First report of Aspergillus parasiticus on Barley Grain in Serbia

Barley (Hordeum vulgare L.) is a secondary grain crop in Serbia used commercially for animal feed, seed, and human food applications. The production of barley in the 2016 to 2017 growing season reached a record yield of almost 400,000 metric tons (USDA 2017). Aspergillus contamination has been rare in the agroecological conditions of cereal-growing areas in Serbia. Changes in climatic factors, such as occurrence of high temperatures and prolonged droughts, increased frequency of Aspergillus spp. Species Aspergillus parasiticus was isolated from maize grain for the first time in Serbia in 2012 and from wheat grains in 2017 (Nikolic et al. 2018). We hypothesized that these pathogens can also be present in barley fields in Serbia. Barley spikes exhibiting bleaching were sampled at the beginning of June 2017 grown in northern Serbia. In severe infections, barley spikes get a dry look with awns that stand upright and firm. The incidence of the disease of the bleached spiked in the field was 15 to 20%. From each representative sample, 100 shriveled grains were collected. After surface sterilization with bleach/distilled water 1:3, 100 grains per sample (10 per Petri dish) were placed on potato dextrose agar and incubated at 25°C for 7 days. After isolation, 50% of isolates were identified as Alternaria spp., 20% as Fusarium spp., approximately 15% as Aspergillus spp. In order to reliably identify individual species of fungi, the fragments of colonies were transferred to malt extract agar (MEA) and Czapek yeast agar (CYA) and incubated in the dark for 7 days. The fungal colonies were dark green. The reverse side was pale yellow. The average colony diameter was 65 mm. Conidia were spherical and rough with thick walls. The average size of conidia was 5.1 µm. Colonies were floccose and thin on MEA and CYA. Based on growth and morphological characteristics, isolates were determined as A. parasiticus (Pitt and Hocking 2009). Molecular detection of Aspergillus species was done by using PCR-restriction fragment length polymorphism analysis of the aflR-aflJ (genes for aflatoxin biosynthesis) intergenic spacer. The restriction enzyme BglII was able to cut the PCR product of A. parasiticus at one restriction site, resulting in two fragments of 363 and 311 bp (El Khoury et al. 2011). A. parasiticus CBS 100926 was used as a reference isolate. The pathogenicity of 20 isolates was verified on a group of 20 randomly selected spikes in four replicates (Mesterházy et al. 1999). A 7-day-old culture of each isolate was used for the preparation of the spore suspension (1 × 10⁶ spores/ml). Inoculation was carried out after 50% of plants reached the anthesis stage. Groups of 20 selected spikes were sprayed from all sides with 20 ml of fungal spore suspension. Control spikes were inoculated by applying an equal amount of sterile distilled water. The infection rate was estimated after 3 weeks on a 1 to 7 scale, with 1 = 0 to 5%, 2 = 5 to 15%, 3 = 15 to 30%, 4 = 30 to 50%, 5 = 50 to 75%, 6 = 75 to 90%, and 7 = 90 to 100% infected spike area. The average infection rate was 3.2. The pathogen was reisolated from the inoculated spikes and identified as A. parasiticus, with the aim to confirm Koch’s postulates. Developed symptoms were similar to those observed on spikes collected from the field. Control spikes did not show any symptoms of the disease. These results confirmed the pathogenicity of A. parasiticus on H. vulgare. To the best of our knowledge, this is the first report of the occurrence of A. parasiticus on barley grain in Serbia. Because A. parasiticus is known to be a severe aflatoxin producer and climatic changes can increase the frequency of this fungus, further studies are necessary to improve strategies for food safety and quality