Microbiology (2013), 159, 1720–1724
DOI 10.1099/mic.0.069039-0
Biological and chemical detection of fumonisins
produced on agar medium by Fusarium
verticillioides isolates collected from corn in Sohag,
Egypt
M. B. Aboul-Nasr and M. R. A. Obied-Allah
Correspondence
M. B. Aboul-Nasr
[email protected]
Received 4 May 2013
Accepted 6 June 2013
Department of Botany, Faculty of Science, University of Sohag, Sohag, Egypt
Fusarium verticillioides (Sacc.) Nirenberg is among the most common Fusarium species corn
pathogens worldwide, and has been recognized as a fumonisin B1 (FB1) and fumonisin B2 (FB2)
producer. In the present work, extracts of 58 F. verticillioides isolates from corn samples collected
from Sohag Governorate, Egypt, were tested for their biotoxicity and production of fumonisin
toxins. Forty-four Fusarium verticillioides isolates out of 58 tested produced FB1 or FB1 and FB2
(15 and 29 isolates, respectively) on potato–sucrose agar medium, detected by TLC, whereas the
other 14 isolates did not produce fumonisin toxins. HPLC crude extract analysis confirmed the
results from TLC plates. Brine shrimp larvae as well as the Gram-negative bacteria Pseudomonas
aeuroginosa showed low bio-sensitivity towards the F. verticillioides crude extract toxicity,
whereas the Gram-positive bacteria Bacillus cereus and Bacillus subtilis, especially B. subtilis,
showed higher sensitivity towards the tested Fusarium crude extracts. These results enabled us to
bio-evaluate and chemically detect fumonisin mycotoxins using a simple agar medium technique.
INTRODUCTION
Several fungi are associated with corn during pre- and
post-harvest periods, of which Fusarium verticillioides
represents the most important fungal species (Marasas
et al., 1984; Fandohan et al., 2005). In addition, F.
verticillioides has a wide geographical distribution and
many researchers have reported fumonisins being implicated in animal and human toxicoses all over the world
(Shephard et al., 1996; Munkvold & Desjardins, 1997;
Marasas, 2001; Taligoola et al., 2004). Wilson & Maronpot
(1971) identified F. verticillioides as the predominant
contaminant of mouldy corn that had caused cases of
equine leukoencephalomalacia (ELEM) in Egypt, and
reproduced ELEM by feeding culture material of the
fungus on corn. Also, fumonisin B1 (FB1) has a postulated
role in human oesophageal cancer and neural tube defects
(Marasas et al., 2004).
Fumonisins are a group of mycotoxins isolated initially
from corn culture of Fusarium moniliforme (syn. F.
verticillioides) (Gelderblom et al., 1988; Fadl, 1997).
Because of the great effect of Fusarium toxins, fumonisins,
on human and animal health, the aim of the present work
was to find an easy, reliable and inexpensive biological and
chemical assay to detect them. The protocols developed in
this study are helpful for the rapid and small-scale
detection of fumonisins from fungal mycelia occurring
Abbreviations: FB1, fumonisin B1; FB2, fumonisin B2.
1720
on contaminated corn samples, rather than using complicated or expensive tools. Many researchers depend on
genomic tools such as PCR analysis, which is no doubt one
of the most accurate methods of detection; however, it is
too complicated to be achieved in developing countries.
METHODS
F. verticillioides isolate collection. A total of 58 F. verticillioides
isolates were collected by the Laboratory of Fungal Physiology,
Department of Botany, Faculty of Science, University of Sohag, Egypt,
from corn samples using the direct plate method to isolate exogenous
Fusarium isolates (Pitt et al., 1992) and the surface disinfection plate
method to isolate endogenous Fusarium isolates (Müllenborn et al.,
2008) on DRBC (dichloran-Rose-Bengal-chloromphenicol) agar
medium (Tournas et al., 2006) and PSA medium (potato–sucrose
agar) (Booth, 1971, 1977). After single-spore isolation, each isolate
was identified on the basis of both macroscopic and microscopic
characteristics using identification keys. Collected cultures were
inoculated onto sterile PSA medium slants and incubated for 1 week
at 25 uC and stored at 5 uC for further chemical and biological
analysis.
Cultivation of F. verticillioides isolates on PSA medium and
extraction of toxins. Fifty-eight different F. verticillioides isolates
were examined for the production of fumonisins. Each isolate was
cultivated on PSA medium under aseptic conditions, using 15 cm
Petri dishes (Bailly et al., 2005), and incubated at 20±2 uC for 5 days.
At the end of the incubation period, all plates were transferred into a
refrigerator for 10 days. The agar media with mycelium were cut into
small pieces (1 cm2), transferred into 250 ml Erlenmeyer flasks
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Detection of fumonisins by Fusarium verticillioides
containing 50 ml 96 % methanol, and the contents were shaken on a
rotary shaker (200 r.p.m., 24 h) and filtered through filter paper
(Whatman no.1). The residue was then washed twice with the same
solvent (25 ml each). The methanol extracts were combined, dried
over anhydrous sodium sulphate and then evaporated to near dryness
under vacuum. The residue was transferred quantitatively to a small
dram vial with methanol and evaporated to near dryness under a
stream of nitrogen.
Biological assays. Brine shrimp (Artemia salina Linnaeus) larvae
(Korpinen, 1974; Ding et al., 2008) and antibacterial activity using the
disc diffusion method (Sleigh & Timburg, 1981) with Bacillus cereus
and Bacillus subtilis (Gram-positive), and Pseudomonas aeruginosa
(Gram-negative) were used to evaluate the bioactivities of the toxin
extracts from all F. verticillioides isolates tested.
Chromatographic analysis of the crude extracts of F. verticillioides isolates.
TLC analysis The thin-layer chromatographic technique adopted by
El-Kady & Moubasher (1982) was employed. The crude extracts
(10 ml) were spotted onto a TLC plate (aluminium sheet, silica gel;
Merck) along with 5 mg FB1 standard (purchased from SigmaAldrich). The spots were dried during application with a flow of
warm air and developed using 96 % methanol/water (80 : 20, v/v). The
developed plates were viewed after spraying with 50 % concentrated
sulphuric acid in methanol under short-wave (254 nm) and longwave (365 nm) UV irradiation. FB1 and fumonisin B2 (FB2) appear as
slight bluish-green spots under both near and far UV (Mubatanhema
et al., 1999) at Rf 0.7 and Rf 0.61, respectively.
HPLC analysis Five filtered fungal extracts (four fumonisin producers
and one non-producer) according to TLC analysis were analysed with
a Waters Binary model 1525 HPLC equipped with a Waters 2475
multi-wavelength fluorescence detector and the data workstation
software Breeze 2. A phenomenex C18 column (25064.6 mm
internal diameter, 5 mm particle size) from Waters was used along
with a mobile phase of methanol/0.1 M NaH2PO4 (75 : 25, v/v),
adjusted to pH 3.35 by the addition of phosphoric acid and filtered
through a membrane filter, in an isocratic system. The separation was
performed at ambient temperature at a flow rate of 1.0 ml min21.
The injection volume was 20 ml for both standard solutions and
sample extracts. The fluorescence detector was operated at 335 nm
(excision) and 440 nm (emission) wavelengths.
Standard solution was prepared from stock standard solution by
transferring 25 ml to a vial and then adding 975 ml acetonitrile/H2O
(1 : 1, v/v). The obtained standard solution contained 25 mg ml21 FB1.
Fifty microlitres of FB1 working standard solution was transferred to
the base of a small vial and then mixed with 225 ml o-phthaldialdehyde (OPA) reagent and 10 ml of reacted mixture was injected into
the HPLC machine within 1 min.
The purified dry film residue of sample extract was dissolved in 200 ml
methanol. Fifty microlitres of this extract was transferred to the base
of a small vial and then 225 ml OPA reagent was added, mixed and
10 ml derivative was injected into the HPLC machine within 1 min of
adding OPA reagent. The mixed solutions of standard as well as
sample extracts after derivatization were filtered through a 0.22 nm
membrane filter. FB1 contents in the sample were calculated from
chromatographic peak areas (AOAC International, 2007).
RESULTS AND DISCUSSION
Fifty-eight F. verticillioides isolates (10 endogenous isolates
and 48 exogenous isolates) were collected from maize
samples from Sohag Governorate in Egypt and tested for
http://mic.sgmjournals.org
fumonisin production. Results of the biological assays are
summarized in Tables 1 and 2. Results of the TLC analysis
of the crude culture extracts of F. verticillioides isolates
tested are also shown. Out of 48 exogenous F. verticillioides
isolates, two were strongly toxic, 11 isolates showed
moderate toxicity, whereas the rest of the tested isolates
ranged from non-toxic to low toxicity. In the case of
endogenous F. verticillioides isolates, one isolate showed
high toxicity and two isolates had moderate toxicity,
whereas the rest had low or non-toxic bioactivity in the
brine shrimp (A. salina) bioassay. Hartl & Humpf (2000)
found that the brine shrimp assay proved to be a
convenient and rapid system for toxicity assessment of
this group of mycotoxins. Mexı́a-Salazar et al. (2008) also
used a white shrimp bioassay to evaluate the biotoxicity of
FB1 and reported that at all FB1 levels tested, the shrimps
were affected. Other researchers have used the brine shrimp
bioassay to evaluate the biotoxicity of FB1 (Hlywka et al.,
1997; Moretti et al., 2007). However, the lower sensitivity
of the Artemia species to several chemical or physical
agents in comparison to other invertebrate test organisms
and the decreased solubility of some chemical substances in
saline or seawater medium, in addition to the various
conditions that control the test, such as temperature, pH,
chemical composition of the medium, oxygen, photoperiod, nutrients, some population effects, type of growth
stage, etc. may affect the toxicity results (Sorgeloos et al.,
1978; Song & Brown, 1998; Soares et al., 1992; Okamura
et al., 2000; Guerra, 2001; Nałecz-Jawecki et al., 2003;
George-Ares et al., 2003; Mayorga et al., 2010).
The Gram-positive bacteria B. cereus and B. subtilis and the
Gram-negative bacterium P. aeruginosa were used to test
the antibacterial activity of the F. verticillioides isolate
extracts. None of the 58 F. verticillioides crude extracts
tested had any antibacterial activity against Gram-negative
bacteria. In contast, 50 (40 exogenous and 10 endogenous)
F. verticillioides isolate extracts showed antibacterial activity
against B. subtilis and 42 (32 exogenous and 10 endogenous) extracts showed activity against B. cereus. Larger
growth inhibition zones were observed with B. subtilis,
indicating greater sensitivity toward the crude extracts of
the F. verticillioides isolates tested (Tables 1 and 2). These
results are in full agreement with those of Hugo & Russell
(1983), Farag et al. (1989), Zohri et al. (1995), Eftekhar
et al. (2005) and Murthy et al. (2006). The tolerance shown
by Gram-negative bacteria could be due to the permeability
barrier provided by the cell wall or the membrane
accumulation mechanism (Adwan & Abu-Hasan, 1998)
or related to lipopolysaccharides in their outer membranes
(Sawer et al., 1997; Gao et al., 1999).
TLC analysis of F. verticillioides culture extracts confirmed the
results of the bioassays, showing that, of the 58 tested isolates,
15 isolates (12 exogenous and 3 endogenous) produced FB1
whereas 29 other isolates (22 exogenous and 7 endogenous)
produced both FB1 and FB2. Several other studies dectected
FB1 and FB2 by TLC as well (Ross et al., 1990; Pepeljnjak et al.,
2003; Bailly et al., 2005; Benedetti et al., 2006).
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M. B. Aboul-Nasr and M. R. A. Obied-Allah
Table 1. Biotoxicity of fumonisins produced by exogenous F. verticillioides isolates grown on PSA medium and TLC analysis of their
crude extracts
Isolate
Toxicity tests
A. salina*
BacteriaD
B. subtilis
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
N
L
N
L
N
L
N
N
N
N
L
L
L
N
L
L
L
L
L
L
M
L
L
L
L
M
M
M
M
N
L
L
N
M
L
M
M
N
H
H
M
N
M
N
L
M
L
N
Mycotoxins detected on TLC
–
1.9
–
1.3
–
1.6
1.2
–
1
1.5
1.2
1.3
1.2
1.1
1.7
1.5
1.2
1.2
1.2
1
1.1
1.2
1.3
1.7
1.7
2.3
0.7
2.2
2.6
1.3
2
1.3
1.3
1.3
1.6
1.4
1.4
–
1.1
1.3
1.8
–
1.4
1.3
1.8
1.3
–
–
Fumonisinsd
B. cereus
P. aeuroginosa
FB1
FB2
–
1
–
–
–
1.4
1.2
–
–
1.4
–
1.3
–
–
1
1.3
–
1.2
–
1
–
1.5
1.3
2
2
2
1.1
1.9
2
1.3
1.8
1.3
1.1
1.5
1.3
1.5
1.4
–
1.5
1.5
1.9
–
1.1
1.1
1.3
1.3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2
+
2
+
2
+
+
2
+
2
+
+
+
2
+
+
+
+
+
2
+
+
+
+
+
+
+
+
+
2
+
2
+
+
2
+
+
2
+
+
+
2
+
2
+
+
+
2
2
+
2
+
2
+
+
2
2
2
2
2
2
2
+
2
+
+
2
2
+
2
2
+
+
+
+
+
+
2
+
2
+
+
2
+
2
2
2
+
+
2
+
2
2
+
2
2
*H, High toxicity (more than 75 % dead larvae); M, moderate toxicity (50–75 % dead larvae); L, low toxicity (25–50 % dead larvae); N, non-toxic (0
dead larvae).
DClear inhibition zone diameter (cm).
d–, Not detected; +, detected.
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Microbiology 159
Detection of fumonisins by Fusarium verticillioides
Table 2. Biotoxicity of fumonisins produced by endogenous F. verticillioides isolates grown on PSA medium and TLC analysis of their
crude extracts
Isolate
Toxicity tests
A. salina*
BacteriaD
B. subtilus
1
2
3
4
5
6
7
8
9
10
N
N
L
M
N
M
N
N
N
H
Mycotoxins detected on TLC
Fumonisinsd
B. cereus
P. aeuroginosa
FB1
FB2
1.3
1.5
1.7
2
2.2
1.5
1.8
1.8
1.3
1.8
–
–
–
–
–
–
–
–
–
–
+
+
+
+
+
+
+
+
+
+
+
2
+
2
+
2
+
+
+
+
1.8
1.8
1.9
2.3
2.3
2
2.2
1.7
1.6
1.7
*H, High toxicity (more than 75 % dead larvae); M, moderate toxicity (50–75 % dead larvae); L, low toxicity (25–50 % dead larvae); N, non-toxic
(0 dead larvae).
DClear inhibition zone diameter (cm).
d–, Not detected; +, detected.
The HPLC analysis of five cleaned extracts of F.
verticilloides isolates (four fumonisin producers and one
non-producer according to TLC analysis) detected FB1
ranging from 0.037 to 1.0 mg ml21 on agar medium, in the
case of the fumonisin producers, and no detectable amounts
of fumonisins in the case of the non-toxic isolate. These
results confirmed the production of fumonisins by the tested
F. verticilloides isolates. This level of production was lower
than those reported in Ghana, where the tested F.
verticillioides strains produced all three types of fumonisins
(B1, B2 and B3) and the total fumonisin levels ranged from
127 to 11 052 mg g21 on maize substrates, not agar medium
(Kpodo et al., 2000). Also, Sydenham et al. (1993) reported
higher levels of fumonisin produced by a strain of F.
verticillioides isolated from Argentinean maize and also grown
on natural medium. Consistent with our results, Visconti &
Doko (1994) and Castellá et al. (1996) showed low fumonisin
production for F. verticillioides isolates from maize and mixed
poultry feeds. Also, a few F. verticillioides isolates from Nepal
(Nelson et al., 1991) did not produce any fumonisin and
several isolates of this species from south-east Asia (Miller et
al., 1993) produced fumonisins at low levels, whereas some
isolates from Australia (Nelson et al., 1991) produced only
trace quantities of FB1. Moreover, Fadl (1997) screened 18
isolates of F. verticillioides from corn samples at Minia
Governorate in Egypt for their ability to produce fumonisins
on polished rice grains. Based on TLC analyses, it was found
that 14 of the 18 isolates tested produced FB1 and FB2,
whereas four isolates were not able to produce fumonisins.
medium technique, which is an easy, inexpensive, timesaving and reliable method.
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