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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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0969-8043/$ - see front matter& 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.apradiso.2009.03.004

Ã 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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R. Braghini et al. / Applied Radiation and Isotopes 67 (2009) 1622–1628 1623



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.

R. Braghini et al. / Applied Radiation and Isotopes 67 (2009) 1622–16281624



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.

ARTICLE IN PRESS

Intact A. alternata with spores in V8 medium

0 kGy 2 kGy

10 kGy

Sunflower

5 kGy

Fig. 2. SEM images of the cultures.




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.

ARTICLE IN PRESS

0 kGy 2 kGy

Corn

Rice

5 kGy 10 kGy

0 kGy 2 kGy

10 kGy5 kGy

Fig. 2. (Continued)




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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Wheat

0 kGy 2 kGy

5 kGy 10 kGy.

Fig. 2. (Continued)




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