Acta agriculturae slovenica, suplement 1(avgust 2004), 233–238. 
12
th
 Int. Symp. “Animal Science Days”, Bled, Slovenia, Sept. 2–4, 2004. 
Original scientific article 
Izvirni znanstveni prispevek 
FUNGAL INFECTION AND OCCURENCE OF ZEARALENONE IN BARLEY 
HARVESTED IN 2003 IN SERBIA 
Marija ŠKRINJAR
 a)
 and Sunčica KOCI Ć-TANACKOV
 b)
 
a)
 Univ. of Novi Sad, Fac. of Technology, Bulevar cara Lazara 1, YU-21000 Novi Sad, Serbia and Montenegro, 
Prof., Ph.D. 
b) 
Same address. 
ABSTRACT 
Mycological investigation in 11 winter barley samples was performed. All of barley samples 
were infected with moulds. The ratio of infected kernels varied from 11.7% (sample SSK8) to 
13.0% (sample SSK9) after 14 days of incubation. The fungi were isolated from kernels and they 
were classified into 6 genera and 11 species. Genus Fusarium was presented with the highest 
number of species (5). F.poae, a toxigenic species of the genus Fusarium, was present in all 
samples tested. The highest ratio (about 66%) of all mycopopulations belonged to genus 
Alternaria spp. All barley samples contained zearalenone, but the concentration was low (from 
5.2 to 52.0 µg kg
–1
). 
Key words: animal nutrition / feed mixtures / barley / fungal infection / zearalenone / Serbia and Montenegro 
OKUŽBE Z GLIVAMI IN POJAV ZERALENONA V VZORCIH JE ČMENA, POŽETIH 
V LETU 2003 V SRBIJI 
IZVLE ČEK 
Preiskali smo 11 vzorcev je čmena na prisotnost plesni in vrsto plesni. Vsi vzorci je čmena so bili 
okuženi s plesnijo. Delež okuženih zrn se je po 14 dnevih inkubacije gibal od 11,7 % (vzorec 
SSK8) do 13,0 % (vzorec SSK9). Iz teh zrn smo glive izolirali in razvrstili v 6 rodov in 11 vrst. 
Rod Fusarium je bil zastopan v najve čjem številu razli čnih vrst (5). F.poae, toksi čno vrsto rodu 
Fusarium, smo našli v vseh testiranih vzorcih. Najve čji delež (okrog 66 %) vseh populacij plesni 
je pripadal rodu Alternaria spp. Vsi vzorci je čmena so vsebovali zearalenone, vendar so bile 
koncentracije majhne (od 5,2 do 52,0 µg kg
–1
). 
Klju čne besede: prehrana živali / krmne mešanice / je čmen / glivice / okužba / zearalenon / Srbija in Črna gora 
INTRODUCTION 
Barley (Hordeum sativum) is a sort of grain (fam. Gramineae) which is used as a component 
of feed mixture. 
Barley kernels can be attacked by microorganisms during vegetative period. But, more often 
microbial infection can occure during the storage of cereals. 
A very important role in micropopulations of cereals, including barley, have fungi. As it is 
known, many of them are producers of varios toxic metabolites, such as aflatoxins, ochratoxins, 
trichothecenes, zearalenone and others (Marasas et al., 1984; Samson and van Reenen Hoekstra, 
1988; Pittet, 1998; Stojanovi ć et al., 2003; Koci ć-Tanackov and Škrinjar, 2004). 
In our country ochratoxin A and zearalenone are quite frequent contaminants of different 
types of agricultural commodities (Bo čarov-Stan či ć et al., 1997; Škrinjar et al., 2002, 2003). The 
contaminations is influenced primarily by climatic conditions (relative humidity and 
http://www.bfro.uni-lj.si/zoo/publikacije/zbornik/suplementi/index.htm

Acta agriculturae slovenica, suplement 1(avgust 2004). 
 
234 
temperature), soil moisture, insect damage, mineral nutrition deficiencies and other factors. But, 
there is no doubt that the highest concentrations of toxins are associated with the postharvest 
fungal growth on poorly stored agricultural products. 
Zearalenone, known as F-2 toxin is one of the main fungal toxin detected in our country. It is 
an estrogenic toxic metabolite produced by different species of genus Fusarium ( F. 
graminearum, F. culmorum, F. avenaceum, F. poae etc.), which frequently colonize cereal 
crops, especially during the storage. 
Despite its biological effects, the presence of zearalenone in feeds has long been a problem in 
animal husbandry, notably by causing infertility and related disorders particularly in swine and 
sheep (Pittet, 1998). 
The natural occurrence of zearalenone in a variety of agricultural commodities (wheat, barley, 
corn) has been reviewed in extensive detail by many investigators (Marasas et al., 1984; Kuiper-
Goodman et al., 1987; Pittet, 1998; Škrinjar et al., 2002, 2003). 
The aim of this study was to investigate a fungal infection of barley kernels during the storage 
and the presence of zearalenone in them, as well. 
MATERIAL AND METHODS 
Mycological investigation (total viable count of moulds, identification of mould species) in 
several winter barley samples (11) was performed. All samples were analyzed on the presence of 
zearalenone, too. 
Determination of total viable count of moulds per one kernel was done as follows. Sample 
(100 kernels) were immersed in a 100% of 0.4% of sodium chypochlorite and shaked on shaker 
for 2 min. After that, chlorine solution was decanted and the sample rinsed with sterile distilled 
water (2 × 100 ml). Under septic conditions kernels were placed on Petri dishes ( φ13 cm) 
containing sterile filter paper (25 kernels/Petri dish) and 10 ml of sterile distilled water. 
Incubation was performed 14 days at 25 °C. 
Identification of isolated fungi was carried out according to Nelson et al. (1983) and Samson 
and van Reenen-Hoekstra (1988). 
Qualitative and quantitative determination of zearalenone was examined by using the 
fluorometric method (Fluorometric “Vicam” series 4), Zearala Test
TM
. 
RESULTS AND DISCUSSION 
Mycological investigation 
As can be seen from Table 1, between 71 (sample SSK9) and 89 barley kernels (sample 
SSK1) was infected with moulds already after 7 days of incubation. It was found that infection 
degree increased about 20% during further incubation time in some cases. After 14 days total 
viable count of moulds per sample (100 kernels) arranged from 115 (sample SSK2) to 130 
(sample SSK9). 
Numerous fungi were isolated from barley samples. They were classified into 6 genera 
(Alternaria, Aspergillus, Fusarium, Rhizopus, Scopulariopsis and Ulocladium) and 11 species 
(Table 2). 
Genus Fusarium was present with the higest number (5) of different species (F. acuminatum, 
F. avenaceum, F. culmorum, F. poae, F. sporotrichioides). All of these species are known as 
producers of various toxic metabolites (Nelson et al., 1983). Except F. acuminatum, all other 
Fusarium species, isolated in these experiments are producers of zearalenone. 
 

Škrinjar,
 
M. and Koci ć-Tanackov,
 
S. Fungal infection … of zearalenone in barley harvested in 2003 in Serbia. 
235
Table 1. Number of infected kernels and total viable count of moulds (per sample/ 100 kernels) 
 
No. of infected kernels after Total count of moulds after Sample- 
Sign 
7 days 14 days 7 days 14 days 
SSK1 89 94 117 123 
SSK2 82 91 101 115 
SSK3 80 90 107 125 
SSK4 87 92 112 120 
SSK5 78 89 104 118 
SSK6 73 87 102 121 
SSK7 84 92 113 121 
SSK8 79 95 88 117 
SSK9 71 92 79 130 
SSK10 83 97 94 123 
SSK12 76 96 84 125 
 
Table 2. Mould species isolated from barley 
 
Genus Species 
alternata (Fr.) Keissler 
Alternaria 
brassicicola (Schw.) Wiltshire 
Aspergillus niger van Tieghem 
acuminatum Ellis & Everhart 
avenaceum (Fr.) Sacc. 
culmorum (W. G. Smith) Sacc. 
poae (Peck) Wollenweber 
Fusarium 
sporotrichioides Sherb. 
Rhizopus stolonifer (Ehrenb.) Lind. 
Scopulariopsis fusca Zach 
Ulocladium chartarum (Preuss) Simmons 
 
It was determined that a share of Fusarium spp. in mycopopulation varried from 11% (sample 
SSK10) to 30% (sample SSK6) (Fig. 1). F. poae was the most frequent species of genus 
Fusarium. It was isolated from all samples tested (Fig. 2). About 27% of barley samples were 
infected with F. acuminatum and F. avenaceum and about 9% with F. culmorum and F. 
sporotrichioides. 
It is necessary to point out, having in mind total mycopopulations isolated from barley (1100 
kernels), that the highest incidence of fungal infection was observed with Alternaria species 
(Fig. 3). Genus Alternaria was presented by two species, A. alternata and A. brassicicola. As it 
was established in our earlier investigations (Škrinjar et al., 1997). A. alternata was extremly 
frequent. Naimely, all samples were infected by it (Table 3). 
 

Acta agriculturae slovenica, suplement 1(avgust 2004). 
 
236 
 
 
Figure 1. Share of Fusarium spp. in mycopopulations isolated from barley samples. 
 
 
 
Figure 2. Frequency of Fusarium spp. in total mycopopulations of genus Fusarium. 
 
Zearalenone 
It was found that all barley samples were contamited with zearalenone at concentrations from 
5.2 to 52.0 µg kg
–1
 (Table 4). In spite of the fact that concentrations were low, so frequent 
occurrence of zearalenone is underisable because of its biological effects. 
 
 
9%
100%
9%
27% 27% 
0 
20 
40 
60 
80 
100 
A B C D E
Fusarium spp. 
Frequency of genus Fusarium, %  
 
F. acuminatum
B = F. avenaceum
C = F. culmorum
D = F 
. poae 
E = F. sporotrichioides
17% 
23% 
27% 
13%
18%
30%
20% 20% 20% 
11% 
17%
0 
10 
20 
30 
40 
50 
SSK1 SSK2 SSK3 SSK4 SSK5 SSK6 SSK7 SSK8 SSK9 SSK10 SSK12
Sample
Share in mycopopulations, % 

Škrinjar,
 
M. and Koci ć-Tanackov,
 
S. Fungal infection … of zearalenone in barley harvested in 2003 in Serbia. 
237
 
 
Figure 3. Share of fungal genera in total mycopopulations. 
 
Table 3. Mycopopulations isolated from barley samples 
 
Sample- Sign Mould species  Sample- Sign Mould species 
Alternaria alternata  Alternaria alternata 
Fusarium avenaceum  Fusarium poae 
F. culmorum  Mycelia sterilia 
F. poae  
SSK7 
Scopulariopsis fusca 
Mycelia sterilia  Alternaria alternata 
SSK1 
Rhizopus stolonifer  A. brassicicola 
Alternaria alternata  Fusarium poae 
Fusarium poae  Mycelia sterilia SSK2 
Mycelia sterilia  Rhizopus stolonifer 
Alternaria alternata  
SSK8 
Ulocladium 
Fusarium poae  Alternaria alternata SSK3 
Mycelia sterilia  Fusarium poae 
Alternaria alternata  Mycelia sterilia 
Fusarium acuminatum  Scopulariopsis fusca 
F. culmorum  
SSK9 
Ulocladium 
F. poae  Alternaria alternata 
Mycelia sterilia  Aspergillus niger 
SSK4 
Ulocladium chartarum  Fusarium poae 
Alternaria alternata  
SSK10 
Mycelia sterilia 
A. brassicicola  Alternaria alternata 
Fusarium acuminatum  A. brassicicola 
F. poae  Fusarium avenaceum 
SSK5 
Mycelia sterilia  F. poae 
Alternaria alternata  
SSK12 
F. sporotrichioides 
Fusarium acuminatum    
F. poae    
SSK6 
Mycelia sterilia    
4.4% 4.7%
0.9%
24.0%
0.2%
68.8% 
0 
10 
20 
30 
40 
50 
60 
70 
A B C D E F
Genus 
Share in total mycopopulations, % 
A = Alternaria
B = Aspergillus
C = Fusarium
D = Rhizopus
E = Scopulariopsis
F = Ulocladium

Acta agriculturae slovenica, suplement 1(avgust 2004). 
 
238 
Table 4. Presence of zearalenone in barley samples 
 
Sample – Sign Zearalenone µg⋅kg
–1
 
SSK1 22.0 
SSK2 5.2 
SSK3 8.6 
SSK4 5.8 
SSK5 7.1 
SSK6 8.2 
SSK7 52.0 
SSK8 24.0 
SSK9 37.0 
SSK10 19.0 
SSK12 16.0 
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