Assessment of Various Nanoprimings for Boosting Pea Germination and Early Growth in Both Optimal and Drought-Stressed Environments.

One of the main climate change-related variables limiting agricultural productivity that ultimately leads to food insecurity appears to be drought. With the use of a recently discovered nanopriming technology, seeds can endure various abiotic challenges. To improve seed quality and initial growth of 8-day-old field pea seedlings (cv. NS Junior) under optimal and artificial drought (PEG-induced) laboratory conditions, this study aimed to assess the efficacy of priming with three different nanomaterials: Nanoplant Ultra (Co, Mn, Cu, Fe, Zn, Mo, and Se), Nanoplant Ca-Si (Ca, Si, B, and Fe), and Nanoplant Sulfur (S). The findings indicate that nanopriming seed treatments have a positive impact on seed quality indicators, early plant growth, and drought resilience in field pea plants established in both optimal and drought-stressed conditions. Nevertheless, all treatments showed a positive effect, but their modes of action varied. Nanoplant Ultra proved to be the most effective under optimal conditions, whereas Nanoplant Ca-Si and Nanoplant Sulfur were the most efficient under drought stress. After a field evaluation, the examined comprehensive nanomaterials may be utilized as priming agents for pea seed priming to boost seed germination, initial plant growth, and crop productivity under various environmental conditions.

First Report of Collar and Root Rot of Lettuce Caused by Plectosphaerella cucumerina in Serbia.

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.

First report of brown spot on stored apple fruits caused by Stemphylium vesicarium in Serbia.

Apple is one of the most economically important fruit crops worldwide, and fungal postharvest diseases can cause significant losses during storage (Petres 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 ß-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 ß-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 ß-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.

The Effect of Cultivation Conditions on Antifungal and Maize Seed Germination Activity of Bacillus-Based Biocontrol Agent.

Aflatoxin contamination is a global risk and a concerning problem threatening food safety. The biotechnological answer lies in the production of biocontrol agents that are effective against aflatoxins producers. In addition to their biocontrol effect, microbial-based products are recognized as efficient biosolutions for plant nutrition and growth promotion. The present study addresses the characterization of the representative of Phaseolus vulgaris rhizosphere microbiome, Bacillus sp. BioSol021, regarding plant growth promotion traits, including the activity of protease, cellulase, xylanase, and pectinase with the enzymatic activity index values 1.06, 2.04, 2.41, and 3.51, respectively. The potential for the wider commercialization of this kind of product is determined by the possibility of developing a scalable bioprocess solution suitable for technology transfer to an industrial scale. Therefore, the study addresses one of the most challenging steps in bioprocess development, including the production scale-up from the Erlenmeyer flask to the laboratory bioreactor. The results indicated the influence of the key bioprocess parameters on the dual mechanism of action of biocontrol effects against the aflatoxigenic Aspergillus flavus, as well on maize seed germination activity, pointing out the positive impact of high aeration intensity and agitation rate, resulting in inhibition zone diameters of 60 mm, a root length 96 mm, and a shoot length 27 mm.

Genetic Diversity of Pectobacterium spp. on Potato in Serbia.

Pectobacterium is a diverse genus which comprises of multiple destructive bacterial species which cause soft rot/blackleg/wilt disease complex in a wide variety of crops by employing high levels of virulence factors. During the 2018, 2019 and 2020 potato growing seasons, numerous outbreaks of bacterial wilt, stem blackleg and tuber soft rot were recorded, and symptomatic plant samples from ten localities in the Province of Vojvodina (Serbia) were collected and analysed. Bacterial soft-rot pathogens were detected in 63 samples using genus and species-specific primers. Through 16S rRNA Sanger sequencing of 19 representative isolates, the identity of P. brasiliense (73.7%), P. punjabense (15.8%), and P. carotovorum (10.5%) species were revealed. To further validate the identification, genotypic profiling of Pectobacterium strains using rep-PCR (ERIC, BOX, REP) was conducted for 25 selected isolates and the phylogenetic assessment based on four selected housekeeping genes (gyrA, recA, rpoA, and rpoS). Physiological and biochemical properties were analysed using basic microbiological tests and VITEK® 2 GN card, and pathogenicity was confirmed on cv. VR808 and cv. Desiree potato tubers and plants. This study confirmed the distinctiveness of the newly described P. punjabense in Serbia as well as the high diversity of Pectobacterium brasiliense and Pectobacterium carotovorum species in Serbia.

Medium for the Production of Bacillus-Based Biocontrol Agent Effective against Aflatoxigenic Aspergillus flavus: Dual Approach for Modelling and Optimization.

One of the leading limiting factors for wider industrial production and commercialization of microbial biopesticides refers to the high costs of cultivation media. The selection of alternative sources of macronutrients crucial for the growth and metabolic activity of the producing microorganism is a necessary phase of the bioprocess development. Gaining a better understanding of the influence of the medium composition on the biotechnological production of biocontrol agents is enabled through bioprocess modelling and optimization. In the present study, after the selection of optimal carbon and nitrogen sources, two modelling approaches were applied to mathematically describe the behavior of the examined bioprocess-the production of biocontrol agents effective against aflatoxigenic Aspergillus flavus strains. The modelling was performed using four independent variables: cellulose, urea, ammonium sulfate and dipotassium phosphate, and the selected response was the inhibition-zone diameter. After the comparison of the results generated by the Response Surface Methodology (RSM) and the Artificial Neural Network (ANN) approach, the first model was chosen for the further optimization step due to the better fit of the experimental results. As the final investigation step, the optimal cultivation medium composition was defined (g/L): cellulose 5.0, ammonium sulfate 3.77, dipotassium phosphate 0.3, magnesium sulfate heptahydrate 0.3.

Distribution, Genetic Diversity and Biocontrol of Aflatoxigenic Aspergillus flavus in Serbian Maize Fields.

Maize is one of the leading export products in the Republic of Serbia. As a country where economic development depends on agriculture, maize production plays a critical role as a crop of strategic importance. Potential aflatoxin contamination of maize poses a risk to food and feed safety and tremendous economic losses. No aflatoxin contamination of maize samples harvested in 2019 and 2020 in different localities in the Republic of Serbia was detected by the Enzyme-Linked Immunosorbent Assay (ELISA) test and High-Performance Liquid Chromatography (HPLC) method. On the other hand, the Cluster Amplification Patterns (CAP) analyses of the isolated Aspergillus flavus strains from 2019 maize samples confirmed the presence of key biosynthesis genes responsible for aflatoxin production. Artificial inoculation and subsequent HPLC analysis of the inoculated maize samples confirmed the high capacity of the A. flavus strains for aflatoxin production, pointing to a high risk of contamination under favorable conditions. Prevention of aflatoxin contamination is primarily based on A. flavus control, where biocontrol agents play a significant role as sustainable disease management tools. In this study, antagonistic activity screening of the novel strains belonging to the Bacillus genus indicated superior suppression of A. flavus strains by two Bacillus strains isolated from the rhizosphere of Phaseolus vulgaris.

Biological Control of Aflatoxin in Maize Grown in Serbia.

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.

Effect of Wheat Milling Process on the Distribution of Alternaria Toxins.

Alternaria toxins are mycotoxins produced by various Alternaria species which, besides the Fusarium species, represent the principal contaminants of wheat worldwide. As currently, only limited information on the behaviour of Alternaria toxins during processing of cereals is available, the objective of this study was to investigate the effect of the dry milling process of wheat on Alternaria toxins distribution. Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) content were analysed by high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in all milling fractions of untreated (control), fungicide-treated, Alternaria tenuissima inoculated and commercial wheat sample. After dry milling process, in last break and milling flows and by-products, increased concentration of examined Alternaria toxins was detected. TeA was quantified in almost all milling fractions in all tested wheat samples, while AOH and AME were detectable mostly in last break and milling flows and by-products. In respect to the contamination with Alternaria toxins, white flour can be considered as relatively safe product. Since Alternaria toxins are concentrated mainly in the peripheral parts of the kernel, a special attention should be given to their content in low-grade flours and milling by-products.

A New Concept to Secure Food Safety Standards against Fusarium Species and Aspergillus Flavus and Their Toxins in Maize.

Commercial maize hybrids are exposed to different degrees of ear infection by toxigenic fungal species and toxin contamination. Their resistance to different fungi and toxin relationships are largely unknown. Without this knowledge, screening and breeding are not possible for these pathogens. Seven- to tenfold differences were found in resistance to Fusarium spp., and there was a five-fold difference in ear coverage (%) in response to A. flavus. Three hybrids of the twenty entries had lower infection severity compared with the general means for toxigenic species. Three were highly susceptible to each, and 14 hybrids reacted differently to the different fungi. Differences were also observed in the toxin content. Again, three hybrids had lower toxin content in response to all toxigenic species, one had higher values for all, and 16 had variable resistance levels. Correlations between infection severity and deoxynivalenol (DON) content were 0.95 and 0.82 (p = 0.001) for F. graminearum and F. culmorum, respectively. For fumonisin and F. verticillioides ear rot, the Pearson correlation coefficient (r) was 0.45 (p = 0.05). Two independent isolates with different aggressiveness were used, and their mean X values better described the resistance levels. This increased the reliability of the data. With the introduction of this methodological concept (testing the resistance levels separately for different fungi and with two isolates independently), highly significant resistance differences were found. The resistance to different fungal species correlated only in certain cases; thus, each should be tested separately. This is very useful in registration tests and post-registration screening and breeding. This would allow a rapid increase in food and feed safety.