Jelena Lević

Effects of Onion (Allium cepa L.) and Garlic (Allium sativum L.) Essential Oils on the Aspergillus versicolor Growth and Sterigmatocystin Production

In the present study the effects of individual and combined essential oils (EOs) extracted from onion (Allium cepa L.) bulb and garlic (Allium sativum L.) clove on the growth of Aspergillus versicolor and sterigmatocystin (STC) production were investigated. The EOs obtained by hydrodistillation were analyzed by GC/MS. Twenty one compounds were identified in onion EO. The major components were: dimethyl-trisulfide (16.64%), methyl-propyl-trisulfide (14.21%), dietil-1,2,4-tritiolan (3R,5S-, 3S,5S- and 3R,5R- isomers) (13.71%), methyl-(1-propenyl)-disulfide (13.14%), and methyl-(1-propenyl)-trisulfide (13.02%). The major components of garlic EO were diallyl-trisulfide (33.55%), and diallyl-disulfide (28.05%). The mycelial growth and the STC production were recorded after 7, 14, and 21 d of the A. versicolor growth in Yeast extract sucrose (YES) broth containing different EOs concentrations. Compared to the garlic EO, the onion EO showed a stronger inhibitory effect on the A. versicolor mycelial growth and STC production. After a 21-d incubation of fungi 0.05 and 0.11 μg/mL of onion EO and 0.11 μg/mL of garlic EO completely inhibited the A. versicolor mycelial growth and mycotoxins biosynthesis. The combination of EOs of onion (75%) and garlic (25%) had a synergistic effect on growth inhibition of A. versicolor and STC production.

The Overview on Toxigenic Fungi and Mycotoxins in Serbia and Montenegro

The available literature data indicate that the importance of toxigenic fungi and mycotoxins is much greater for the area of Serbia than for the area Montenegro. The knowledge on the occurrence of moulds (Gibberella saubinetii), as well as on animal and human diseases caused by mouldy food, especially by maize that has been traditionally grown as food and feed in the area of Serbia, dates as far back as the 19th and the beginning of the 20th century (Radic, 1872; Lozanie, 1904; Ranojevie, 1914). However, the largest outbreaks of epidemic moulds, mostly caused by Fusarium species, were registered in maize in 1955, 1968, 1972, 1974 and 1984 and in wheat in the early 1970s (Levic, 2004). The greatest outbreaks of animal diseases, especially diseases of pigs such as oestrogenism, vomiting and feed refusal, dermatoxicity, haemorrhages and others were recorded during the same and/or subsequent years. Alimentary intoxication of horses, described in some cases in 1955, indicates, to a great extent, clinical signs of eleucoencephalomalacia. This type of horse illness could be a result of the use of mouldy feedstuffs, mostly husked maize ears and shelled kernels (Raj ic, 1963), or of the occurrence of F. monilifoorme that prevailed in epidemic ear rot in 1955 (Marie, 1981). Nevertheless, it has never been completely observed nor identified.

Antifungal activities of basil ( Ocimum basilicum L.) extract on Fusarium species

The basil extract composition was determined by the GC-MS method and 38 different components were identified. The major components of the basil extract were estragol (86.72%), trans-α-bergamotene (2.91%), eucalyptol (2.67%), trans-ocimene (1.04%), linalool (0.72%), methyl-eugenol (0.71%), etc. The antifungal potential of the basil extract was tested against Fusarium oxysporum, F. proliferatum, F. subglutinans , and F. verticillioides isolated from cakes, using the agar plate method. Extract concentrations of 0.35 and 0.70% (v/v) significantly inhibited the growth of F. proliferatum (33.37 and 44.30%, respectively) and F. subglutinans (24.74 and 29.27%, respectively) whereas other investigated Fusarium species exhibited much lower sensitivity. The basil extract completely inhibited the growth of investigated Fusarium spp. at the concentration of 1.50% (v/v). Higher concentrations (0.35 and 0.70% (v/v)) reduced growth of aerial mycelium in all tested species. Strong medium pigmentation in the case of F. proliferatum and F. verticillioides was observed. The microscopic examination of the samples confirmed the presence of hyphae deformations with a frequent occurrence of fragmentations, thickenings and diminished sporulation. In addition to the basic, sensory, role the extract of basil has in the food product, it exerted significant antifungal properties, depending on its concentration. Key words : Basil (Ocimum basilicum L.) extract, components, antifungal activity, Fusarium spp.

distribution Frequency and Incidence of Seed-borne Pathogens of Some Cereals and Industrial Crops in Serbia

SUMMARy A total of 41 species of fungi were isolated from seed samples of barley, maize, soybean, and sunflower collected at different locations in Serbia. The majority of detected species occurred on barley (35 of 41 species or 87.8%) comparing to soybean (17 of 41 species or 41.5%), sunflower (16 of 41 species or 39.0%) and maize (15 of 41 species or 36.9%). Species belonging to genera Alternaria, Chaetomium, Epicoccum, Fusarium, Penicillium and Rhizopus were present on seeds of all four plant species. Alternaria species were dominant on soybean, barley and sunflower seeds (85.7%, 84.7% and 76.9%). F. verticillioides and Penicillium spp. were mainly isolated from maize seeds (100 and 92.3% respectively), while other species were isolated up to 38.5% (Chaetomium spp. and Rhizopus spp.). F. graminearum, F. proliferatum, F. poae and F. sporotrichioides were the most common Fusarium species isolated from barley (51.1-93.3%), while on the soybean seeds F. oxysporum (71.4%), F. semitectum (57.1%) and F. sporotrichioides (57.1%) were prevalent. Frequency of Fusarium species on sunflower seeds varied from 7% (F. equiseti, F. graminearum, F. proliferatum and F. subglutinans) to 15.4% (F. verticillioides). Statistically significant negative correlation (r = –0.678*) was determined for the incidence of F. graminearum and Alternaria spp., as well as, Fusarium spp. and Alternaria spp. (r = –0.614*), on barley seeds. The obtained results revealed that seedborne pathogens were present in most seed samples of important cereals and industrial crops grown under different agroecological conditions in Serbia. Some of the identified fungi are potential producers of mycotoxins, thus their presence is important in terms of reduced food safety for humans and animals. Therefore, an early and accurate diagnosis and pathogen surveillance will provide time for the development and the application of disease strategies.

An outbreak of Aspergillus species in response to environmental conditions in Serbia.

SUMMARY The frequency and incidence of A. flavus and A. niger on barley, maize, soybean, sunflower and wheat grain, the abundance of European corn borer (Ostrinia nubilalis) moths and their interaction depending on weather conditions in the 2008-2012 period were studied. Under the agroecological conditions of Serbia, the species A. niger is more frequent than A. flavus, and concerning the crop species, its frequency is highest in kernels of sunflower, than soybean, maize, barley and wheat. A. flavus was extremely dominant on all plant species in 2012 regarding its frequency: 100% on soybean, 95.3% on maize, 65.2% on barley, 57.1% on sunflower and 45.8% on wheat. Furthermore, the incidence of A. flavus was higher in 2012 than in previous years. The uncommonly high frequency and incidence of A. flavus infestation of maize grain in 2012 were caused by extremely stressful agrometeorological conditions, high temperatures and drought over the period from flowering to waxy maturity of maize. The precipitation factor (Pf = precipitation sum / average monthly temperature) showed that 2012 was extremely arid in June (Pf = 0.57), July (Pf = 1.45), August (Pf = 0.15) and September (Pf = 1.42). European corn borer (ECB) was a second factor causing intensive occurrence of A. flavus on maize grain in 2012. The maximum flight of ECB moths was recorded as early as in July (5,149) and, as a result of this, high damage and numerous injuries were detected at harvest. Those injuries were covered by visible olive-green powdery colonies typical of A. flavus. In the chronology of A. flavus occurrence, these are the first data on its very high frequency and incidence under the agroecological conditions of Serbia. As intensive infections with A. flavus were rare in the past 50 years, the level of aflatoxins in maize grain was low.

Investigation of toxigenic potential of fungal species by the use of simple screening method.

Potential for the biosynthesis of aflatoxin B1 (AFLB1), ochratoxin A (OTA), diacetoxyscirpenol (DAS), T-2 toxin (T2), and zearalenone (ZON) was investigated in different fungal species belonging to the genera: Aspergillus, Fusarium and Penicillium. The majority of investigated isolates originated from cereal grains, crushed oil soybean seed and fodder mixtures. The simple screening method developed by Filtenborg et al. (1983) was applied with few modifications concerning the type of the medium and cultivation temperature. In order to optimise the biosynthetic conditions for different mycotoxins, the following control cultures, known as mycotin producers were used: OTA - A. ochraceus CBS 108.08, DAS - F. semitectum (SL-B i SL-C), T2 - F. sporotrichioides (ITM-391, M-1-1, R-2301) and ZON - F. graminearum (GZ-LES). The fungi were cultivated on the standard medium (YESA - 2% yeast extract, 15% sucrose and 2% agar, pH 6.5), three modifications of the basic medium (YESAZn - the standard medium supplemented with 0.23 mg/l ZnSO4 x 5 H2O; PPSA - the medium in which yeast extract was replaced with peptone-1; PPSAZn - the medium in which yeast extract was replaced with peptone-1 and supplemented with 0.23 mg/l ZnSO4 x 5 H2O), and the potato-dextrose agar (PDA). The earlier biosynthesis of tested mycotoxins was recorded under the following cultivation conditions of fungal species: AFLB1 - after 14 days on PDA at 27±1°C, OTA - after 10 days on YESA and YESAZn at 27±1°C, DAS - after 10 days on PPSA and PPSAZn at 27±1°C, T2 - after 7 days on PPSAZn and PPSA at room temperature (20-24°C), and ZON - after 1 week on YESA and YESAZn at room temperature (21-24°C).

Genetic, pathogenic and toxigenic variability by F. proliferatum isolated from maize kernels

Genetic (mating population and vegetative compatibility), pathogenic and toxigenic variability of F. proliferatum isolated from maize kernels originating from different localities in Serbia were studied. Investigated isolates of F. proliferatum were fertile and most of them (8/13) belonged to the mating type MATD-2. Based on the complementary test isolates, F. proliferatum were grouped into 11 vegetative compatibility groups (VCGs) and two isolates were self-incompatible. All tested field isolates of F. proliferatum and nit mutants were pathogenic and most expressed medium pathogenicity. Field isolates of F. proliferatum showed a higher potential for biosynthesis of FB1 (up to 1,246.00 µg g -1 ) in relation to their nit1 (up to 53.41 µg g -1 ) and NitM mutants (up to 56.17 µg g -1 ). Based on genetic, pathogenic and

Fusariotoxins in wheat grain in Serbia.

Samples of wheat grain (41), collected during the 2010 harvest from seven localities in Serbia, were analyzed for the presence of zearalenone (ZEA), T-2 toxin, deoxynivalenol (DON) and fumonisine B1 (FB1). Results of Enzyme-Linked Immunosorbent Assay (ELISA) showed that all analysed samples were positive for the presence of at least one of four observed fusariotoxins. The most distributed mycotoxins were ZEA (90.2%, with the average concentration of 442.6μg kg-1) and T-2 (90.2%, with the average concentration of 24.2 μg kg-1). DON (73.2%) and FB1 (84.4%) were detected in a somewhat smaller number of samples, but their average concentrations were higher (1988.1 μg DON kg-1 and 882.7 μg FB1 kg-1). The established correlations between concentrations of DON and FB1 (r = 0.32) or DON and ZEA (r = 0.22) were not statistically significant. A negative correlation was established between concentrations of T-2 and FB1 (r= -0.24), as well as, between T-2 and DON (r = -0.36). Detected concentrations of ZEA and T-2 were bellow the level prescribed by the World Health Organisation (WHO), while concentrations of FB1 and DON detected in five that is, 17 samples, respectively, were above the permissible limit for human consumption.

Dynamics of deoxynivalenol and zearalenone production by Fusarium graminearum under laboratory conditions.

Toxicological investigations encompassed two cultures of Fusarium graminearum: (i) D2 isolate, originating from air was obtained on Sabouraud medium during a routine control of laboratory sterility conditions at the Department of Microbiology of the Center for Bio-Ecology in 2006, and (ii) GZ-LES control isolate, a well known producer of zearalenone (ZON) and deoxynivalenol (DON), was isolated from maize kernel collected at Leskovac in 1975. Preliminary analysis of fungal potential for the production of DON and ZON were performed by the modified rapid screening method of Filtenborg et al. (1983). Dynamics of DON and ZON biosynthesis was tested under different conditions of isolate cultivation: (i) in a basic liquid semi-synthetic medium with 2% yeast extract and 15% sucrose, pH 6.5 (YES), (ii) in broth with same concentrations of yeast extract and sucrose supplemented with 0.23 mg/l ZnSO4 x 5 H2O, pH 6.5 (YESZn) and (iii) on natural solid substrates such as wet sterilized maize and rice kernels. The quantitative determination of DON and ZON was performed in both liquid and natural solid substrates with thin-layer chromatographic methods (TLCs). The maximum yield of DON was recorded after three weeks of cultivation on maize kernels at 27±1°C. Contrary to the D2 isolate, which did not show the potential for the DON biosynthesis, the control isolate GZ-LES produced 645.6 ppb of the same type B trichothecene under previously mentioned conditions. The ZON biosynthesis by the isolate D2 (1.2 ppb) was observed after 2 weeks of the stationary cultivation in YES and YESZn at room temperature (17-19°C). The same isolate produced 0.74 ppb and 17.35 ppb ZON on maize and rice kernels after only 7 and 28 days of cultivation at the room temperature ranging from 17 to 19°C and from 15 to 23°C, respectively.

IN VITRO DEGRADATION OF DIACETOXYSCIRPENOL AND T-2 TOXIN BY USE OF MUCOR RACEMOSUS FRESEN. F. RACEMOSUS ISOLATE *

Under controlled in vitro conditions the capacity of the Mucor racemosus f. racemosus 1215/09 isolate to degrade type A trichothecenes (diacetoxyscirpenol - DAS and T-2 toxin) was observed in the liquid nutritive medium. According to previously performed experiments it was proved that the selected isolate, originating from sunflower meal, had the ability to degrade these fusariotoxins when growing on the modified Vogel’s agar supplemented with crude extracts of DAS and T-2 toxin. In order to determine biodegradation of fusariotoxins, the liquid nutritive medium - SPY (5% sucrose + 0.1% peptone + 0.1% yeast extract, pH 6.2) was simultaneously inoculated with the isolate M. racemosus f. racemosus 1215/09 and: a) Fusarium semitectum SL-B (DAS producer) or b) F. sporotrichioides R-2301 (T-2 toxin producer). The SPY media, inoculated with single fungal isolates, were used as a control of toxin biosynthesis. The cultures were incubated at room temperature (21-26oC) on the rotary shaker (175 rpm). After the 3-5-day incubation, the filtration of liquid cultures and the extraction of fusariotoxins from filtrates with ethyl-acetate were performed. Determinations of DAS and T-2 toxin were done by thin layer chromatography using silica gel G. Depending on the incubation duration, M. racemosus f. racemosus in the mixed culture with F. semitectum degraded from 90.0 to 99.97% of DAS present in the medium (40,000- 120,000 µg l-1), while in the mixed culture with F. sporotrichioides it degraded from 95.0 to 96.7% of T-2 toxin present in the medium (240,000 µg l-1). Sterile filtrates of mixed cultures and single culture of M. racemosus f. racemosus, obtained by passing liquid cultures through the 0.45-µm membrane filter and added to the SPY medium, did not affect degradation of type A trichothecenes that had been biosynthesised by isolates F. semitectum SL-B and F. sporotrichioides R-2301 in the liquid medium.