In Serbia, aspergillus ear rot caused by the disease pathogen Aspergillus parasiticus (A. parasiticus) was first detected in 2012 under both field and storage conditions. Global climate shifts, primarily warming, favour the contamination of maize with aflatoxins in temperate climates, including Serbia. A five-year study (2012–2016) comprising of 46 A. parasiticus strains isolated from maize kernels was performed to observe the morphological, molecular, pathogenic, and toxigenic traits of this pathogen. The HPLC method was applied to evaluate mycotoxin concentrations in this causal agent. The A. parasiticus isolates synthesised mainly aflatoxin AFB1 (84.78%). The percentage of isolates synthesising aflatoxin AFG1 (15.22%) was considerably lower. Furthermore, the concentration of AFG1 was higher than that of AFB1 in eight isolates. The polyphase approach, used to characterise isolates, showed that they were A. parasiticus species. This identification was verified by the multiplex RLFP-PCR detection method with the use of restriction enzymes. These results form an excellent baseline for further studies with the aim of application in the production, processing, and storage of cereal grains and seeds, and in technological processes to ensure the safe production of food and feed.

The species of the genus Aspergillus, A. flavus and A. parasiticus, are the most aflatoxin-producing fungi. All previous studies carried out under the production conditions of Serbia showed no presence of A. parasiticus on wheat kernel. On the basis of changes in climatic factors, such as occurrence of high temperatures and prolonged droughts, which favour increased frequency of Aspergillus spp., we assumed that this pathogen can also be present in Serbia. The significance of direct losses as a consequence of wheat kernel infection, as well as potential contamination with aflatoxins, have pointed out to the need to determine the presence of toxigenic potential of A. flavus and A. parasiticus isolates originating from Serbia. For that purpose, wheat kernel samples were collected in nine locations. According to morphological, toxicological and molecular traits of isolated fungi, the presence of A. flavus and A. parasiticus was confirmed. This is the first time that A. parasiticus was identified on wheat under climatic conditions in Serbia. This study indicates that these pathogens may be a potential danger in wheat production in the region of Serbia. This danger will be much more certain if global climatic changes continue as they will provide more intensive development of these pathogens.

Barley (Hordeum vulgare L.) is a secondary grain crop in Serbia used commercially for animal feed, seed and for human food applications. The production of barley in the 2016/17 growing season reach...

Species of the genus Aspergillus, section Flavi are facultative plant pathogens with the capability to contaminate primary agricultural products in the field, at harvest, in warehouses, and during processing. Infection is done through silk or injuries on maize ears. The objective of this study was to test pathogenicity of 25 A. parasiticus isolates on barley leaves. A parasiticus was isolated from maize kernels collected from 22 locations in Serbia during 2013-2015. Inoculation of the first barley leaf was done by the spore suspension of the fungus A. parasiticus. The spot sizes were statistically compared and this comparison showed that the isolate MRIZP 3930 was the least virulent. The isolate MRIZP 3819 was the most virulent seven days after inoculation and together with the isolate MRIZP 4265 was classified into the most aggressive group rated as 5. Pathogenicity was confirmed in all isolates.

In recent years, countries in Southeast Europe are facing climate changes characterized by extreme hot weather, which contribute to the increased frequency of Aspergillus species. Because of these changes, Aspergillus parasiticus was isolated, for the first time, from maize grain in Serbia (Nikolic et al, 2018). The presence of black powdery mycelia on maize ears indicated occurrence of species of the genus Aspergillus section Nigri, which led to the need for detailed identification of these fungi. Disease incidence ranged from 10 and 15% in August 2013. Maize ears with black powdery symptoms were collected from field in Zemun Polje, Serbia. Symptomatic kernels were surface sterilized with 1% sodium hypochlorite solution for 3 min, rinsed three times with sterilized water, then incubated at 25°C in the dark for 7 days on potato dextrose agar (PDA). Twenty isolates were identified as genus Aspergillus section Nigri. Monospore cultures formed black cottony colonies with a yellowish border on PDA. The average colony diameter was 50 mm. In order to reliably identify, isolates were transferred to Malt Extract agar (MEA) and Czapek Yeast Autolysate agar (CYA) (Samson et al, 2014). On CYA fungal colonies consisted of a white mycelium, covered by a layer of black conidiophores. On MEA fungal colonies were dense, black, with yellowish border. The reverse side was colorless to pale yellow, with a yellow ring in the middle. The average size of conidia was 4.3 µm. The conidia were globose to sub-globose, smooth to roughened, which coincides with previous research (Silva et al, 2020). Given that the fungi Aspergillus niger and Aspergillus welwitschiae are morphologically indistinguishable (Susca et al, 2016), species level identification was completed by analysis of a partial sequence of the internal transcribed spacer (ITS) region (ITS1/ITS4 primers) and calmodulin gene (CMD5/CMD6 primers) (Samson et al., 2014). The sequences were compared with the sequences of A. welwitschiae strains registered in the GenBank database based on nucleotide similarity, and results showed 99,64 and 100% similarity with ITS (OL711714) and calmodulin (KX894585), respectively. The sequence was deposited in GenBank with accession numbers OQ456471 (ITS) and OQ426518 (calmodulin). We also confirmed the presence of this species with specific primers (AWEL1/AWEL2) designed by Susca et al. 2020. Pathogenicity test was performed in Zemun Polje on the same maize hybrid from which the fungal species was isolated. Using artificial inoculations by the injecting conidial suspension into the silk channel, three days after 50% of plants reached the silking stage. Twenty ears were inoculated with each isolate, in four replicates (Reid et al, 1996). Inoculum was prepared from 7-day-old colonies on PDA, and 2 ml of a conidial suspension (1×106 spores/ml) was used. Control plants were inoculated with sterile water. All inoculated ears showed symptoms, similar to those from field infections. Control ears were symptomless. The fungus was reisolated and was morphologically identical to the original isolates, thus completing Koch's postulates. Based on molecular, morphological and pathogenic properties, the isolates were identified as A. welwitschiae. This is the first report of A. welwitschiae as the causal agent of black maize ear rot not only in Serbia, but also in the other countries of the Western Balkans. Given that the fungus A. welwitschiae synthesizes both ochratoxin A (OTA) (Battilani et al, 2006) and fumonisin (FB) (Frisvad et al, 2011), further studies should be focused on assessment its aggressiveness and toxicological profile.

The objective of the present study was to investigate the susceptibility of maize hybrids to the natural mycotoxins contamination: aflatoxin total (AFLA), deoxynivalenol (DON), zearalenon (ZEA) and fumonisins (FB). Hybrids were grown during two production years (2019 and 2020) in two locations. Mycotoxin contamination of maize grains was evaluated in five sweet maize hybrids. Contamination level of investigated hybrids of first cluster was mainly by below average values of ZEA (all equal to zero) and DON. Hybrids PK4 (S) 2020, PK6 (MS) 2020 and PK4 (MS) 2020 had below average values for AFLA, while remaining treatments of this cluster have mostly elevated values of this mycotoxin. Contamination level of investigated hybrids of second cluster mostly had increased values of mycotoxins ZEA and DON and below average values of AFLA. Samples did not contain fumonisins. Mycotoxin contamination were significantly affected by hybrids and years. We have established that DON and ZEA levels were influenced by the environmental conditions. There were no significant effects of location on the level of AFLA in the sweet maize hybrids. The variation in the properties of mycotoxin content (DON) was significantly influenced by hybrids, and there was no significance of hybrids x location interaction. Differences were more expressed for the content of ZEA and AFLA compared to the content of DON. Hybrid PK1 had the lowest content of DON, while it had the highest content of ZEA. Mycotoxin analyses showed that in all tested hybrids, levels of AFLA, DON, ZEA and FBs were below the maximum permissible levels stipulated by the legislation of the European Union and the Republic of Serbia in maize intended for direct human consumption. These results confirmed that the susceptibility of hybrids is one of the important risks, in addition to climatic factors, for the appearance of toxigenic fungi and their mycotoxins. Genotype tolerance is very important as a preventive measure, which indicates that breeders have to pay attention to it in sweet maize breeding programs.