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Effects of g-radiation on the fungus Alternaria alternata in artificiallyinoculated cereal samples
R. Braghini a,Ã, C.R. Pozzi b, S. Aquino c, L.O. Rocha a, B. Correa a
a Departamento de Microbiologia, Instituto de Ciencias Biomedicas II, Universidade de Sao Paulo, Av. Prof. Lineu Prestes, 1374, CEP 05508-900 Sao Paulo, Brazilb Instituto de Zootecnia, Rua Heitor Penteado 56, CEP 13460-000, Nova Odessa, Sa o Paulo, Brazilc Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355 , CEP 01246-902 ,Sa o Paulo, Brazil
a r t i c l e i n f o
Article history:Received 13 August 2008
Received in revised form
9 March 2009
Accepted 9 March 2009
Keywords:
Foods
Cereals
Alternaria alternata
g-radiation
Fungi
a b s t r a c t
The objective of this study was to evaluate the effects of different g-radiation doses on the growth of Alternaria alternata in artificially inoculated cereal samples. Seeds and grains were divided into four
groups: Control Group (not irradiated), and Groups 1, 2 and 3, inoculated with an A. alternata spore
suspension (1Â106 spores/mL) and exposed to 2, 5 and 10kGy, respectively. Serial dilutions of the
samples were prepared and seeded on DRBC (dichloran rose bengal chloramphenicol agar) and DCMA
(dichloran chloramphenicol malt extract agar) media, after which the number of colony-forming units
per gram was determined in each group. In addition, fungal morphology after irradiation was analyzed
by scanning electron microscopy (SEM). The results showed that ionizing radiation at a dose of 5 kGy
was effective in reducing the growth of A. alternata. However, a dose of 10 kGy was necessary to inhibit
fungal growth completely. SEM made it possible to visualize structural alterations induced by the
different g-radiation doses used.
& 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Fungi can contaminate foods from cultivation to harvest,
during transportation and storage, and in various production
phases, whenever the fungus is under favorable conditions of
temperature and humidity (Frisvad and Samson, 1991). The effects
of fungal invasion include a reduced germination potential,
development of visible moldiness, discoloration, unpleasant odor,
loss of dry matter, heating, chemical and nutritional changes,
loss of quality, and production of mycotoxins (Christensen and
Kaufmann, 1969).
Mycotoxins are a group of toxic substances produced by
filamentous fungi, which, depending on their concentrations in
foods and feeds, may pose serious problems to human and animal
health (Moss, 1998).Species of the genus Alternaria are abundant in nature. These
fungal species invade cereals, oleaginous plants and other crops.
They are able to produce a wide variety of mycotoxins under
favorable conditions of temperature and humidity (Chulze et al.,
1995). The genus Alternaria produces about 71 known mycotoxins
(Montemurro and Visconti, 1992). A. alternata, the most toxigenic
species of the genus, is known to produce seven toxins in foods,
with alternariol (AOH) and alternariol monomethyl ether (AME)
being the most studied (Combe et al., 1970; Pero et al., 1973;Rosett et al., 1957; Visconti et al., 1986). Cereal grains are
frequently contaminated with various Alternaria species, particu-
larly A. alternata (Conner and Thomas, 1985). They occur naturally
in sunflower seeds (Dalcero et al., 1997; Pozzi et al., 2005), wheat
(Li et al., 2001; Logrieco et al., 1990; Aziz et al., 2006), corn (Aziz
et al., 2006; Torres et al., 1998), and rice (Broggi et al., 2007; Tonon
et al., 1997), among others.
Irradiation has been used to preserve foods and to produce
foods free of pathogenic microorganisms (Rustom, 1997). Irradia-
tion inactivates microorganisms that decompose foods, particu-
larly bacteria, molds and yeast. This treatment also destroys
pathogenic organisms, including worms and insects, which
degrade the quality of stored foods (OMS, 1989). Ionizing radiation
has been widely recognized as a method of decontaminationof foodstuffs. Many reviews have summarized the nutritional
adequacy of irradiated foods. They clearly demonstrate that
irradiation results in minimal, if at all noticeable, changes in the
taste, provided that the optimal dose for each type of food is not
exceeded (Diehl and Josephson, 1994). In general, irradiation
to the recommended doses changes the chemical composition of
foods very little. According to Diehl (1992, 1995), at doses below
1 kGy, nutritional losses are considered to be insignificant, and
none of the chemical changes found in irradiated foods is harmful,
dangerous or even lying outside of the limits normally observed
(Satin, 1993; Delincee et al., 1998). Aziz et al. (2006) concluded
that doses of up to 10 kGy are highly effective in microbial
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Applied Radiation and Isotopes
0969-8043/$ - see front matter& 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.apradiso.2009.03.004
à Corresponding author. Tel.: +551130917295; fax: +551130917354.
E-mail address: [email protected] (R. Braghini).
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decontamination and have no adverse effects on the nutritional
quality of cereal grains (see also World Health Organization,
1994).
In view of these facts, we have made an attempt to evaluate the
effects of a range of g-radiation doses on the growth of Alternaria
alternata in artificially inoculated cereal samples.
2. Materials and methods
2.1. Cereal samples
Samples of sunflower (Catissol 1), corn (Agromen 2012
Hybrid), rice (BRS Atlanta), and wheat (IAC 370) were used.
2.2. Determination of the natural fungal microflora in seeds and
grains
The samples were triturated, and 10 g aliquots of each sample
were transferred to Erlenmeyer flasks containing 90 mL of sterile
distilled water. The mixtures were then homogenized by shaking
for 30 min, and their 1 mL portions were used for serial dilutions
in sterile test tubes.
Two Petri dishes (90 mmÂ15mm) containing dichloran 18%
glycerol agar (DG 18, a medium used for analysis of foods with a
water activity, Aw, below 0.90 ( Jarvis et al., 1983) were used
for each dilution. A 0.1 mL aliquot of each sample was then
transferred to a Petri dish and evenly distributed over its surface
with a Drigalski spatula. The plates were incubated in an oven at
25 1C for 7 days. The numbers of colony-forming units per gram
(CFU/g) were determined thereafter (Pitt et al., 1983). The colonies
were identified to the genus levels according to Pitt and Hocking
(1997).
2.3. Irradiation of seeds and grains
The seeds and grains were divided into four groups: Control
Group, Groups 1, 2, and 3. Each group consisted of eight 200 g
samples, which were stored separately in plastic bags sealed with
adhesive tape and preliminarily irradiated to 20 kGy in order to
eliminate the natural contaminating microbiota. Irradiations were
carried out at Instituto de Pesquisas Energeticas e Nucleares
(IPEN–CNEN/SP) at temperatures between 25 and 28 1C using a
calibrated cobalt-60 source (Gammacell 220, MDS Nordion,
Ottawa, Canada) with the dose rate declining from 4.84 to
4.74kGy/h.
2.4. Humidity and temperature control
The samples were stored in sterile receptacles, and Aw was
adjusted to 0.98. A relative humidity 97.5% was maintained with a
solution saturated with saline and containing 30% of potassium
sulfate (K2SO4) (Winston and Bates, 1960).
2.5. Preparation of the spore suspension
Inoculum of A. alternata (isolated from CATI sunflower seeds
cultivated at Experimental Station of Zootechny, Nova Odessa)
was added to a Roux flask containing V8 agar (Stevenson, 1974)
and kept under continuous illumination with cold light for 15
days. After this period, the fungal surface was gently scraped with
a cell scraper and washed with sterile distilled water and Tween
80 (2 drops of Tween 80 in 100 mL of the solution).
Spores were counted in a Neubauer chamber, and the
concentration of the final solution was adjusted to 1Â106
spores/mL, according to the paper by Aziz et al. (1991).
2.6. Inoculation of the spore suspension into cereal samples
The cereal samples were inoculated with 1-mL portions of the A. alternata suspension containing 1Â106 spores/mL. The inocu-
lated samples were stored in a plastic container sealed withadhesive tape at 25 1C for 21 days in a BOD incubator, with
humidity and temperature controlled with a thermohygrometer.
After this period, samples of Groups 1, 2 and 3 were irradiated to
2, 5 and 10kGy, respectively. Samples of the control group were
not irradiated.
2.7. Determination of the fungal mycoflora in the control group and
irradiated samples
After incubation, the samples were triturated, irradiated, and
10 g aliquots of each sample were transferred to an Erlenmeyer
flask containing 90 mL of sterile distilled water. The flasks were
shaken for 30 min, and 1 mL portions of the solutions were used
for serial dilutions.For each dilution, we used two Petri dishes (90 mmÂ15mm)
containing dichloran rose bengal chloramphenicol agar (DRBC,
recommended for the enumeration of fungi commonly present in
foods; Pitt and Hocking, 1985) and dichloran chloramphenicol
malt extract agar (DCMA, recommended for the isolation of Alternaria species; Andrews, 1992). A 0.1 mL aliquot of each
dilution was added to the Petri dish and evenly distributed over
the surface with a Drigalski spatula. The plates were incubated in
an oven at 25 1C for 7 days, and the number of CFU/g was then
determined (Pitt and Hocking, 1997).
2.8. Determination of water activity
Water activity of the samples was determined with anAQUALAB CX-2 apparatus (Decagon, Pullman, WA, USA).
2.9. Scanning electron microscopy (SEM)
After the incubation, the samples were irradiated, and
three seeds or grains from each sample were put into a 40%
glutaraldehyde solution for 24 h. Next, the seeds or grains were
dried at 42 1C for at least 48 h and fixed to appropriate aluminum
bases. The material was then sputtered with gold, and the sample
was examined and photographed under a scanning electron
microscope.
2.10. Statistical analysis
Statistical analysis of variance of replicate measurements
with the Huynh–Feldt correction was performed with the SPSS
program, Version 12.0. The level of significance was 5%.
3. Results and discussion
3.1. Fungal mycoflora in unirradiated samples
Fungi were detected in all sunflower seed samples, as well as in
samples of corn, wheat and rice grain, with the genus Aspergillus
being the most frequent. Fungal counts ranged from 2 Â104 to
10Â104 CFU/g in sunflower seeds, from 3Â104 to 7Â104 CFU/g in
corn grains, from 3Â104
to 6Â104
CFU/g in wheat grains, and
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from 2Â104 to 105 CFU/g in rice grains. Water activities of the
samples were 0.58 for sunflower, 0.75 for corn, 0.70 for wheat, and
0.51 for rice. A. alternata was not isolated, probably due to the low
water activity of the analyzed samples. According to Sautour et al.
(2001), the minimal, optimal and maximal Aw levels necessary for
the growth of A. alternata are 0.88, 0.98, and 0.99, respectively.
Studies of the occurrence of fungi in sunflower seeds in Brazil
are few. Mentem (1985) found 22 potentially pathogenic fungal
species, with A. alternata being one of the most frequent.
The importance of A. alternata as a contaminant of sunflower
was confirmed by Pozzi et al. (2005), who detected the fungus in
46% of sunflower seed samples, with the highest frequencies
observed at water activities from 0.89 to 0.95. Aspergillus spp. and Penicillium spp. were isolated from corn
grain samples. The absence of Alternaria spp. and Fusarium spp. in
corn grain might be attributed to the low Aw levels in the corn
samples (0.75), which favor the growth of Aspergillus spp.
According to Sautour et al. (2001) and Lacey and Magan (1991),
the minimal water activity necessary for the growth of Alternaria
spp. and Fusarium spp. is close to 0.88. In Brazil, several studies
have found a high frequency of the genera Fusarium, Aspergillus
and Penicillium in corn grains, with Alternaria spp. detected in only
0.2% of the samples (Almeida et al., 2002). In Argentina, Gonzalez
et al. (1995) frequently isolated A. alternata from corn grains of the
1990 harvest. Torres et al. (1998) hypothesized that alternariol
and alternariol monomethyl ether occur in corn naturally.
In our study, the genera Aspergillus and Penicillium were
isolated from wheat samples. These results agree with findings
of Li and Yoshizawa (2000), who reported a higher frequency
of species of the genera Drechslera, Penicillium, Fusarium, and
Aspergillus, among others. In Brazil, Lima et al. (2000) analyzed
wheat samples stored for 3 months and identified species of the
genus Alternaria as predominant. Studies conducted in Australia
have also demonstrated a prevalence of A. alternata and A. infectoria
in wheat samples (Webley et al., 1997).Our isolation of genera Aspergillus and Penicillium from the rice
grain samples is in line with the findings of Tonon et al. (1997)
and Nunes (2001). However, A. alternata was the fungus most
frequently isolated from rice grains in Argentina (Broggi et al.,
2007).
3.2. Fungal mycoflora in irradiated samples
Fungal contamination of the four substrates studied (sun-
flower, corn, wheat and rice) was found to decrease with
increasing g-radiation dose (Table 1, Fig. 1). Water activity was
the same (0.98) before and after irradiation in all the substrates.
For all substrates, the largest number of CFU/g was observed in
the control group (unirradiated) on both the culture media. A
comparison between the control group and the groups irradiated
to 2, 5 and 10 kGy showed a reduction in contamination at 2 and
5 kGy and a complete absence of growth at 10kGy for all the four
substrates. We found fungi (0.1Â103 CFU/g) in only one of the
samples irradiated to 5 kGy. Similar results have been reported by
Ferreira-Castro et al. (2007), who studied the effects of g-radiation
on corn samples artificially contaminated with Fusarium verticil-
lioides. The authors observed fungal growth in 80% of the samples
irradiated to 5 kGy and the absence of growth at 10 kGy (the
maximal dose used). Aquino et al. (2005), evaluating the effectsof g-radiation on the growth of Aspergillus flavus, demonstrated
a higher resistance of the fungus to radiation as compared withF. verticillioides and A. alternata, which showed no growth after
exposure to 10 kGy.
According to Aziz et al. (1991), a g-radiation dose of 3 kGy was
sufficient to completely eliminate contamination of tomato juice
with A. alternata; however, Aw was 0.98. In another study using
medicinal plants, Aziz et al. (1997) showed that a dose of 5 kGy
was sufficient to eliminate fungi in the samples completely. The
genus Alternaria is known for its resistance to radiation, and some Alternaria species survived doses of 4.0 kGy (Beraha et al., 1960;
Maity et al., 2004).
According to Salama et al. (1977), fungi are resistant to
radiation due to mycelial water and the natural radioprotective
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Table 1
Numbers of colony-forming units per gram (CFU/g) in the irradiated substrates cultured on DRBC and DCMA.
Substrate CFU/g (Â103)
Dose
0 kGy 2 kGy 5 kGy 10 kGy
DRBC DCMA DRBC DCMA DRBC DCMA DRBC DCMA
Sunflower 18.979.7 (8) 10.575.6 (8) 4.472.1 (8) 3.671.8 (8) 0.01 (1) 0.01 (1) – –
Corn 27.3711.6 (8) 25.679.2 (8) 5.873.8 (8) 4.973.7 (8) 0.01 (1) 0.01 (1) – –
Wheat 6.975.4 (8) 3.671.2 (8) 1.770.7 (8) 1.370.6 (8) – 0.01 (1) – –
Rice 9.476.3 (8) 5.374.0 (8) 2.471.8 (8) 1.770.9 (8) – 0.025 (1) – –
The results are reported as means7single standard deviations; the numbers of positive samples are given in the parentheses.
0
5
10
15
20
25
30
D R B C
D C M A
D R B C
D C M A
D R B C
D C M A
D R B C
D C M A
Sunflower
0 kGy
2 kGy
5 kGy
10 kGy
Corn Wheat Rice
Fig. 1. Numbers of colony-forming units per gram (CFU/g) in the irradiated
substrates cultured on DRBC and DCMA.
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agents. Some investigators postulated that fungi produce numer-
ous metabolites, such as alcohols, acids, enzymes, pigments,
polysaccharides, and steroids, as well as some complex com-
pounds, such as ergotinine, and antibiotics, including penicillin,
notatin, flavicin, and fumigacin. In addition, intracellular fungal
components (sulfhydric compounds, pigments, amino acids,
proteins and fatty acids) have been reported to be responsible
for radioresistance of fungi (Aziz et al., 1997; Silveira, 1995).
Melanin, a polymer that protects live organisms against UV raysand ionizing radiation, has also been associated with fungal
radioresistance, especially among dematiaceous fungi. Aquino
(2007), analyzing medicinal plant samples, demonstrated a
higher resistance of Phoma spp. to a radiation dose of 5 kGy.
Other studies have also shown a higher resistance of dematiac-
eous fungi ( A. alternata, Cladosporium cladosporioides, Curvularia
lunata, and C. geniculata) to g-radiation (Saleh et al., 1988). In a
subsequent study, Aziz and Moussa (2002) investigated the effects
of g-radiation on the fungal mycoflora of fruits stored at
refrigeration temperatures (below 10 1C) and observed a progres-
sive reduction of fungal contamination in samples treated with
1.5 and 3.5 kGy. Ladaniya et al. (2003), who exposed three citrus
species to lowg-radiation doses (0.25, 0.5,1, and 1.5 kGy) and thenstored them at 6–71C for 75–90 days, showed that the dose
of 1.5 kGy was not sufficient to completely control fungi: it only
delayed fungal growth and, thus, increased the shelf-life of the
fruits.
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Intact A. alternata with spores in V8 medium
0 kGy 2 kGy
10 kGy
Sunflower
5 kGy
Fig. 2. SEM images of the cultures.
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According to the World Health Organization (1994), a dose of
2 kGy markedly reduces the number of microorganisms present in
foods, and higher doses (4–6 kGy) completely inhibit the presence
of fungi in foods (Saleh and Aziz, 1996; Abd El-Aal and Aziz, 1997).
These findings agree with the results of our study, which revealed
fungi in samples treated with a dose up to 5 kGy.
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0 kGy 2 kGy
Corn
Rice
5 kGy 10 kGy
0 kGy 2 kGy
10 kGy5 kGy
Fig. 2. (Continued)
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ANOVA and the Huynh–Feldt correction test ( po0.05) showed
a statistically significant difference in the fungal counts between
the unirradiated samples and the samples irradiated to 2 kGy, for
all the substrates. There was no statistically significant difference
between the counts in the two culture media (DRBC and DCMA)
for the unirradiated and irradiated (2 kGy) samples of corn, rice
and wheat ( p40.05); however, there was a significant difference
for sunflower ( po0.05).
3.2.1. SEM
The advantages of SEM, as compared with light microscopy, are
a better resolution, higher magnification, greater depth of fieldand greater versatility (Goodhew and Humpreys, 1998). It makes
fungal structures on the substrate and fungal growth more visible
(Bacon et al., 1992). SEM studies conducted by Torres et al. (2003)
have shown that water activity and temperature affect the growth
of Aspergillus ochraceus, A. alternata and Fusarium verticillioides in
maize grains. Murillo et al. (1999) used SEM to observe hyphal
penetration of F. moniliforme in maize grains (Fig. 2).
In this study, we analyzed the samples irradiated to 2, 5, and
10kGy, as well as control samples, with SEM in order to evaluate
morphological changes resulted from the exposure to ionizing
radiation. The magnitude of the changes was proportional to the
radiation dose.
The structures of the fungal mycelium were found unchanged
in the control samples and in the samples irradiated to 2 kGy. In
contrast, twisted filamentous forms with marked alterations in
the shape and surface of the hyphae and an aspect of ‘‘dehydra-
tion’’ and ‘‘rupture of the filaments’’ were found in the samples
exposed to 5 and 10 kGy. Filamentous forms featuring a melting-
like aspect and apparent adhesion to the seed surface were
observed in sunflower seeds treated identically, probably due to
rancification. However, the numbers of intact hyphae in corn, rice
and wheat grains were smaller. There were no spores in
inoculated grains. Ferreira-Castro et al. (2007) observed similar
alterations increasing with dose.
4. Conclusions
g-Irradiation to 5 kGy was effective in slowing the growth of A. alternata. However, a dose of 10 kGy was necessary to inhibit
fungal growth completely. DRBC medium was more effective in
the isolation of A. alternata in sunflower seeds. SEM made it
possible to identify structural changes induced by irradiation
to the different doses, which confirmed the CFU counting results.
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Fig. 2. (Continued)
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