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Allelopathy Journal 23 (2): 357-368 (2009) International Allelopathy Foundation 2009
Tables: 3, Figs : 2
Antifungal and allelopathic effects of Asafoetida against
Trichoderma harzianum and Pleurotus spp.
P. ANGELINI*, R. PAGIOTTI, R. VENANZONI and B. GRANETTI
Dipartimento di Biologia Applicata, Borgo XX Giugno, 74 - 06121 Perugia, Italy
E. Mail: paolaangelini@ymail.com
(Received in revised form: November 8, 2008)
ABSTRACT
Methanol extract (MeOH) of Asafoetida oleogum-resin was assayed for its
in-vitro ability to control Trichoderma harzianum. The thirty-two components of
MeOH-extracted resin were identified by GC-MS analysis. The antifungal and
allelopathic effects of the MeOH extracts concentrations against T. harzianum and
Pleurotus spp., were investigated in dual culture experiments on an agar-based
medium. MeOH extract showed fungistatic and fungicidal properties against T.
harzianum strains and Pleurotus spp. at higher concentrations. In dual culture, all
strains of T. harzianum were antagonistic to Pleurotus spp. than in control. When
MeOH extracts concentrations was added to the substrate culture, the antagonistic
activity of T. harzianum against the Pleurotus spp. was moderate (0.625 μg/ml of
MeOH extract) or weak (1.25 μg/ml of MeOH extract) against the Pleurotus spp. that
either completely or partially replaced T. harzianum. TEM observations revealed that
fungal growth inhibition from the MeOH extract was accompanied by marked
morphological and cytological changes.
Key words: Antagonism, GC/MS, green moulds, MIC, MFC, transmission electron
microscopy.
INTRODUCTION
Oyster mushroom spp. belonging to Pleurotus eryngii spp.-complex [P. eryngi
(DC:Fr.) var. eryngii, P. eryngi(DC:Fr.) var. ferulae Lanzi, P. nebrodensis (Inzenga)
Quél., and P. hadamardii Costantin] are well known for their fruiting bodies that have
excellent organoleptic qualities (31). In recent years, the improved edible fungus cultures
have become of great interest due to economic importance of mushroom production (7).
Mycelial growth of Pleurotus eryngii spp.-complex is fast and various lignocellulosic
waste products can be used as culture substrate (32). The aim of commercial mushroom
substrate preparation is to produce a substrate that is optimal and selective for vegetative
mycelial growth (23,30).
Trichoderma harzianum Rifai (filamentous soil fungi), is an antagonist that
causes extensive losses in Apiaceae oyster mushroom production. Pleurotus compost or
casings infected with T. harzianum do not produce mushrooms and the crop loss is
proportional to the infected area and this infection is called green mould disease (19).
Pleurotus eryngii spp.-complex, colonise the roots and stems of some Umbelliferous
aromatic plants (Apiaceae), rich in active materials (resins and essential oils), that plays
*Correspondence author
Angelini et al
358
important role in plant-plant, plant-animal and plant microbe interactions and are primary
source of potential allelochemicals (2, 28). The Ferula genus of Apiaceae family is rich
source of oleogum-resin (8). Ferula assa-foetida (Asafoetida) grows in Kashmir, Iran and
Afghanistan. It is an herbaceous, perennial plant of 2 m height (15). Ethanolic extract of
Asafoetida oleogum-resin has shown antifungal activity against Mucor dimorphosphorous,
Penicillium commune and Fusarium solani (24).
In this in-vitro study, the potential effects of Asafoetida oleogum-resin extract to
inhibit T. harzianum strains encountered in Apiaceae oyster mushroom cultivation has
been investigated. The possible allelopathic dose of Asafoetida oleogum-resin extract was
investigated in dual culture to inhibit the T. harzianum and stimulation of Pleurotus spp.
mycelium growth. Transmission electron microscopy techniques were used to observe the
action of Asafoetida oleogum-resin extract on the ultrastructure of T. harzianum and
Pleurotus spp. fungal cells.
MATERIALS AND METHODS
T. harzianum strains, A, B, and C, were isolated from a naturally-contaminated
Apiaceae oyster mushrooms straw-based cultivation substrate in 2002. P. eryngii var.
ferulae strains 1 and 2, were isolated from basidiocarps in Tarquinia (VT, Italy) in 2001;
P. eryngii var. eryngii strains were isolated from basidiocarps in Senise (MT, Italy) in
2001; P. nebrodensis strains 529 and 193 were obtained from the Mycothèque du Museum
National d’Histoire Naturelle de Paris), P. hadamardii strain was isolated from basidiocarp
in Predazzo (TN, Italy) in 2002. All fungi were maintained on malt extract agar (MEA; 2%
malt extract and 1.5% agar) and stored at 4±1°C in the dark. Voucher cultures are kept in
the DBVBAZ culture collection (University of Perugia, Italy) and are accessible.
The hardened oleogum-resin of Asafoetida used in this study was supplied by
Aboca Erbe, San Sepolcro, AR, Italy. Using 250 ml glass bottles with screw caps, a
sample (30 g) of oleogum-resin was macerated with 100 ml of 96% (v/v) MeOH
(methanol) at room temperature for 7 days. The sample was filtered and concentrated in a
rotary evaporator under reduced pressure at 50° C. The dried MeOH extract was dissolved
in dimethyl sulfoxide (DMSO, Sigma Chemical Company) and used for antifungal assays.
GC and GC-MS analysis: The GC analyses were carried out using a Varian 3300
instrument equipped with a FID and an HP-InnoWax capillary column (30 m x 0.25 mm,
film thickness 0.17 μm), starting at 60°C (3 min) and increasing to 210°C (15 min) at
4°C/min or an HP-5 capillary column (30 m x 0.25 mm, film thickness 0.25 μm) starting at
60°C (3 min) and increasing to 300°C (15 min) at 4°C/min; injector and detector
temperatures, 250°C; carrier gas, helium (1 ml/min); split ratio, 1 : 10.GC-MS analyses
were carried out using a Hewlett Packard 5890 GC-MS system operating in the EI mode at
70 eV. The operating conditions were the same as those reported in the GC analysis
section. Injector and transfer line temperatures were 220°C and 280°C, respectively.
Helium was used as the carrier gas, flow rate 1
ml/min. Split ratio, 1 : 10.
The components of MeOH extract of Asafoetida oleogum-resin were identified by
matching the spectra with those from mass spectral libraries and the identity of each
Antifungal and allelopathic effects of Asafoetida 359
component was confirmed by comparing the retention indices, relative to the C6-C22 n-
alkanes from both columns, with those from the literature (1). When reported, coelution
gas chromatography with reference compounds was used as an additional confirmation of
the compound identity.
The percentage composition of the MeOH extract was obtained by the
normalisation method from the GC peak areas, without using correction factors.
Antifungal assay: The antifungal activity of Asafoetida oleogum-resin extract was tested
using the macrodilution technique (13). The mycelium growth inhibition (MGI),
fungistatic and fungicidal oleogum-resin extract concentrations were determined against
strains of T. harzianum and Pleurotus spp..
A known amount (0.625, 1.25, 2.5, 5, 10, 20, 30 and 40 μg/ml) of dried extract,
mixed with dimethyl sulfoxide (DMSO, Sigma Chemical Company), was then added to
Petri dishes containing 15 ml of Sabouraud Dextrose Agar (SDA, Oxoid) medium. The
final concentration of DMSO in these assays was <1%. The growth medium was
inoculated the next day at the centre of plates, using 5 mm cores taken from mycelial stock
culture plates and incubated at 25° C for 21 days.
The mycelium growth inhibition (MGI) percentage was calculated as per
following equation:
MGI = (dc-dt)/dc x 100,
Where, dc is fungal colony diameter in control, dt is fungal colony diameter in treatment
sets, 21 days after incubation.
The minimal inhibitory concentration (MICs) values of tested MeOH extract
were the lowest concentrations that did not exhibit any visible growth of fungal mycelium,
but which remained viable and grew when plated on SDA medium, after 21 days of
incubation. The minimum fungicidal concentration (MFCs) values were determined by
method of Garber and Huston (9). This was done by subculturing the inhibited fungal discs
at MICs on SDA medium. Observations were recorded 7 days after incubation at 25°C.
Fungal growth on day 7 was indicative of a fungistatic nature, while the absence of fungal
growth denoted a fungicidal action of the MeOH extract.
Every experiment was done in triplicate.
Allelophatic assay: Allelophatic effects of MeOH extract on competitive interactions
between Pleurotus spp. and T. harzianum were studied in dual-culture experiments (6).
The Asafoetida oleogum-resin extract dissolved in DMSO, was added to autoclaved SDA
at 0.625 μg/ml and 1.25 μg/ml and then poured into Petri dishes (9 cm dia) at 40–45 °C.
Sterile double-distilled H2O alone was added to the SDA of control plates. In each dish,
two 2-mm dia mycelial disks, one from Pleurotus spp. colony and one from T. harzianum
were placed on the agar surface 30 mm apart. The Pleurotus spp. and T. harzianum strains
were paired in all possible combinations. Three replicates were prepared for each pairing.
Paired cultures were incubated on a laboratory bench at ambient room temperature of 26 ±
2 °C, for 21 days and examined daily under a stereomicroscope to study the interaction
process. A rating scale with 3 types (A, B and C) and 4 sub-types (CA1, CB1, CA2 and
CB2) of reactions was used for each fungus. Type A and B were deadlock (mutual
Angelini et al
360
inhibition, in which neither organism was able to overtake the other) at mycelial contact
(A), or at distance (B); type C replacement, able to over take without initial deadlock. The
intermediate subtypes scored were: CA1 partial, and CA2 complete, replacement after
initial deadlock with mycelial contact; CB1 partial, and CB2 complete, replacement after
initial deadlock at a distance.
The following score was assigned to each type or sub-type of reaction:
A=1; B=2; C=3; CA1=3.5; CB1=4; CA2=4.5; CB2=5.
The antagonism index (AI) was calculated for each fungal spp. using the formula:
AI = ∑n x i
Where, n: number (frequency) of each type or sub-type of reaction; i: corresponding score.
Transmission electron microscopy: After 21 and 74 days of Asafoetida oleogum-resin
extract treatment (1.25 μg/ml) by macrodilution technique, small pieces of agar from the
fungal colony edge were taken from the treated and control Petri dishes for transmission
electron microscope (TEM) studies.
Samples from single cultures of each fungus were fixed in 2% (v/v)
glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) with 1 mM CaCl and 1% (w/v) sucrose
for 3 h at room temperature. Samples were then rinsed six times with the same buffer and
postfixed with 1% (w/v) osmium tetroxide in the same buffer for 2 h at room temperature.
After being rinsed thoroughly with 0.1 M cacodylate buffer (pH 7.4), samples were
dehydrated in a graded ethanol series. Fully dehydrated samples were moved from
absolute ethanol through a 1:1 mixture of ethanol and propylene oxide to pure propylene
oxide. Samples were infiltrated through a series of Epon-Araldit-Mixture resin in
propylene oxide, then embedded in blocks with fresh 100% resin and polymerised at 65°C
for 36 h. More than three replicate experiments were performed. Ultrathin sections, cut
with a glass knife, were collected on formvar-coated slot grids. After drying, the grids
were contrasted with uranyl acetate and lead citrate and examined with an EM 10 CR
electron microscope (Zeiss, Oberkochen, Germany) at 60 KV.
Statistical analyses: The effects of antifungal activity of Asafoetida oleogum-resin extract
were analysed by two factorial analysis of variance (ANOVA), followed by LSD post hoc
determinations (p≤ 0.05). All computations were done using the statistical software
SuperAnova for Mac Plus (1989-90, Abacus Concepts, Inc).
RESULTS AND DISCUSSION
GC-MS was used to identificaty and determine the percentage composition of
compounds found Asafoetida oleogum-resin extract (Table 1). Thirty - two compounds
were identified, representing 79.75 % of the extract. The main constituents were: sec-butyl
propenyl disulfide (19.53%), vinil-4-guaiacol (17.43%), ferulic acid (11.18%) and ß-
pinene (6.17 %).
Antifungal and allelopathic effects of Asafoetida 361
Table 1. Composition of MeOH extract of Asafoetida oleogum-resin (%).
Constituents Relative peak area (%)
sec-Butyl propenyl disulfide 19.53
Vinil-4-guaiacol 17.43
Ferulic acid 11.18
β-Pinene 6.17
Acetaldehyd diethyl acetal 5.08
α-Pinene 3.11
Guaiol 2.05
Vanillin 1.23
Elemol 1.09
Cariophyllene oxide 0.87
α-Cariophyllene 0.83
Farnesil acetate 0.64
cis-β-Ocimen 0.62
Sativene 0.58
Methyl chavicol 0.53
trans-Pinocarvyl acetate 0.53
trans-β-Ocimen 0.52
Aristolen 0.50
β-Eudesmol 0.47
Farnesol 0.30
di-sec-Butyl disulfide 0.23
Limonene 0.21
α-Terpineol 0.21
Pinocarveol 0.20
Propyl sec-butyl disolfide 0.20
Fenchol 0.13
Myrcen 0.12
2,3,4-Trimethylthiophene 0.06
Isovalerianic acid 0.05
Canphene 0.05
p-Cimene 0.02
α-Fenchene 0.01
Others 25.25
Antifungal effects of Asafoetida oleogum-resin extract on T. harzianum and Pleurotus spp.
The mycelium growth inhibition, fungistatic and fungicidal activity values of
MeOH extract against the tested fungi are reported in Table 2 and Figure 1. T. harzianum
strains exhibited fungal growth inhibition at the lowest concentrations of MeOH extract
(0.625 – 1.25 μg/ml), while Pleurotus spp. showed mycelial growth stimulation at the
same concentrations.
The MeOH extract of Asafoetida oleogum-resin showed fungistatic and
fungicidal properties against P. nebrodensis 529, P. eryngii var. ferulae 1 and 2 and
P. eryngii var. eryngii 1 at the highest concentrations used, i.e. 30 μg/ml MIC and 40
μg/ml MFC. The MeOH extract had fungistatic activity against P. nebrodensis 193 and
Angelini et al
362
Table 2. Mycelial growth inhibition, fungistatic and fungicidal activity of asafoetida oleogum-resin extract on Pleurotus species
Asafoetida oleogum-
resin extract (µg/ml)
P. n. 529 P. n. 193 P. f. 1 P. f. 2 P. e.1 P. e. 2 P. h.
0.625 - 46.7 ± 0.5 (b) - 86.2 ± 0.7 (b) - 33.4 ± 0.6 (b) - 64.4 ± 1.1 (b) -20.0 ± 0.6 (b) -46.1 ± 0.6 (b) -5,1 ± 0.1 (a)
1.25 - 60.0 ± 2.7 (a) - 135.8 ± 0.6 (a) - 48.9 ± 1.0 (a) - 82.3 ± 0.6 (a) -46.6 ± 0.3 (a) -76.9 ± 0.2 (a) -2,2 ± 0.1 (b)
2.5 -36.4 ± 0.2 (c) - 56.7 ± 0.4 (c) - 6.4 ± 0.2 (c) -29.8 ± 0.5 (c) 2.2 ± 0.1 (c) - 18.7 ± 0.6 (c) 21,2 ± 0.6 (c)
5 6.9 ± 0.4 (d) - 2.3 ± 0.2 (d) 12.7 ± 0.4 (d) 10.2 ± 0.6 (d) 43.8 ± 1.1 (d) 8.5 ± 0.3 (d) 36.4 ± 0.5 (d)
10 32.8 ± 1.0 (e) 8.8 ± 0.6 (e) 41.3 ± 0.6 (e) 52.4 ± 1.1 (e) 78.6 ± 0.6 (e) 39.1 ± 0.6 (e) 46,7 ± 1.0 (e)
20 71.4 ± 0.5 (f) 47.6 ± 0.9 (f) 97.4 ± 1.1 (f) 92.3 ± 1.1 (f) 98.1 ± 0.6 (f) 78.2 ± 0.7 (f) 61,7 ± 0.4 (f)
30 100 (g) Fs 84.3 ± 1.1 (g) 100 (g) Fs 100 (g) Fs 100 (g) Fs 94.6 ± 1.1 (g) 69,6 ± 1.1 (g)
40 101 (g) Fc 100 (h) Fs 100 (g) Fc 100 (g) Fc 100 (g) Fc 100 (h) Fs 87.4 ± 0.6 (h)
P.n. - Pleurotus nebrodensis; P.f. - P. eryngii var. ferulae; P.e. - P. eryngii var. eryngii; P.h. - P. hadamardii. Data in the column followed by different letters in the
parentheses are significantly different in LSD post hoc test (p≤ 0.05). The values are means of three repetitions ± standard error. Fs - Fungistatic activity; Fc -
Fungicidal activity.
Table 3. Types and sub-types of hyphal reactionsa between Pleurotus species and Trichoderma harzianum in pairings on PDA medium without asafoetida
oleogum-resin extract (control), with 0.625 µg/ml and 1.25 µg/ml of asafoetida oleogum-resin extract
Pleurotus species Control Asafoetida oleogum-resin extract
(0.625 µg/ml)
Asafoetida oleogum-resin extract
(1.25 µg/ml)
T. h. A T. h. B T. h. C Total A.I.b T. h. A T. h. B T. h. C Total A.I.b T. h. A T. h. B T. h. C Total A.I.b
P. nebrodensis 529 CA1* CA1* CA1* 0 CB1* CB1 C
B1 8 CB1 C
B1 C
B2 13
P. nebrodensis 193 CA1* CA1* CA2* 0 B CB2 C
B2 12 CB2 C
B2 C
B2 15
P. eryngii var. ferulae 1 CA1* CA1* CB1* 0 CB1* CB1 C
B1* 4 CB1 C
B2 C
B2 14
P. eryngii var. ferulae 2 CA2* CA1* CB1* 0 CB1* CB1 B 6 B CB2 C
B1 11
P. eryngii var. eryngii 1 CA2* CA2* CA1* 0 CB2* B B 4 CB2 C
B1 C
B2 14
P. eryngii var. eryngii 2 CA2* CB1* CB1* 0 CB1* CB1 B 6 CB2 C
B2 C
B2 15
P. hadamardii CA1* CA1* CA1* 0 CA1* A A 2 CA1* A CA1* 1
Total A.I.a 27.5 23 27 26.5 3 11 5.5 1 3.5
a, A - deadlock, mutual inhibition, in which neither organism was able to overgrow the other after mycelial contact; B - deadlock at a distance without mycelial
contact; C - replacement, overgrowth without initial deadlock; CA1 - Partial replacement after initial deadlock; CB1 - Partial replacement after initial deadlock at
adistance; CA2 - Complete replacement after initial deadlock; CB2 - Complete replacement after initial deadlock at a distance. b
, A.I. = Antagonism Index. *, T.
harzianum overgrew Pleurotus species. In the other replacement reactions Pleurotus species overgrew T. harzianum.
Antifungal and allelopathic effects of Asafoetida 363
0
10
20
30
40
50
60
70
80
90
100
Mycelial growth inhibition
0,63 1,25 2,5 5 10 20 30 40
Asafoetida oleogum-resin extract conc.
T. harzianum A
T. harzianum B
T. harzianum C
Figure 1. Mycelial growth inhibition of Asafoetida oleogum-resin extract on T. harzianum strains.
P. eryngii var. eryngii 2 at a concentration of 30 μg/ml (MIC). P. hadamardii was the least
sensitive to the MeOH extract; no fungistatic or fungicidal properties were observed.
T. harzianum A was the most resistant strain among the fungi tested with MIC and MFC
values of 40 and >40 μg/ml, respectively, while T. harzianum B was the most sensitive
strain with MIC and MFC values of 10 and 20 μg/ml, respectively. MIC and MFC value of
MeOH extract against T. harzianum C were 20 and 30 μg/ml, respectively.
Our results showed that at the lowest concentrations, Asafoetida oleogum-resin
MeOH extract exerts a semispecific antifungal effect on the growth of T. harzianum
mycelium and stimulated the mycelial growth in Pleurotus spp. Although oleogum-
resins/essential oils are well known antimicrobial agents, they stimulates some
microorganisms and use them as carbon energy sources (18,29). Thus we suggest that the
weak parasitism of P. eryngii spp.-complex on roots and stems of umbellifers (family
Apiaceae, genera Eryngium, Ferula, Ferulago, Cachrys, Laserpitium, Diplotaenia and
Elaeoselinum) is mediated by allelopathic interactions. The oleogum-resin/essential oils
(or their components) shifts the microrganism balance in favour of those microrganisms
(e.g. Pleurotus spp.) that can tolerate them. Some even use them as a carbon and energy
source (4, 14).
Allelopathic effects of Asafoetida oleogum-resin extract on hyphal interactions
between Pleurotus spp. and T. harzianum in dual-culture
The interactions between T. harzianum and Pleurotus spp. in dual-culture on
PDA with Asafoetida oleogumresin MeOH extract are shown in Table 3. Three types of
competitive reactions were observed: CB1, partial replacement after initial deadlock at a
distance, CB2, complete replacement after initial deadlock at a distance and B, deadlock at
a distance without mycelial contact. All T. harzianum strains were antagonistic to
Pleurotus spp. in control, when the MeOH extract was not included in the culture
Mycelial growth inhibition
Asafoetida oleogum-resin extract concentration
Angelini et al
364
substrate. After the initial physical contact (4 to 7 days after culture), all T. harzianum
strains overtook, sporulated on and completely inhibited the mycelial growth of Pleurotus
spp.
When the MeOH extract was added to the culture substrate at 0.625 μg/ml, the
Pleurotus spp. were divided into three groups based on the AI values: I – active (AI > 10):
P. nebrodensis 193, II –moderately active (AI = 5 - 10): P. nebrodensis 529, P eryngii var.
ferulae 2, P eryngii var. eryngii 2 and III – weakly active (AI < 5) P eryngii var. ferulae 1,
P eryngii var. eryngii 1 and P. hadamardii. At the 1.25 μg/ml concentration Pleurotus spp.
were divided as follows: I – active (AI > 10): P. nebrodensis 529, P. nebrodensis 193, P
eryngii var. ferulae 1, P eryngii var. ferulae 2, P eryngii var. eryngii 1, P eryngii var.
eryngii 2 and II – weakly active (AI < 5): P. hadamardii. In the presence of the MeOH
extract, all strains of T. harzianum were moderately active (0.625 μg/ml of MeOH extract)
or slightly active (1.25 μg/ml of MeOH extract) against the Pleurotus spp. that either
completely or partially replaced T. harzianum (Table 3). T. harzianum A showed greater
competitive activity. In most pairings, the fungus partially or completely overtook the
Pleurotus spp. mycelium, after initial deadlock at a distance.
The antifungal and allelopathic activities of oleogum-resin/oils were correlated
with their chemical structure (27), hence, it is necessary to isolate the active component.
Ferulic acid (one of the main constituents of MeOH extract of Asafoetida oleogum-resin)
is an allelochemical found in soil (16). Asiegbu et al. (5), reported that 5 g/l ferulic acid
severely suppressed the growth of T. harzianum, while at 0.5 g/l, it slightly stimulated the
growth of Trametes versicolor and Pleurotus sajor-caju. Granetti (10,11) and Angelini et
al. (3) reported that ferulic acid did not effect the growth of some strains of Umbelliferae
oyster mushrooms but increased their colony diameter and dry weight. In contrast higher
concentrations of ferulic acid, inhibited and in same cases, totally blocked the growth of
test spp.
Fine structural modifications of T. harzianum and Pleurotus spp. hyphae induced by
Asafoetida oleogum-resin extract
Transmission electron microscopy (TEM) sections of Pleurotus spp. and
T. harzianum control hyphae and hyphae treated with MeOH extract at a concentration of
1.25 μg/ml medium are shown in Figure 2. The cell wall of non-treated healthy mycelia is
composed of a uniform layer, with a definite plasma-membrane and periplasm region with
normal thickness. A septum, typical of ascomycetes and basidiomycetes, can be seen in
Figure 2a and 2d, respectively. Treatment with the Asafoetida oleogum-resin MeOH
extract disturbed the normal ultrastructure of the fungal cells. Cell structure alterations
were observed in all fungal spp. 21 and 74 days after treatment with MeOH extract. After
21 days, the most frequent change in T. harzianum strains was an increase in number and
size of vacuoles in cells of same age (Figure 2b). While after 74 days, the strains showed
marked thickening of cell walls (up to twice of controls) and degeneration of hyphal
cytoplasm (Figure 2c). No adverse effects were observed in Pleurotus spp., 21 day after
treatment. However after 74 days, there was increased vacuolization and an alteration in
the cytoplasmic membrane that was partially detached from the cell wall (Figure 2f ).
The cytomorphological modifications (cell wall thickening and undulation of
plasmalemma) of T. harzianum and Pleurotus spp., induced by MeOH extract of
Antifungal and allelopathic effects of Asafoetida 365
Figure 2. Transmission electron micrographs. T. harzianum C: (a) hyphae of control, bar 1 µm; (b)
hyphae treated with Asafoetida oleogum-resin extract 0.125 µg/ml for 21 days showing
increase in the number and size of the vacuoles in the cells, bar 2 µm; (c) hyphae treated
with Asafoetida oleogum-resin extract 0.125 µg/ml for 74 days with a marked thickening
in the cell walls and degeneration of hyphal cytoplasm, bar 2 µm. P. nebrodensis 193: (d)
hypha of control, bar 1 µm; (e) particular of hypha treated with 0.125 µg/ml of Asafoetida
oleogum-resin extract after 21 days showing no alterations of cell structure, bar 1 nm; (f)
hypha treated with 0.125 µg/ml of Asafoetida oleogum-resin extract after 74 days showing
undulation of the cytoplasmic membrane, bar 1 µm.
Asafoetida oleogum-resin, are similar to those observed in other fungal spp. during
treatments with sterol-biosynthesis-inhibiting fungicides (12). Cell membrane alterations
after treatment with fungitoxic products could be caused by change in the composition of
the bi-lipid layer of same membrane (22). These alterations could, in turn, modify the
activity of membrane enzymes involved in cell wall formation causing anomalous
development.
CONCLUSIONS
The lower concentrations of MeOH extract of Asafoetida oleogum-resin,
corresponding to the allelopathic concentrations tested, could be used to stop proliferation
of green mould, which is currently being treated with other sanitizing agents. Solutions
Angelini et al
366
and emulsions used as sprays with or without a carrier are the preferred forms in which the
compounds can be applied with minimal effort to large areas of casing soil. Evaporation
by heating should also be considered. Ingestion of Asafoetida oleogum-resin has not been
associated with toxicity in adults (26). It is a potent antioxidant (21) and therefore ferulic
acid has shown some promise as a chemopreventive agent (17); Asafoetida may offer
some protection against carcinogenesis. Till now use of synthetic fungicides to control
green mould is discouraged due to negative effects on food: carcinigenocity,
teratogenicity, high and acute residual toxicity, longer degradation and side-effects in
humans (20). One of the major problems related to the use of these synthetic chemicals is
that the fungi can develop resistance. The use of higher concentrations of chemical, to
overcome microbial resistence increases the risk of high levels of toxic residues in
products. In contrast, the use of natural products to control green moulds does not seem to
foster the development of resistance by the contaminants. This is due to the presence of a
mixture of extract components which, apparently, have different mechanisms of
antimicrobial activity (25).
Further studies are needed to determine the strategies that can be used for
practical application. Consumer acceptance of mushrooms that have been treated with
Asafoetida oleogum-resin MeOH extract must also be tested.
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