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.

Bacillus-Loaded Biochar as Soil Amendment for Improved Germination of Maize Seeds.

Biochar is considered one of the most promising long-term solutions for soil quality improvement, representing an ideal environment for microorganisms' immobilization. Hence there is a possibility to design microbial products formulated using biochar as a solid carrier. The present study was aimed at development and characterization of Bacillus-loaded biochar to be applied as a soil amendment. The producing microorganism Bacillus sp. BioSol021 was evaluated in terms of plant growth promotion traits, indicating significant potential for production of hydrolytic enzymes, indole acetic acid (IAA) and surfactin and positive tests for ammonia and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production. Soybean biochar was characterised in terms of physicochemical properties to evaluate its suitability for agricultural applications. The experimental plan for Bacillus sp. BioSol021 immobilisation to biochar included variation of biochar concentration in cultivation broth and adhesion time, while the soil amendment effectiveness was evaluated during maize germination. The best results in terms of maize seed germination and seedling growth promotion were achieved by applying 5% of biochar during the 48 h immobilisation procedure. Germination percentage, root and shoot length and seed vigour index were significantly improved when using Bacillus-biochar soil amendment compared to separate treatments including biochar and Bacillus sp. BioSol021 cultivation broth. The results indicated the synergistic effect of producing microorganism and biochar on maize seed germination and seedling growth promotion, pointing out the promising potential of this proposed multi-beneficial solution for application in agricultural practices.

Genome Sequence Resource of Fusarium graminearum TaB10 and Fusarium avenaceum KA13, Causal Agents of Stored Apple Rot.

The filamentous fungus Fusarium graminearum is a well-known cereal pathogen and F. avenaceum is a pathogen with a wide host range. Recently, both species were reported as causal agents of apple rot, raising concerns about postharvest yield losses and mycotoxin contamination. Here, we report genome assemblies of F. avenaceum KA13 and F. graminearum TaB10, both isolated from fruits with symptoms of apple rot. The final F. avenaceum KA13 genome sequence assembly of 41.7 Mb consists of 34 scaffolds, with an N50 value of 2.2 Mb and 15,886 predicted genes. The total size of the final F. graminearum TaB10 assembly is 36.76 Mb, consisting of 54 scaffolds with an N50 value of 1.7 Mb, and it consists of 14,132 predicted genes. These new genomes provide valuable resources to better understand plant-microbe interaction in stored apple rot disease. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

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.

Dairy and Wine Industry Effluents as Alternative Media for the Production of Bacillus-Based Biocontrol Agents.

Food industry effluents represent one of the major concerns when it comes to environmental impact; hence, their valorization through different chemical and biological routes has been suggested as a possible solution. The vast amount of organic and inorganic nutrients present in food industry effluents makes them suitable substrates for microbial growth. This study suggests two valorization routes for whey as dairy industry effluent and flotation wastewater from the wine industry through microbial conversion to biocontrol agents as value-added products. Cultivations of the biocontrol strain Bacillus sp. BioSol021 were performed in a 16 L bioreactor to monitor the bioprocess course and investigate bioprocess kinetics in terms of microbial growth, sugar substrate consumption and surfactin synthesis, as an antimicrobial lipopeptide. The produced biocontrol agents showed high levels of biocontrol activity against mycotoxigenic strains of Aspergillus flavus, followed by a significant reduction of sugar load of the investigated effluents by the producing microorganisms. With proven high potential of whey and winery flotation wastewater to be used as substrates for microbial growth, this study provides grounds for further optimization of the suggested valorization routes, mostly in terms of bioprocess conditions to achieve maximal techno-economical feasibility, energy saving and maximal reduction of effluents' organic and inorganic burden.

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.

Taguchi Grey Relational Analysis for Multi-Response Optimization of Bacillus Bacteria Flocculation Recovery from Fermented Broth by Chitosan to Enhance Biocontrol Efficiency.

Degradation of environment is a challenge to crop production around the world. Biological control of various plant diseases using antagonistic bacteria is an encouraging alternative to traditionally used chemical control strategies. Chitosan as a well-known natural flocculation agent also exhibits antimicrobial activity. The goal of this study was to investigate a dual nature of chitosan in flocculation of Bacillus sp. BioSol021 cultivation broth intended for biocontrol applications. Experiments were performed based on L18 standard Taguchi orthogonal array design with five input parameters (chitosan type and dosage, pH value, rapid and slow mixing rates). In this study, the grey relational analysis was used to perform multi-objective optimization of the chosen responses, i.e., flocculation efficiency and four inhibition zone diameters against the selected phytopathogens. The results have indicated a great potential of a highly efficient method for removal of the Bacillus bacteria from the cultivation broth using chitosan. The good flocculation efficiency and high precipitate antimicrobial activity against the selected phytopathogens were achieved. It has been shown that multiple flocculation performance parameters were improved, resulting in slightly improved response values.

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.

First Report of Sclerotinia minor on lettuce in Serbia.

Lettuce (Lactuca sativa L.) is the world's most popular leafy salad vegetable. One of the major challenges facing lettuce producers are fungal diseases that could, under favorable conditions, devastate the harvest (Raid, 2004). During February 2021, poor growth, plant stunning and blanching of leaves of lettuce plants, cultivated in unheated plastic tunel in Potocanje (Zlatibor region), Serbia, were observed. The crowns were softened with spreading decaying lesions covered with white mycelium, particulary on the leaves near the soil surface. Approximately 2 to 3 weeks before harvest, diseased plants began to wilt and collapse. Estimated disease incidence was 50-55%. In order to identify the causal agent, symptomatic tissues from diseased plants were cut into small pieces, surface sterilized with 70% ethanol for 1 min, rinsed three times in sterile distilled water and placed on potato dextrose agar (PDA). Five isolates with uniform morphology were derived from infected tissue. The colonies had fast-growing, white, cottony aerial mycelium, producing profuse numbers (184 sclerotia/ Petri plate in average) of small, black, irregularly shaped sclerotia, less than 2 mm in diameter. Based on morfological features, the isolates were identified as Sclerotinia minor Jagger (Kohn, 1979). To confirm the species identity, the internal transcribed spacer region of nuclear rDNA of a representative isolate 15-2 was amplified using the primer pair ITS1/ITS4 (White et al. 1990). Sequence analysis of ITS region revealed 100% nucleotide identity between the isolate 15-2 (GenBank Accession No. OL423632.1) and 14 isolates of S. minor from different parts of the world (e.g., accession Nos. MK356551.1, KY707828.1, JF2798801.1). Pathogenicity tests were conducted by artificial inoculation of 55-day-old lettuce plants cv. 'Majska kraljica', grown on commercial growth substrate in l L pots. The obtained isolates were grown on PDA for 7 days and mycelial plugs, 5 mm in diameter, were cut from the margin of the colony and placed mycelium-side down on undamaged ground-level leaves of lettuce plants. Two plugs per isolate were placed onto five plants each for a total of ten replicates per isolate. Negative control plants (5) were inoculated similarly with sterile PDA plugs. Inoculated plants were covered with transparent plastic bags, sprayed with water (under the plastic) twice a day for 3 to 5 days to maintain high humidity, and kept in a growth chamber at 22°C (13 h light). After 7 to 10 days, all pathogen-inoculated plants developed lettuce drop disease symptoms, whereas the control plants remained symptomless. The pathogen was reisolated from symptomatic leaves and Koch's postulates were completed by confirming the identity of the isolates. To our knowledge, this is the first report of S. minor on lettuce in Serbia. More research is needed to better understand this disease, establish control strategies and minimize the spread of the pathogen to other lettuce-producing areas of the country. References: Kohn, L. M. 1979. Delimitation of the economically important plant pathogenic Sclerotinia species. Phytopathology 69: 881-886. White, T. J., et al. 1990. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Raid, R.N., 2004. Lettuce diseases and their management. In Diseases of Fruits and Vegetables: Volume II (pp. 121-147). Springer, Dordrecht. Founding: This work was funded by the Ministry of Education, Science and Technological Development (contract 451-03-9/2021-14/200214).

Utilization of waste glycerol for the production of biocontrol agents nigericin and niphimycin by Streptomyces hygroscopicus: bioprocess development.

Search for more environment-friendly methods for controlling plant diseases that would contribute to the goal of sustainability in agriculture is in focus. In the present study, the potential of Streptomyces hygroscopicus isolated from soil sample in the production of biocontrol agents, nigericin and niphimycin, effective against Alternaria alternata storage apple pathogen was examined. Also, modelling and optimization of medium composition for biocontrol agent biosynthesis was performed. The results showed that the optimum amount of C3H8O3, (NH4)2SO4 and K2HPO4 in the medium for Streptomyces hygroscopicus biosynthesis is 20, 0.25 and 1.46 g/L, respectively. Scale-up and validation of the obtained results performed in the 3 L laboratory-scale bioreactor showed that on the optimized medium at an aeration rate of 0.7 vvm and an agitation speed of 200 rpm, produced nigericin and niphimycin, showed high activity. Under the same conditions, cultivation of S. hygroscopicus was performed in a 7 L laboratory bioreactor in a medium with waste glycerol instead of pure glycerol. Results showed that the methanol extract of S. hygroscopicus cultivation liquid, containing nigericin and niphimycin, was high effective against two Alternaria isolates. This was confirmed in vitro by obtaining large inhibition zone diameters on A. alternata KA10 (47 mm) and T1Jg3 (44.33 mm) isolates. After successful in vitro analysis, in planta testing was performed. It was found that necrosis diameters that were measured on artificially inoculated apple fruits with A. alternata compared to necrosis diameter measured on untreated, control fruits, were 4.47 and 3.56 times smaller.

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.

Pepper Bacterial Spot Control by Bacillus velezensis: Bioprocess Solution.

Pepper bacterial spot is one of the most severe plant diseases in terms of infection persistence and economic losses when it comes to fresh pepper fruits used in nutrition and industrial processing. In this study, Bacillus velezensis IP22 isolated from fresh cheese was used as a biocontrol agent of pepper bacterial spot, whose main causal agent is the cosmopolitan pathogen Xanthomonas euvesicatoria. After optimization of the cultivation medium composition aimed at maximizing of the antimicrobial activity against X. euvesicatoria and validation of the optimized medium at the scale of a laboratory bioreactor, in planta tests were performed. The results have showed significant suppression of bacterial spot symptoms in pepper plants by the produced biocontrol agent, as well as reduction of disease spreading on the healthy (uninoculated) pepper leaves. Furthermore, HPLC-MS (high pressure liquid chromatography-mass spectrometry) analysis was employed to examine antimicrobial metabolites produced by B. velezensis IP22, where lipopeptides were found with similar m/z values compared to lipopeptides from fengycin and locillomycin families. The bioprocess solution developed at the laboratory scale investigated in this study represents a promising strategy for production of pepper bacterial spot biocontrol agent based on B. velezensis IP22, a food isolate with a great perspective for application in plant protection.

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.

Monilinia spp. Causing Brown Rot of Stone Fruit in Serbia.

Brown rot is one of the most important pre- and postharvest fungal diseases of stone fruit worldwide. In Serbia, where production of stone fruit is economically important, Monilinia laxa and M. fructigena are widely distributed. In surveys from 2011 to 2013, 288 isolates of Monilinia spp. were collected from 131 localities in 16 districts and from six hosts in Serbia. Using multiplex polymerase chain reaction, phylogenetic analysis, and morphological characterization, three species of Monilinia were identified as the causal agents of brown rot of stone fruit: M. laxa (89% of isolates), M. fructigena (3%), and M. fructicola (8%). In 2011, M. fructicola was reported for the first time on stone fruit in Serbia, with only one isolate detected. More isolates of M. fructicola were detected in 2012 (2 isolates) and 2013 (20 isolates). The presence of M. fructicola, as well as its increased frequency of detection during the survey, may indicate a change in the population structure of these pathogens, which could have an important impact on brown rot disease management in Serbia.