
cylinders E2, E6 and E7. Growth
of
around
Aspergillus
fumigatus w a s n o t i n h i b i t e d b y a n y o f t h e s e v e n Epicoccum s t r a i n s , e v e n a t i n o c u l u m c o n c e n t r a t i o n s o f 1 • l 0 s a n d 1 • 10 3 c e l l s m 1 - 1 . T h e a n t i fungal activity of the
R e s u l t s
two
giving The results obtained
using the diffusion method
are shown
1. E. purpurascens s t r a i n s
in Table
E2, E6 and E7 showed
activity against Gram+ve
yeasts slightly
Epicoccum w a s w e a k e r a g a i n s t
tested, positive
only
strains
results
E2
with
and
E6
Candida
pseudotropicalis. A s i m i l a r p a r t i a l i n h i b i t i o n w a s a l s o o b s e r v e d w i t h C. albicans a t d i l u t i o n s o f 1 • 10 3 c e l l s m l
~.
86 Table 2. Antibiotic activities of fractions I, [I, II1 prepared from strains E2 and E7 of Epicoccum purpurascens against two microorganisms.
E2
E7
Quantity applied to disk
I
II
III
II
III
S. aureus (106 cells ml -j)
250 txg 500/xg
6 12
8 12
0 0
I 4 8
6 10
0 0
T. rnentagrophytes
250/xg 500 txg
8 12
12 14
0 0
6 [2
10 12
0 0
Average diameter of inhibition zone (mm, less disk diameter) with two quantities (250/*g and 500 p,g).
Results were almost identical when tests were carried out with 6- or 10-d old Epicoccum cultures. However 20-d old Epicoccum cultures were much less active, and were inactive against microorganisms tested at concentrations of 1 x 103 cells m1-1. Results of the activity tests done with crude ethanolic extracts (fraction I) show that only those prepared from strains E2 and E7 inhibited the growth of S. aureus and T. mentagrophytes at the two quantities used. Inhibitory activity was also observed in the chloroform extracts (fraction II), whereas fraction III had no effect on the growth of either of the two micro-organims (Table 2). Several compounds were observed by TLC of the ethanol extracts. Examination under uv showed four spots which were common to all the extracts (Rf = 0.18, 0.6, 0.8 and 0.9; Fig. 1). However, with E2 and E7, m o r e spots were observed (Fig. 1). Compounds present in fraction I of strains E2 and E7, which migrate using TLC, were also detected in fraction II, and the most polar compounds were found in fraction III (Fig. 2). Preparative TLC revealed that only 3 compounds showed growth inhibition: compound at Rf = 0.57 inhibited the growth of S. aureus and compounds at Rf = 0.18 and 0.90 inhibited that of T. mentagrophytes (Fig. 3).
different micro-organisms tested. Only the most active strains, E2 and ET, sporulated strongly in agar medium while E6 and E7 developed a few sporodochia in culture after more than one month. E3, E4 and E5 remained at the sterile. The amount of pigment released into the agar was highest in the case of spoulating strains E2, E7, E6 and E~. TLC revealed spots which were present in all the ethanol extracts of 10-d old culture filtrates. The yellow compound detected at Rf = 0.18 is believed to be the phenolic compound flavipin [1], since it reacted with 2-4 dinitrophenylhydrazine [4] and it showed the same migration pattern using TLC under our experimental conditions and
Rf
0.5
c'_'- cb
,2)
,..D
C2
c:D
o
- -
vO 0
'8"
0
'
0
I
II
III
E2 Discussion
The biological activity of the seven Epicoccum strains investigated was not identical against the
' !
II
III
E7
Fig. 1. Thin layer chromatography (TLC) of crude ethanolic extracts of Epicoccum purpurascens on silica gel (samples 1 to 7). Solvents: chloroform/methanol (94:6, v/v). Detection uv at 254nm O, at 360 nm ,22L --- Spots revealed by 2,4dinitrophenylhydrazine~ Y: Yellow spots.
87
Rf
Q 0.5
o
o@O I
2
9
GO
4
3
5
0 6
7
F/g. 2, Thin layer chromatography (TLC) of Epicoccum purpurascens extracts (samples E2 and E7). I Crude ethanol extracts; II Soluble chloroform fraction: III Insoluble chloroform fraction recovered in ethanol, Solvents: chloroform/ methanol (94:6, v/v). Detection: uv at 254nm 9 at 360nm @. --- Spots revealed by 2-4 dinitrophenylhydrazine; Y: Yellow spots.
Rf
under the same conditions as those described by B r o w n et al. [7]. This c o m p o u n d is active against T. mentagrophytes and is also r e p o r t e d to be a grown inhibitor of Botrytis all& Phytophtora spp and Pythium spp (3). Besides flavipin, o t h e r compounds were detected at Rf = 0.6, 0.8 and 0.9. These c o m p o u n d s do not seem to have been identified before. A t t e n t i o n was first drawn to t h e m by B r o w n et al. [7] in culture filtrates over 12 days old in the case of the first c o m p o u n d (Rf = 0.6) and less than 12 days old in that of the other two. Only the third c o m p o u n d (Rf = 0.9) was active against
T. mentagrophytes. E2, E6 and E7 inhibited the growth of S. aureus (Table 1). B a u t e et al. [6] have isolated Epicorazines A and B which are active against this bacterium. W h e n tested using bio-autography, these two products were active against p h y t o p a t h o g e n i c fungi [7]. O n e of the spots on T L C detected at Rf = 0.5 using uv absorption at 254 n m and 2 - 4 dinitrophenylhydrazine spray reagent corre-
1
C~
J
0.5 t.
Y
Fig. 3. Preparative thin layer chromatography (PTLC) of EpicoccumpllrpurasEetTs chloroform fraction (ET) on silica gel Solvents: chloroform/methanol (95:5, v/v). Detection uv at 254 nm 9 at 360 nm ':~. --- Spots revealed by 2,4 dinitrophenylhydrazine; Y: Yellow spots.
88 s p o n d e d v e r y closely to the R f v a l u e p r e v i o u s l y o b t a i n e d with e p i c o r a z i n e B [6, 7]. A s d e m o n s t r a t e d b y t h e a n t i b a c t e r i a l tests p e r f o r m e d on the e l u a t e s f r o m the p r e p a r a t i v e c h r o m a t o g r a m (Fig. 3), this c o m p o u n d was r e s p o n s i b l e for t h e i n h i b i t o r y activity o f E2 c h l o r o f o r m e x t r a c t against S. aureus. A l t h o u g h all the strains w e r e f o u n d to b e active on T. mentagrophytes with the c y l i n d e r m e t h o d ( T a b l e 1), o n l y the e t h a n o l a n d c h l o r o f o r m e x t r a c t s o f E2 a n d E7 i n h i b i t e d the g r o w t h of this fungus. This m i g h t be a t t r i b u t a b l e to the fact t h a t t h e e x t r a c t s (250/_tg a n d 500/~g, which a r e the q u a n t i t i e s g e n e r a l l y u s e d in s c r e e n i n g tests) set o n t h e cellulose disks c o r r e s p o n d e d to q u a n t i t i e s o f fungus (E. purpurascens) which a r e at least 4 a n d 8 t i m e s l o w e r t h a n t h o s e u s e d with t h e c y l i n d e r t e c h n i q u e . In c o n c l u s i o n , t h e results o f t h e p r e s e n t s t u d y s h o w t h a t d i f f e r e n c e s exist in t h e activity o f t h e v a r i o u s strains i n v e s t i g a t e d , that t h e activity s e e m s to d e p e n d on the d e g r e e of s p o r u l a t i o , a n d t h a t t h e r e exist c o m p o u n d s which a r e active a g a i n s t d e r m a t r o p h y t e s which to o u r k n o w l e d g e have never previously been tested.
2. Eka OU. A note on properties of pigments produced by Epicoccurn nigrum. Experientia 1970: 25: 924. 3. Eka OU. Fungistatic action of the pigment secreted by the fungus Epicoccum nigrum link. Experientia i970; 26: 1278-9. 4. Burge WR~ Buckley LJ, Sullivan JD, McGrattan CJ, Ikawa M. Isolation and biological activity of the pigments of the Mold Epicoccum nigrum. Journal of Agricultural and Food Chemistry 1976; 24: 555-9. 5. Ikawa M, McGrattan CJ, Burge WR, Iannitelli RC, Uebel J J, Noguchi T. Epirodin, a polyene antibiotic from the mold Epicoccum nigrurn. The Journal of Antibiotics 1978; 3i: 159-161. 6. Baute MA, Deffieux G, Baute R, Neveu A. New antibiotics from the fungus Epicoccum nigrum, I: fermentation, isolation and antibacterial properties. The journal of Antibiotics 1978; 31: 1099-1101. 7. Brown AE, Finlay R, Ward JS. Antfungal compounds produced by Epicoccum purpurascens against soil-borne plant pathogenic fungi. Soil Biology and Biochemistry 1987; 19: 657-64. 8. Webber JF, Hedger JN. Comparison of interactions between Ceratocystis ulmi and elm bark saprobes in vitro and in vivo. Transactions of the British Mycological Society i986; 86: 93-101. 9. Frederick CB, Szaniszlo PJ, Vickrey PE, Bentley MD, Shive W. Production and isolation of siderophores from the soil fungus Epicoccum purpurascens. Biochemistry 1981; 20: 2432-6.
References Address for correspondence: Monique Mallea, Laboratoire de 1. Bamford PC, Norris GLF, Ward G. Flavipin production by Epicoccum spp. Transactions of the British Mycological Society 1961; 44: 354-6.
Microbiologie, Case 452, Facult6 des Sciences de St-J6r6me, Av. Escadrille Normandie-Niemen, F-I3397 Marseille Cedex 13. Tel. 91.28.85.3i.
Comparison between antifungal and antibacterial activities of several strains of Epicoccum purpurascens from the Mediterranean area M. Mallea, 1 D. Pesando, 2 P. Bernard 2 & B. Khoulalene t 1Laboratoire de Microbiologie, Facultd des Sciences de Saint Jdr6rne, 13397 Marseille Cedex 13, France 2Institut National de la Santd et de la Recherche Mddicale, INSERM, Unitd 303, 06230 Villefranche-surMer, France Received 17 July 1990; accepted in revised form 10 February 1991
Key words: Antimicrobial activity, cultural characteristics, Epicoccum purpurascens, intraspecies comparison, metabolites, thin layer chromatography
Abstract
The antimicrobial activities of seven Epicoccum purpurascens strains isolated either from evergreen oak leaves (Quercus ilex) collected over a period of one year, or from the atmosphere were compared in vitro. Two strains sporulated and conspicuously inhibited the growth of Staphylococcus aureus and Trichophyton mentagrophytes. Thin-layer chromatographic studies showed the existence of some compounds, such as flavipin, which were common to all the strains. Epicorazine B was present in the extracts of only the two most active strains.
Introduction
Epicoccum purpurascens Link ( = E . nigrum Ehrenb. ex Schlecht) is an ubiquitous hyphomycete which grows saprophytically on many substrates, particularly decaying plants. It has also been isolated from a wide variety of soils. This fungus produces the slightly phytotoxic substance flavipin, which is capable of inhibiting the germination of Botrytis allii conidia at pH = 3.5 [1]. It also has a pH-dependent fungistatic action [2, 3]. The A and B pigments, which have antibacterial properties, were isolated by Burge et al. [4]. These yellow alcohol soluble pigments (epirodins) have antifungal effects [5]. Moreover epicorazines A and B, which are also produced by Epicoccum purpurascens, inhibit the growth of
Staphylococcus aureus [6] and that of plant pathogenic fungi [7, 8]. The metabolic products include a new compound, triornicin, which has anti-tumor properties [9]. The purpose of this study was to compare the activities of seven strains of Epicoccum purpurascens from various biotopes on several opportunist pathogens, and to observe the compounds which are present in these strains.
Material and m e t h o d s
Origin of the strains. The strains (El to E7) were isolated either from the litter (E5 and E7) or from oak leaves, Quercus ilex at different times of year
84 ( E l in May; E2 in October; E3 and E4 in December 1988), or from the atmosphere (E6). All of these strains came from the state forest of the Gardiole de Rians, Var, France. They were cultured on 2% malt extract agar (MEA) at p H = 6 and incubated at 25~ in daylight. Maintainance was on M E A at 4~
Test micro-organisms. Epicoccum purpurascens strains were tested against some fungal species that are opportunist pathogens or pathogenic to humans (capable of causing mycosis). Two yeasts, Candida aIbicans (IPC, 1283), C. pseudotropicalis (IPC, 842), and three dermatophytes, Microsporum canis, M. gypseum (IPC, 1296), and Trichophyton mentagrophytes (IPC 1273), were obtained from the Pasteur Institute Collection in Paris. Aspergillus fumigatus was obtained from Sainte Marguerite hospital in Marseille. They were cultured on Sabouraud medium (20 g Merck glucose, 10 g Difco peptone, 20 g Difco agar, l l distilled water; p H = 6.0-6.3). Tests were also performed against three bacteria, Staphylococcus aureus (IPC, 53146), Micrococcus luteus (IPC, 5345) and Escherichia coli (Sainte Marguerite hospital), maintained on trypticase soy agar (Bio-Merieux; pH = 7.3).
Fractionation of compounds. Each strain of Epicoccum purpurascens was cultured on 100 ml of MEA. After 10d, the agar was finely crosssectioned with a scalpel and extracted in an Erlenmeyer flask with 250 ml of ethanol on a magnetic stirrer for 3h at 4 ~ The ethanol was removed and retained, and the residue was extracted again overnight at 4 ~ in an equal volume of alcohol. The combined ethanol mixture (100 ml of medium containing mould and 500 ml of alcohol) was filtered on a porosity 3 fritted disk and the filtrate vacuum-dried at 35 ~ (Rotavapor). The residue was dissolved in ethanol to yield fraction I at 1 0 m g m l i Part of fraction I was further dried and extracted with a mixture of chloroformmethanol ( 9 5 : 5 , v/v) to obtain fraction II (10 mg ml 1). the chloroform-methanol insoluble
residue was once again dissolved in ethanol to obtain fraction III, which was concentrated to 10 mg ml i using the same procedure. With this method it was possible to separate non-polar compounds (fraction II) from more polar ones (fraction III). Chromatography of the extracts was on 0.2 mm thick silica gel 60 F25o (Merck) plates in chloroform-methanol ( 9 5 : 5 , v/v) [6]. After migration, the plates were examined under uv light at 254 nm and 360 nm and were then sprayed with 2 - 4 dinitrophenylhydrazine and examined under visible light. Partial purification of detected metabolites was performed using preparative thin-layer chromatography (TLC) silica gel 60 F254 0 . 5 m m (Merck), previously washed with methanol. Aliquots of the chloroform fraction, 1 ml, were deposited on the plate (200 • 200 ram), which was developed using the same solvent system as above. Each band, localized under uv light (254 and 360 nm), was scraped off, eluted with methanol, and the methanol removed in a vacuum. The residue was redissolved with 1 ml of methanol and tested for antibacterial and antifungal activities.
Anti-microbial tests. Anti-microbial tests were carried out by measuring the growth inhibition zone of each micro-organism, using two techniques. First, cylinders, 8 mm diam, were punched out of 6, 10 and 20 d old cultures of Epicoccum purpurascens on agar, and transferred into a well of the same diameter in a nutritive agar, the surface of which had been previously seeded with a suspension of bacterial and fungal spores or mycelial fragments. The spore suspensions, prepared in distilled water, were adjusted to 1 • 103, 1 x 105 and 1 • 106 cells ml * with a Thoma chamber and homogenized with a vortex mixer. Suspensions of filamentous fungi were homogenized (Tenbroeck homogenizer; Kontes Glass Co, Vineland N.J.). Cultures of dermatophytes, Microsporum canis, M. gypseum and Trichophyton mentagrophytes, were incubated at 25~ those of Candida
albicans, C. pseudotropicalis, Aspergillus fumi-
85
Table 1. Activity of 7 strains Epicoccum purpurascells against some pathogenic micro-organisms. to
o
E1
~
6 i0 20
. .
6 10 20
6 6 6
6 10 20
. .
E4
6 10 20
.
E5
6 10 20
.
E6
6 10 20
2 4 2
+ + .
E7
6 10 20
2 4
+ +
E2
E3
.
.
.
.
.
.
.
.
.
+ + . .
.
. .
+
.
-
.
.
2 2
24 21 2O
-
3
. .
10 11 4
.
.
.
.
.
+ +
-
.
2 +
8 7 10
.
.
4
.
.
.
.
.
. .
.
. . .
.
.
-
. . .
.
12 8
.
-
.
+ +
.
. -
.
-
+ + +
.
. + +
+ +
-
.
.
.
.
-
.
-
.
.
.
9 14.5 8
. -
-
Average diameter of growth inhibition zone (ram) around an 8 mm cylinder. ( - ) No Epicoccum effect on the growth of the micro-organisms tested; (+) Zone around the Epicoccum cylinder with weak microbial growth. Results obtained on test microorganism suspensions of 1 x 106 cells ml ~.
gatus, Staphylococcus aureus, Escherichia coli a n d Micrococcus luteus a t 37 ~
bacteria.
S. aureus b e i n g m o r e s e n s i t i v e t h a n M .
500/~g) of the fungal extracts and set on surfaces
luteus. T h e a n t i f u n g a l a c t i v i t y o f t h e s e v e n E . pupurascens s t r a i n s w a s p a r t i c u l a r l y strong a g a i n s t T. rnentagrophytes. A l l t h e r e s u l t s w e r e n e g a t i v e w i t h M. canis b u t a t a h i g h e r d i l u t i o n , a
previously
zone
Second,
cellulose disks 6 mm in diameter
impregnated
with seeded
test suspension
25/xl with
and lml
50/zl
were
( 2 5 0 p,g a n d
of a homogeneous
of the micro-organism.
A l l t e s t s w e r e c a r r i e d o u t i n t r i p l i c a t e in P e t r i d i s h e s (14 c m d i a m ) .
of partial
inhibition
was observed
cylinders E2, E6 and E7. Growth
of
around
Aspergillus
fumigatus w a s n o t i n h i b i t e d b y a n y o f t h e s e v e n Epicoccum s t r a i n s , e v e n a t i n o c u l u m c o n c e n t r a t i o n s o f 1 • l 0 s a n d 1 • 10 3 c e l l s m 1 - 1 . T h e a n t i fungal activity of the
R e s u l t s
two
giving The results obtained
using the diffusion method
are shown
1. E. purpurascens s t r a i n s
in Table
E2, E6 and E7 showed
activity against Gram+ve
yeasts slightly
Epicoccum w a s w e a k e r a g a i n s t
tested, positive
only
strains
results
E2
with
and
E6
Candida
pseudotropicalis. A s i m i l a r p a r t i a l i n h i b i t i o n w a s a l s o o b s e r v e d w i t h C. albicans a t d i l u t i o n s o f 1 • 10 3 c e l l s m l
~.
86 Table 2. Antibiotic activities of fractions I, [I, II1 prepared from strains E2 and E7 of Epicoccum purpurascens against two microorganisms.
E2
E7
Quantity applied to disk
I
II
III
II
III
S. aureus (106 cells ml -j)
250 txg 500/xg
6 12
8 12
0 0
I 4 8
6 10
0 0
T. rnentagrophytes
250/xg 500 txg
8 12
12 14
0 0
6 [2
10 12
0 0
Average diameter of inhibition zone (mm, less disk diameter) with two quantities (250/*g and 500 p,g).
Results were almost identical when tests were carried out with 6- or 10-d old Epicoccum cultures. However 20-d old Epicoccum cultures were much less active, and were inactive against microorganisms tested at concentrations of 1 x 103 cells m1-1. Results of the activity tests done with crude ethanolic extracts (fraction I) show that only those prepared from strains E2 and E7 inhibited the growth of S. aureus and T. mentagrophytes at the two quantities used. Inhibitory activity was also observed in the chloroform extracts (fraction II), whereas fraction III had no effect on the growth of either of the two micro-organims (Table 2). Several compounds were observed by TLC of the ethanol extracts. Examination under uv showed four spots which were common to all the extracts (Rf = 0.18, 0.6, 0.8 and 0.9; Fig. 1). However, with E2 and E7, m o r e spots were observed (Fig. 1). Compounds present in fraction I of strains E2 and E7, which migrate using TLC, were also detected in fraction II, and the most polar compounds were found in fraction III (Fig. 2). Preparative TLC revealed that only 3 compounds showed growth inhibition: compound at Rf = 0.57 inhibited the growth of S. aureus and compounds at Rf = 0.18 and 0.90 inhibited that of T. mentagrophytes (Fig. 3).
different micro-organisms tested. Only the most active strains, E2 and ET, sporulated strongly in agar medium while E6 and E7 developed a few sporodochia in culture after more than one month. E3, E4 and E5 remained at the sterile. The amount of pigment released into the agar was highest in the case of spoulating strains E2, E7, E6 and E~. TLC revealed spots which were present in all the ethanol extracts of 10-d old culture filtrates. The yellow compound detected at Rf = 0.18 is believed to be the phenolic compound flavipin [1], since it reacted with 2-4 dinitrophenylhydrazine [4] and it showed the same migration pattern using TLC under our experimental conditions and
Rf
0.5
c'_'- cb
,2)
,..D
C2
c:D
o
- -
vO 0
'8"
0
'
0
I
II
III
E2 Discussion
The biological activity of the seven Epicoccum strains investigated was not identical against the
' !
II
III
E7
Fig. 1. Thin layer chromatography (TLC) of crude ethanolic extracts of Epicoccum purpurascens on silica gel (samples 1 to 7). Solvents: chloroform/methanol (94:6, v/v). Detection uv at 254nm O, at 360 nm ,22L --- Spots revealed by 2,4dinitrophenylhydrazine~ Y: Yellow spots.
87
Rf
Q 0.5
o
o@O I
2
9
GO
4
3
5
0 6
7
F/g. 2, Thin layer chromatography (TLC) of Epicoccum purpurascens extracts (samples E2 and E7). I Crude ethanol extracts; II Soluble chloroform fraction: III Insoluble chloroform fraction recovered in ethanol, Solvents: chloroform/ methanol (94:6, v/v). Detection: uv at 254nm 9 at 360nm @. --- Spots revealed by 2-4 dinitrophenylhydrazine; Y: Yellow spots.
Rf
under the same conditions as those described by B r o w n et al. [7]. This c o m p o u n d is active against T. mentagrophytes and is also r e p o r t e d to be a grown inhibitor of Botrytis all& Phytophtora spp and Pythium spp (3). Besides flavipin, o t h e r compounds were detected at Rf = 0.6, 0.8 and 0.9. These c o m p o u n d s do not seem to have been identified before. A t t e n t i o n was first drawn to t h e m by B r o w n et al. [7] in culture filtrates over 12 days old in the case of the first c o m p o u n d (Rf = 0.6) and less than 12 days old in that of the other two. Only the third c o m p o u n d (Rf = 0.9) was active against
T. mentagrophytes. E2, E6 and E7 inhibited the growth of S. aureus (Table 1). B a u t e et al. [6] have isolated Epicorazines A and B which are active against this bacterium. W h e n tested using bio-autography, these two products were active against p h y t o p a t h o g e n i c fungi [7]. O n e of the spots on T L C detected at Rf = 0.5 using uv absorption at 254 n m and 2 - 4 dinitrophenylhydrazine spray reagent corre-
1
C~
J
0.5 t.
Y
Fig. 3. Preparative thin layer chromatography (PTLC) of EpicoccumpllrpurasEetTs chloroform fraction (ET) on silica gel Solvents: chloroform/methanol (95:5, v/v). Detection uv at 254 nm 9 at 360 nm ':~. --- Spots revealed by 2,4 dinitrophenylhydrazine; Y: Yellow spots.
88 s p o n d e d v e r y closely to the R f v a l u e p r e v i o u s l y o b t a i n e d with e p i c o r a z i n e B [6, 7]. A s d e m o n s t r a t e d b y t h e a n t i b a c t e r i a l tests p e r f o r m e d on the e l u a t e s f r o m the p r e p a r a t i v e c h r o m a t o g r a m (Fig. 3), this c o m p o u n d was r e s p o n s i b l e for t h e i n h i b i t o r y activity o f E2 c h l o r o f o r m e x t r a c t against S. aureus. A l t h o u g h all the strains w e r e f o u n d to b e active on T. mentagrophytes with the c y l i n d e r m e t h o d ( T a b l e 1), o n l y the e t h a n o l a n d c h l o r o f o r m e x t r a c t s o f E2 a n d E7 i n h i b i t e d the g r o w t h of this fungus. This m i g h t be a t t r i b u t a b l e to the fact t h a t t h e e x t r a c t s (250/_tg a n d 500/~g, which a r e the q u a n t i t i e s g e n e r a l l y u s e d in s c r e e n i n g tests) set o n t h e cellulose disks c o r r e s p o n d e d to q u a n t i t i e s o f fungus (E. purpurascens) which a r e at least 4 a n d 8 t i m e s l o w e r t h a n t h o s e u s e d with t h e c y l i n d e r t e c h n i q u e . In c o n c l u s i o n , t h e results o f t h e p r e s e n t s t u d y s h o w t h a t d i f f e r e n c e s exist in t h e activity o f t h e v a r i o u s strains i n v e s t i g a t e d , that t h e activity s e e m s to d e p e n d on the d e g r e e of s p o r u l a t i o , a n d t h a t t h e r e exist c o m p o u n d s which a r e active a g a i n s t d e r m a t r o p h y t e s which to o u r k n o w l e d g e have never previously been tested.
2. Eka OU. A note on properties of pigments produced by Epicoccurn nigrum. Experientia 1970: 25: 924. 3. Eka OU. Fungistatic action of the pigment secreted by the fungus Epicoccum nigrum link. Experientia i970; 26: 1278-9. 4. Burge WR~ Buckley LJ, Sullivan JD, McGrattan CJ, Ikawa M. Isolation and biological activity of the pigments of the Mold Epicoccum nigrum. Journal of Agricultural and Food Chemistry 1976; 24: 555-9. 5. Ikawa M, McGrattan CJ, Burge WR, Iannitelli RC, Uebel J J, Noguchi T. Epirodin, a polyene antibiotic from the mold Epicoccum nigrurn. The Journal of Antibiotics 1978; 3i: 159-161. 6. Baute MA, Deffieux G, Baute R, Neveu A. New antibiotics from the fungus Epicoccum nigrum, I: fermentation, isolation and antibacterial properties. The journal of Antibiotics 1978; 31: 1099-1101. 7. Brown AE, Finlay R, Ward JS. Antfungal compounds produced by Epicoccum purpurascens against soil-borne plant pathogenic fungi. Soil Biology and Biochemistry 1987; 19: 657-64. 8. Webber JF, Hedger JN. Comparison of interactions between Ceratocystis ulmi and elm bark saprobes in vitro and in vivo. Transactions of the British Mycological Society i986; 86: 93-101. 9. Frederick CB, Szaniszlo PJ, Vickrey PE, Bentley MD, Shive W. Production and isolation of siderophores from the soil fungus Epicoccum purpurascens. Biochemistry 1981; 20: 2432-6.
References Address for correspondence: Monique Mallea, Laboratoire de 1. Bamford PC, Norris GLF, Ward G. Flavipin production by Epicoccum spp. Transactions of the British Mycological Society 1961; 44: 354-6.
Microbiologie, Case 452, Facult6 des Sciences de St-J6r6me, Av. Escadrille Normandie-Niemen, F-I3397 Marseille Cedex 13. Tel. 91.28.85.3i.
