First report of Aspergillus welwitschiae causing maize ear rot in Serbia.

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.

Effect of Ultrasonic and Hydrothermal Treatment on Digestibility and Antioxidant Properties of Whole Wheat Flour with Different Amylose Content§.

Research background: The consumption of whole grain cereal flour contributes to increased intake of dietary fibre and phenolic compounds beneficial to human health. However, whole grain flour also has some disadvantages, such as poor baking properties and lower technological quality. Applying ultrasonic and hydrothermal treatments can provide new opportunities to modify and improve the baking and biofunctionality of flour as well as the quality of baked goods. Experimental approach: The whole grain flour samples of six wheat varieties with different amylose content were studied. The original chemical composition and viscosity profiles of the flour were determined. The flour samples were subjected to ultrasonic treatment at a frequency of 30 kHz and temperature of 40 °C for 10 min and hydrothermal treatment on a magnetic stirrer with heating for 3 min after reaching the boiling point. The treatments were carried out to determine their influence on the studied digestible and antioxidant properties of the flour. A multistep in vitro enzymatic digestibility protocol simulating the digestion process in the human gastrointestinal tract was applied to the untreated and treated whole grain flour samples. Total free phenolic compound content and total antioxidant capacity were also determined. Results and conclusions: Hydrothermal treatment had a positive effect on the digestibility of the whole grain flour, especially in waxy wheat genotypes compared to those with high amylose content, due to the formation of resistant starch. The hydrothermal treatment had an overall negative effect on the antioxidant capacity of the flour samples, while ultrasonic treatment generally increased the analytical values of total free phenolic compounds by enhancing their extractability. These findings can provide valuable information for the development of new whole wheat foods. Novelty and scientific contribution: To the best of our knowledge, this type of study of the effects of ultrasound and hydrothermal treatment on the digestibility and antioxidant properties has not yet been performed on whole wheat flour with different amylose content. Waxy and high-amylose wheat varieties are considered novel raw materials because of their unique properties in bread making, such as improved bread texture and increased dietary fibre content.

Genetic and association mapping study of wheat agronomic traits under contrasting water regimes.

Genetic analyses and association mapping were performed on a winter wheat core collection of 96 accessions sampled from a variety of geographic origins. Twenty-four agronomic traits were evaluated over 3 years under fully irrigated, rainfed and drought treatments. Grain yield was the most sensitive trait to water deficit and was highly correlated with above-ground biomass per plant and number of kernels per m(2). The germplasm was structured into four subpopulations. The association of 46 SSR loci distributed throughout the wheat genome with yield and agronomic traits was analyzed using a general linear model, where subpopulation information was used to control false-positive or spurious marker-trait associations (MTAs). A total of 26, 21 and 29 significant (P < 0.001) MTAs were identified in irrigated, rainfed and drought treatments, respectively. The marker effects ranged from 14.0 to 50.8%. Combined across all treatments, 34 significant (P < 0.001) MTAs were identified with nine markers, and R(2) ranged from 14.5 to 50.2%. Marker psp3200 (6DS) and particularly gwm484 (2DS) were associated with many significant MTAs in each treatment and explained the greatest proportion of phenotypic variation. Although we were not able to recognize any marker related to grain yield under drought stress, a number of MTAs associated with developmental and agronomic traits highly correlated with grain yield under drought were identified.

Toxigenic Species Aspergillus parasiticus Originating from Maize Kernels Grown in Serbia.

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.