Emerging mycotoxins such as moniliformin (MON), enniatins (ENs), beauvericin (BEA), and fusaproliferin (FUS) may contaminate maize and negatively influence the yield and quality of grain. The aim of this study was to determine the content of emerging Fusarium mycotoxins in Serbian maize from the 2016, 2017, and 2018 harvests. A total of 190 samples from commercial maize production operations in Serbia were analyzed for the presence of MON, ENs, BEA, and FUS using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The obtained results were interpreted together with weather data from each year. MON, BEA, and FUS were major contaminants, while other emerging mycotoxins were not detected or were found in fewer samples (<20%). Overall contamination was highest in 2016 when MON and BEA were found in 50–80% of samples. In 2017 and 2018, high levels of MON, FUS, and BEA were detected in regions with high precipitation and warm weather during the silking phase of maize (July and the beginning of August), when the plants are most susceptible to Fusarium infections. Since environmental conditions in Serbia are favorable for the occurrence of mycotoxigenic fungi, monitoring Fusarium toxins is essential for the production of safe food and feed.

Aspergillus flavus is the main producer of aflatoxin B1, one of the most toxic contaminants of food and feed. With global warming, climate conditions have become favourable for aflatoxin contamination of agricultural products in several European countries, including Serbia. The infection of maize with A. flavus, and aflatoxin synthesis can be controlled and reduced by application of a biocontrol product based on non-toxigenic strains of A. flavus. Biological control relies on competition between atoxigenic and toxigenic strains. This is the most commonly used biological control mechanism of aflatoxin contamination in maize in countries where aflatoxins pose a significant threat. Mytoolbox Af01, a native atoxigenic A. flavus strain, was obtained from maize grown in Serbia and used to produce a biocontrol product that was applied in irrigated and non-irrigated Serbian fields during 2016 and 2017. The application of this biocontrol product reduced aflatoxin levels in maize kernels (51–83%). The biocontrol treatment had a highly significant effect of reducing total aflatoxin contamination by 73%. This study showed that aflatoxin contamination control in Serbian maize can be achieved through biological control methods using atoxigenic A. flavus strains.

Penicillium expansum is one of the main postharvest pathogens of several fruits including apples, resulting in decay and causing severe economic losses. Furthermore, it might represent a safety issue as the main producer of the mycotoxin patulin. In this study, we investigated the efficacy and related mode of action of the strain BBJ of the yeast Meyerozyma caribbica in controlling blue mold by P. expansum and consequent patulin contamination. The yeast M. caribbica BBJ was selected within a screening of the epiphytic population of apple fruit. It was found to significantly control the incidence of blue mold in apples, apricots, and sweet cherries, reducing in vitro P. expansum growth and patulin presence, showing toxin-degrading abilities. In addition, the results of this study showed that M. caribbica strain BBJ induced resistance in the host tissues, causing an up-regulation of apple defense-related genes, particularly coding chitinase enzyme. These experiments also indicated that yeast BBJ's biocontrol ability against P. expansum relied on competition for nutrients and production of volatile compounds. When tested on a semi-commercial scale, M. caribbica BBJ proved to be effective against apple rots, particularly as a preharvest treatment. In summary, although further large-scale trials are needed, M. caribbica BBJ has the potential to be used as antagonistic yeast against postharvest blue mold of apples and other fruits susceptible to P. expansum infection.

Apple is one of the most economically important fruit crops worldwide, and fungal postharvest diseases can cause significant losses during storage (Petreš et al. 2020). Apple fruits (cultivar Fuji) with necrosis symptoms were collected during the fall of 2022 from the cold storage facility (ULO - Ultra Low Oxygen) in Titel, Serbia. The fruits originated from the apple orchard in Titel, Serbia (45°12'47.1"N, 20°15'23.6"E). The pathogens were isolated from collected fruit samples using standard phytopathological techniques. Fruits were surface-sterilized, rinsed with sterile water, aseptically cut in half, and small fragments collected from the border of healthy and diseased tissue were placed into Petri dishes on Potato Dextrose Agar medium (PDA) and incubated at 25±1 °C in dark for seven days. The obtained 11 isolates were identified to the genus level as Alternaria (incidence 46%), Penicillium (36%), Fusarium (9%) and Stemphylium (9%) based on morphological characteristics. Pathogenicity of all isolates was confirmed on apple fruits of cultivars Fuji and Golden Delicious. The fruits were surface-sterilized, sprayed with 5 ml conidial suspension (1×10⁵ conidia/ml) and incubated at room temperature for 21 days. Symptoms developed on inoculated fruits were the same as symptoms observed on apple fruit samples collected from cold storage. Reisolation from artificially inoculated fruits resulted in colonies that morphologically corresponded with the colonies used for inoculation. Stemphylium isolate was the only one included in further research. Initial symptoms and symptoms on artificially inoculated apple fruits caused by Stemphylium sp. occurred as circular dark brown necrosis located near the calyx, without visible sporulation on the fruit surface. The isolate and reisolate formed aerial, white to light brown mycelia. The pigmentation of the culture medium was pale to dark brown. Conidia were singular, cylindrical and multicellular, brown to dark brown, 22-35.1 long and 12.6-18.9 μm wide. Based on morphological properties, isolate and reisolate were identified as Stemphylium vesicarium which is in line with the description reported by Sharifi et al. (2021) and Gilardi et al. (2022). The identification of S. vesicarium isolate was confirmed by polymerase chain reaction (PCR) by amplifying and sequencing three regions using following primer pairs: Bt2a (5'- GGT AAC CAA ATC GGT GCT GCT TTC -3') and Bt2b (5'-ACC CTC AGT GTA GTG ACC CTT GGC-3') for β-tubulin region (Nasri et al. 2015), ITS1 (5'-TCC GTA GGT GAA CCT GCG G - 3') and ITS4 (5'- TCC TCC GCT TAT TGA TAT GC-3') for ITS region (White et al. 1990), and EF1 (5' - ATG GGT AAG GAG GAC AAG AC - 3') and EF2 (5'- GGA AGT ACC AGT GAT CAT GTT - 3') for TEF-1α region (O'Donnell et al. 1998). PCR products were separated by horizontal gel electrophoresis in 1.5% agarose gel, stained with ethidium bromide, and visualization under UV light revealed amplified fragments of the expected size of 500 bp for Bt2a/ Bt2b primer pair, 600 bp for ITS1/ITS4 primer pair, and 700 bp for EF1/EF2 primer pair. The obtained amplicons were Sanger sequenced (Macrogen Europe BV) in both directions. BLASTn analysis showed the identity of amplified fragments of the isolates with sequences of S. vesicarium present in the GenBank of 100% (MT881940.1 and JQ671944.1) for the β-tubulin region, 99.40% (MT520589.1 and OR256793.1) for the ITS region, and 99.49% (DQ471090.2 and MT394642.1) for the TEF-1α region. The sequences were deposited to NCBI GenBank (Accession No. OQ653540 for the β-tubulin region, OQ678016 for the ITS region, and OR232710 for the TEF-1α region). To our knowledge, this is the first finding of S. vesicarium on apple fruits in the Republic of Serbia, and the finding of a new causal agent of postharvest apple fruit rot.

Apple blue mold, caused by the Penicillium species, is a significant postharvest disease, leading to food loss and impacting food safety due to mycotoxin contamination. This study aimed to identify the Penicillium species associated with apple blue mold in Serbia, assess their pathogenicity, and evaluate their patulin production potential. A total of 70 Penicillium isolates were collected from symptomatic apple fruit and identified as P. expansum (92.9%), P. crustosum (4.3%), P. solitum (1.4%), and P. chrysogenum (1.4%). The pathogenicity assay revealed P. expansum strains as the most virulent. Molecular detection of msas gene and HPLC analysis confirmed patulin production exclusively in P. expansum isolates. Principal Component Analysis (PCA) grouped P. expansum strains in two distinctive clusters, while P. crustosum strains clustered separately with P. solitum and P. chrysogenum, yet in distinct positions. This is the first report of P. solitum and P. chrysogenum as causal agents of apple blue mold in Serbia. The results of the study provide insights that might be useful in the development of effective control strategies for apple blue mold, ensuring consumption of healthy and safe apple fruit and apple-based products.

In March 2021, unusual plant stuning, collar, and wet root rot of lettuce (Lactuca sativa L.) during the rosette stage was observed in two commercial fields in Serbia (44°58'N, 20°32'E; 44°45'N, 20°43'E). Disease incidence in the fields (≈ 0.9 ha each) was approximately 15 and 20%, respectively. Initial above-ground symptoms were yellowing and wilting of leaves, while below-ground symptoms were collar, wet root rot, and lesions becoming necrotic. Eventually, whole plants wilted, collapsed, and died. A total of 35 symptomatic plants were collected from the fields, and diseased tissues were cut into small pieces, surface sterilized, and plated on potato dextrose agar (PDA). Isolation resulted in 20 morphologically uniform monoconidial isolates. The isolates formed white to creamy colonies, gradually becoming salmon pink, slimy, or moist in appearance, with sparse aerial mycelia. Numerous hyphal coils with conidiophores and hyaline, smooth-surfaced, ellipsoid to ovoid, septate or aseptate conidia were formed (4.5 to 10.1×1.2 to 3.7 μm (n = 100)). To confirm the species identity, the internal transcribed spacer (ITS) region and the D1/D2 region of a selected representative isolate 13-3-c were amplified and sequenced by using primer pairs ITS1/ITS4 (White et al. 1990) and N1/N2 (O'Donnell and Gray 1995), respectively. The sequences were deposited in GenBank (ITS: OR880564 and D1/D2: OR880567). Sequence analysis revealed 100% nucleotide identity with P. cucumerina isolates from different countries deposited in the NCBI GenBank, including isolate MH860704 (Vu et al. 2019) (ITS region) and isolate KY662256 (Su et al. 2017) (D1/D2 region). Neighbor-joining analysis was conducted based on the combined ITS and D1/D2 regions, and the tree was constructed with the substitution models (1,000 bootstrap). The combined phylogeny confirmed that the sequences shared a common clade with P. cucumerina. Hence, morphological, microscopic, and molecular characterization confirmed the pathogen as P. cucumerina (Palm et al., 1995; Carlucci et al., 2012). In a pathogenicity assay, 10 isolates were tested. Five 30-day-old lettuce plants (cv. Majska Kraljica) per isolate were root-dipped in the conidial suspensions (1×105 conidia/ml). The 10 inoculated plants were transplanted into 1 L pots containing sterile substrate (Floragard, Germany). Plants treated with sterile distilled water were used as controls. Plants were maintained in a greenhouse at 25 to 28°C under a 12-hour photoperiod (Cai et al., 2021). Four weeks after inoculation, stunting, chlorosis, and wilting of plants were observed, while collars and roots exhibited typical decaying symptoms. No symptoms were observed on the control plants. The pathogen was reisolated from symptomatic tissue as previously described. Koch's postulates were completed by confirming the identity of reisolates based on morphological features. To our knowledge, this is the first report of P. cucumerina on lettuce or any other crop in Serbia. P. cucumerina is already known as a pathogen of lettuce and other hosts grown in many countries worldwide, as well as in some European countries (Belgium, England, Italy, the Netherlands, and Switzerland) (Zhang et al. 2019). This emerging pathogen may cause significant economic losses in lettuce production in Serbia and in the entire Balkan region. Our results may help to develop effective management strategies based on accurate and timely identification and regular pathogen monitoring.